Thesis Reference - Archive ouverte UNIGE

Thesis
Characterization of α-actinin as a member of the spectrin superfamily
of proteins in "Neurospora crassa"
COTADO-SAMPAYO, Marta
Abstract
Nous avons étudié le rôle de l' α-actinine dans le développement de "Neurospora crassa". A
l'aide des outils bioinformatiques, nous avons trouvé un gêne codant pour l'α-actinine puis
nous avons caractérisé ce dernier biochimiquement. Nous avons déterminé sa localisation "in
situ" et "in vivo" pendant la germination et pendant la croissance hyphale. Bien que le rôle
exact de l'α-actinine n'ait pas pu être totalement élucidé, l'étude phénotypique du transformant
("knock-out" partiel de l'α-actinine) nous amène à penser que cette protéine participe avec
l'actine à la coordination de la germination, la formation des septa et l'établissement des
ramifications hyphales lors de la croissance du champignon. En outre, nous avons pu
déterminer que l'α-actinine est la seule protéine appartenant à la superfamille des spectrines
chez les champignons filamenteux, les levures et les Oomycètes et représente un membre
"primitif" de cette superfamille.
Reference
COTADO-SAMPAYO, Marta. Characterization of α-actinin as a member of the spectrin
superfamily of proteins in "Neurospora crassa". Thèse de doctorat : Univ. Genève, 2008,
no. Sc. 3976
URN : urn:nbn:ch:unige-18247
Available at:
http://archive-ouverte.unige.ch/unige:1824
Disclaimer: layout of this document may differ from the published version.
[ Downloaded 14/10/2016 at 16:45:29 ]
UNIVERSITÉ DE GENÈVE
Département de botanique et biologie végétale
Laboratoire de bioénergétique et microbiologie
FACULTÉ DES SCIENCES
Professeur Reto J. Strasser
Dr François Barja
Characterization of α-actinin as a member of the spectrin
superfamily of proteins in Neurospora crassa
THÈSE
présentée à la Faculté des sciences de l’Université de Genève
pour obtenir le grade de Docteur ès sciences, mention biologie
par
Marta COTADO-SAMPAYO
de
Ourense (Espagne)
Thèse n° 3976
Genève
Repromail, Université de Genève
2008
Les minutes passées à réfléchir au contenu d’une erreur
marquent plus profondément les esprits que les heures
passées à ingurgiter des théorèmes exacts. Car ces
minutes sont accompagnées d’une émotion, d’une
révolte intérieure……..
Albert Jacquard « L’Équation du nénuphar »
REMERCIEMENTS
Je désire d’abord remercier les membres du Jury: le Dr. Roland Beffa, le Professeur William
Broughton, le Professeur Reto Strasser et le Dr. Francisco Barja qui ont accepté de lire et
d’évaluer ce travail. Je tiens à exprimer ma gratitude au Dr. R. Beffa qui a soigneusement
veillé à la bonne organisation et rédaction de ce manuscrit et au Professeur R. Strasser pour
son soutien.
L’aboutissement de cette thèse a été rendu possible par le soutien porté par mon superviseur
de thèse, Francisco Barja. Arrivée à Genève comme étudiante Erasmus, un peu désorientée et
avec un projet incertain, Francisco m’a ouvert les portes de son laboratoire et m’a donné sa
confiance pour entreprendre une thèse. J’apprécie que malgré toutes les difficultés
rencontrées, il m’ait toujours soutenue dans tous mes efforts et ma motivation pour mener à
bien à ce travail. Ce projet de recherche qui se termine maintenant est le résultat de tout le
«groupe de Francisco». Grâce à la bonne entente de l’équipe propice à un travail fructueux, il
est encore possible de nos jours de travailler dans une ambiance de convivialité et de
confiance.
C’est donc avec une particulière gratitude que je remercie Malou Chappuis et Ariane Fehr
pour leur aide efficace. Merci à Cristina Andrés pour tous les bons moments partagés et pour
son si précieux soutien dans les moments difficiles.
Le mérite de cette thèse est à partager avec le Dr. Ruben Ortega qui m’a aidée par ses conseils
judicieux et ses critiques constructives tant dans la réalisation pratique que dans la discussion.
Et encore plus important il ne m’a pas laissé « achicopalarme ».
Je remercie aussi vivement le Dr. Mukti Ojha pour son aide savante et amicale, et qui m’a
sans cesse encouragée en me guidant et me donnant des conseils avisés.
Un grand merci aussi aux membres du groupe de Bioimagerie, le Dr. Christophe Bauer et
Jérôme Bosset pour sa disponibilité. À Mike Parkan pour m’avoir auxilié avec beaucoup de
patience avec mes « lacunes » dans le domaine d’informatique et bien sur pour sa bonne
humeur.
Je tiens aussi à remercier tous mes collègues du laboratoire de Bioénergétique (Abdallah
Oukarroum, Georgina Ceppi, Madeleine Fontana, Marie-France Blanc, Gert Schansker et
Dina Hanggraini) ainsi que ceux du département de Biologie végétale, spécialement
Christophe Dunand et Sonia Guimil, pour tout le soutien qu’ils m’ont apporté.
Enfin, ma reconnaissance va aussi aux nombreux membres du laboratoire, aussi bien anciens
qu’actuels, qui ont également participé plus au moins directement à ce travail. Ainsi, je
remercie Arlette Cattanéo, Pilar Okenve, Edurne Martinez, Idoia Alonso, Javier Remiro,
Greta Rubio, Enrique Raposo, Loreto Naya, Lucia Soliño, Marta Alonso, Sibylle Baruchel
(Schindhelm), Aurélia Weber, Catherine Wilson, Marco Dias, Géraldine Martinelli et
Fabrizio Molino pour leur aide scientifique mais aussi pour tous les bons moments passés
ensembles.
Bien sûr une pensée pour mes amis, spécialement Maria del Mar, Ana “pajaros”, Txema,
Cristina “bailarina”, Urko, Ana “Barja”, Gorge Faustino et Sébastien qui ont suivi de très près
l’aventure de ma thèse.
Un grand merci à Manu pour tout l’amour qu’il m’a donné malgré les heures que mon travail
lui a volé.
Enfin, merci à ma famille pour leur soutien et leurs encouragements.
Con gran alegría y satisfacción, dedico este trabajo a mis padres.
CONTENTS
Résumé
3
Summary
5
List of original publications
6
Abbreviations
7
1. GENERAL INTRODUCTION
9
1.1. CYTOSKELETON
9
1.2. ACTIN CYTOSKELETON
11
1.2.1. Actin
11
1.2.2. Actin binding proteins
13
1.2.3. Spectrin superfamily
13
1.2.4. α-Actinin
17
1.2.4.1. Functions
1.2.4.2. Isoforms
1.2.4.3. “Atypical” α-actinins
1.3. WHY STUDY FUNGI AS A MODEL OF TIP GROWTH? 20
1.3.1. Filamentous fungi
21
1.3.2. Cytoskeleton in fungi
22
1.3.3. Actin cytoskeleton in fungi
23
1.3.3.1. Actin
1.3.3.2. Actin binding proteins
2. BACKGROUND AND AIMS OF THE STUDY
25
3. MATERIALS AND METHODS
27
4. RESULTS
37
4.1. Identity of anti-αβ-spectrin immunoreacting peptides
37
in fungi and Oomycetes (Publications I and II)
4.2. α-Actinin orthologs in fungi (Publication III)
51
4.3. Characterization of α-actinin from Neurospora crassa
53
(Publication IV)
5. DISCUSSION, CONCLUSIONS AND PERSPECTIVES
67
6. REFERENCES
75
ANNEXE
95
Résumé
Caractérisation
de
l’α-actinine,
une
protéine
membre
de
la
superfamille des spectrines chez Neurospora crassa
La superfamille des spectrines est composée par des protéines qui lient l’actine. Elles participent à
l’organisation du cytosquelette et interagissent avec d’autres protéines ou structures comme la
membrane plasmique. Les protéines de ce groupe possèdent trois domaines: N-terminal « CHdomain » (Calponin Homology domain), C-terminal « EF-hand motifs » et un domaine central
composé d’un nombre variable de « spectrin repeats ». Avec ces caractéristiques, la spectrine, l’αactinine et la dystrophine/utrophine ont toujours été considérées comme des membres de la
superfamille des spectrines. Ces protéines ont été décrites dans la plupart des cellules eucaryotes.
Néanmoins, chez les plantes et les champignons, la présence de spectrine a été démontrée sur la base
des résultats obtenus par des techniques immunologiques, souvent en utilisant des anticorps
commerciaux polyclonaux. Ainsi, la présence des protéines «spectrin-like » chez les champignons
filamenteux, les levures et les Oomycetes est mise en doute car ces protéines ont seulement été
identifiées à l’aide d’anticorps dont la spécificité n’était pas suffisamment établie. De plus, chez ces
organismes, après l’analyse des génomes complètement séquencés, aucun gène codant pour la
spectrine n’a été trouvé. Par analyse de spectrométrie de masse, nous avons identifié la protéine
« spectrin-like » comme étant le facteur d’élongation 2 (EF 2) chez Neurospora crassa. Par ailleurs,
nous avons aussi identifié une protéine correspondant à la protéine de choc thermique (Hsp70) chez
l’oomycete Phytophthora infestans. La caractérisation du peptide reconnu chez N. crassa par
l’anticorps anti-αβ-spectrine ainsi que la réactivité croisée de cet anticorps ont été amplement traités
dans la partie 4.1 de cette thèse, ainsi que dans les publications I et II.
De plus, en utilisant les outils de bioinformatique (BLAST) nous avons trouvé dans la base de données
génomique de N. crassa (http://www.broad.mit.edu/annotation/fgi/) un gène (ncu06429.4) qui code
pour une protéine similaire à l’α-actinine. Nous avons utilisé la séquence de cette protéine pour
chercher leurs orthologues chez les champignons. L’α-actinine semble être le seul membre de la
superfamille des spectrines. Néanmoins, cette protéine n’est pas présente dans tous les champignons ;
chez certaines levures du groupe des Saccharomycotina, il n’y a pas d’évidence prouvant l’existence
d’α-actinine. Dans ces champignons cette protéine semblerait avoir été perdue lors de l’évolution du
fait que d’autres protéines liant l’actine pourraient complémenter sa fonction. Ce sujet a été traité en
détail dans la partie 4.2 de cette thèse et dans la publication III.
Suite à notre étude bioinformatique nous avons pu identifier un gène codant pour l’α-actinine, membre
le plus « primitif » de la superfamille des spectrines. Cette protéine a été identifiée, localisée in situ et
in vivo et caractérisée biochimiquement chez N. crassa.
3
Bien que le rôle exact de l’α-actinine chez N. crassa n’aie pas pu être totalement élucidé, l’étude
phénotypique du transformant (« knock-out » partiel de l’α-actinine) nous amène à penser que l’αactinine participe avec l’actine à la coordination d’activités telles que la germination, la formation des
septa et l’établissement des ramifications hyphales lors de la croissance. En outre, à la différence
d’autres organismes, chez N. crassa l’α-actinine est une protéine essentielle, car son absence
(« knock-out » complet de l’α-actinine) est létale pour le champignon.
4
Summary
Cell shape, cell division, polarization and tip growth are processes that have been widely studied using
fungi as models. The organization of the cytoplasm and the coordination of cell activity during the life
cycle of fungi are poorly understood but are believed to depend on a cytoskeletal system. The
cytoskeleton in fungi is composed principally of microfilaments and microtubules. The dynamics and
function of these structures are regulated by associated proteins. These proteins were first described
using immunological techniques but their existence can now be verified by the availability of a
number of complete fungal and plant genome sequences. The existence of an actin-binding “spectrinlike” protein in plants, fungi, Oomycetes and lower eukaryotic organisms has been reported. However,
these reports were solely based on immunological studies and evidence for a gene coding for spectrin
was not presented. In fungi and Oomycetes another member of the spectrin superfamily, α-actinin,
was first proposed by us to be the immunoreactive peptide and we gave this the name “spectrinrelated” protein. However, further studies demonstrated that α-actinin is not related to the protein that
is recognized by anti-spectrin antibodies in fungi and Oomycetes. Instead, this immunoreactive
peptide turned out to be a cross-reacting protein not related to the spectrin superfamily.
Assessing the characteristics of α-actinin in fungi may provide further insights into the biology of
fungi and also help to establish new links between developmental complexity and genome evolution.
In the work described here we studied the features of the α-actinin orthologous group in fungi. Using
Neurospora as a model for filamentous fungi we studied the ability of this protein to bind actin and
calcium. Our results on the localization of α-actinin and the phenotype of the α-actinin knock-out
strain suggest that α-actinin is essential for conidial germination and septum formation during hyphal
growth. The fact that α-actinin is also localized along the peripheral region suggests that this protein
may have additional functions.
5
List of original publications
The present thesis is based on the following original articles (see annexe), which are referred to in the
text by their Roman numerals:
I. Cotado-Sampayo M., Ojha M., Ortega Perez R., Chappuis M-L., Barja F., 2006. Proteolytic
cleavage of a spectrin-related protein by calcium-dependent protease in Neurospora crassa. Curr.
Microbiol. 53: 311-316.
II. Cotado-Sampayo M., Okenve Ramos P., Ortega Perez R., Ojha M., Barja F., 2008. Specificity of
commercial anti-spectrin antibody in the study of fungi and oomycetes: cross-reaction with
proteins other than spectrin. Fungal Genet. Biol. 45 (6): 1008-1015.
III. Cotado-Sampayo M., 2008. Features of α-actinin in fungi and Oomycetes. Under revision.
IV. Cotado-Sampayo M., Ortega Perez R., Seum C., Ojha M., Barja F., 2008. Characterization of
Neurospora crassa α-actinin. Under revision.
6
Abbreviations
aa
ABD
ABP
Aip1
Amp
ARP
ATP
BLAST
BCIP
bp
BSA
°C
CBD
cDNA
CDP
GCY
GFP
GST
CH
CIP
cm
2D
Da
DAB
dH2O
dist.
DMSO
DNA
DNAse
dNTP
DTT
EDTA
EF
e.g.
EGTA
EST
et al.
FGSC
Fig.
FITC
g
GFP
GST
GTP
h
HEPES
His
HMW
Hsp
i.e.
IPTG
k
kDa
Amino acid
Actin-binding domain
Actin-binding protein
Actin-interacting protein 1
Ampicillin
Actin related protein
Adenosine-5'-triphosphate
Basic local alignment search tool
Bromo-chloro-indolyl phosphate
Base pairs
Bovine serum albumin
Degree Celsius
Calcium-binding domain
Complementary DNA
Calcium-dependent protease
Glucose Casein hydrolase Yeast extract
Green fluorescent protein
Glutathione S-transferase
Calponin homology
Calf intestinal phosphatase
Centimeter
Two dimensional
Dalton
3,3'-Diaminobenzidine tetrahydrochloride
Distilled water
Distilled
Dimethylsulfoxide
Deoxyribonucleic acid
Deoxyribonuclease
Deoxyribonucleotide triphosphate
Dithiothreitol
Ethylenediaminetetraacetic acid
Elongation factor
“exempli gratia” (for example)
Ethyleneglycol-bis(2-aminoethylether)-N,N'-tetraacetic acid
Expressed sequence tag
“et alii” (and others)
Fungal genetics stock center
Figure
Fluorescein-isothiocyanate
Gram
Green fluorescence protein
Glutathione S-transferase
Guanosin-5'-triphosphate
Hour
4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid
Histidine
High molecular weight
Heat shock protein
“id est” (that is)
Isopropyl-β-D-thiogalactopyranoside
Kilo
Kilodalton
7
KIF
KO
l
LB
LMW
M
MALDI
min
μF
µm
MIPS
MOPS
Mr
MS
MYA
nm
NMDA
OD
PAGE
PBS
PCR
PDA
pH
pI
PMSF
pNA
PVDF
PVP
RNA
RNAse
rpm
SDS
SEM
sec
SR
siRNA
TBS
TCA
TOF
Tris
TRITC
Triton X-100
Tween 20
U
V
v/v
w/v
WT
YpSs
Kinesin family protein
Knock-out
Liter
Luria Bertani
Low molecular weight
Mol/l
Matrix-assisted laser desorption/ionization
Minute
Microfaraday
Micrometer
Munich center for protein sequences
Morpholinopropanesulfonic acid
Relative molecular mass
Mass spectrometry
Million years ago
Nanometer
N-methyl-D-aspartate
Optical density
Polyacrylamide gel electrophoresis
Phosphate buffer saline
Polymerase chain reaction
Potato Dextrose Agar
Potential of hydrogen
Isoelectric point
Phenylmethylsulfonyl fluoride
para-Nitroaniline
Polyvinylidene difluoride
Polyvinyl polypyrrolidone
Ribonucleic acid
Ribonuclease
Revolutions per minute
Sodium dodecyl sulfate
Simple and efficient method for transformation
Second
Spectrin repeat
Small interfering RNA
Tris buffer saline
Trichloroacetic acid
Time-of-Flight
Tris-hydroxymethyl-aminomethane
Tetramethylrhodamine isothiocyanate
tert-Octylphenoxypolyethoxyethanol
Polyoxyethylene(20) sorbitan monolaureate
Units
Volt
Volume per volume
Weight per volume
Wild-type
Yeast protein Soluble starch
8
1. GENERAL INTRODUCTION
1.1. CYTOSKELETON
The cytoskeleton is fundamental to the intracellular organization, plays an important role in cell
division and also in the communication of the cell with its environment. A large number of studies
have been performed in recent years in order to identify proteins involved in the cytoskeleton and to
understand its role in the physiology of eukaryotic cells. Processes such as the establishment of
cellular shape, cell locomotion, endo- and exocytosis, signaling, intracellular transport and cell
division depend on this complex network of protein filaments that extends throughout the cytoplasm
(Schliwa, 1986; Lloyd, 1991; Qualmann and Kessels, 2002; Smythe and Ayscough, 2006; Lanzetti,
2007). A substantial amount of molecular, biochemical and physiological information has been
obtained on the cellular organization of the cytoskeleton. Knowledge of cytoskeletal and associated
proteins is also important to gain a better understanding of numerous human diseases that depend on
cytoskeleton dysfunction. Abnormal phosphorylation of a microtubule associated protein, Tau, is
associated with Alzheimer neurofibrillary pathology (Goedert et al., 1996; Strong et al., 2006; von
Bernhardi, 2007) and point mutations in cardiac actin have been detected in familial hypertrophic
cardiomyopathy (Mogensen et al., 1999; Vang et al., 2005; Monserrat et al., 2007).
The cytoskeleton is composed of three major types of protein filaments: actin filaments
(microfilaments), microtubules and intermediate filaments (Figure 1). Each type of filament is formed
as a chain of protein monomers and can be built into a variety of structures depending on its associated
proteins.
Intermediate filaments are polymers of elongated fibrous protein monomers, such as vimentin,
keratin and lamin, which belong to a family of structurally and genetically related proteins.
Intermediate filaments are found in metazoans. Different families of intermediate filaments are
expressed in different cell types. One essential role of these filaments is to distribute tensile forces
across cells in a tissue.
9
A
B
Figure 1. Cytoskeleton in eukaryotic cell. A. Organization of actin, microtubules, and intermediate filaments
within a cell. B. Confocal image of an endothelial cell where actin filaments are shown in red, microtubules in
green, and the nuclei in blue (web images in the public domain).
Microtubules are hollow tubes formed by the assembly of heterodimers of α- and β-tubulin. The
heterodimers are arranged in longitudinal rows called protofilaments. Thirteen protofilaments are
assembled in a parallel fashion around a hollow core that is approximately 25 nm in diameter. The
microtubules are polar structures with two distinct ends: a fast-growing plus end and a slow-growing
minus end. This polarity is an important consideration in determining the direction of movement along
microtubules. The “minus” end in the cell starts in the MTOC (microtubule organizing center). Oakley
and Oakley (1989) identified a third type of tubulin, named γ-tubulin. Microtubules participate in
chromosome segregation during cell division, transport of vesicles and organelles, and cilia and
flagella movements.
Microfilaments are 4 to 7 nm wide filaments formed by actin monomers (G-actin), a globular protein
of 43 kDa. Microfilaments (F-actin) are the principal components of the actin cytoskeleton. The actin
cytoskeleton shows an organizational flexibility that allows cells to assume many shapes. In motile
cells like fibroblasts actin participates in locomotion. In mammals and many other organisms, actin is
required for muscle contraction, cytokinesis, cell-substrate interactions, endocytosis and secretion.
10
1.2. ACTIN CYTOSKELETON
1.2.1. Actin
Microfilaments are polymers of actin monomers that, together with a large number of actin-binding
proteins, form the actin cytoskeleton (dos Remedios et al., 2003; Dominguez, 2004).
Actin monomer structure
Monomeric G-actin has dimensions of ~67 × 40 × 37 Å and a molecular mass of about 43 kDa. About
40% of the structure consists of α-helices (Otterbein et al., 2001; dos Remedios et al., 2003). The actin
monomer contains four subdomains (Figure 2), a central cleft contains a high-affinity binding site for a
nucleotide (ATP or ADP) and a cation (usually Ca2+ or Mg2+). Many of the known actin-binding
proteins bind to the same loci, in the hydrophobic cleft between subdomains 1 and 3, and therefore can
be expected to compete for this binding site (Dominguez, 2004). Subdomain 2 contains a DNAse-Ibinding loop that participates in the intra-strand interactions between F-actin subunits. The function of
the DNAase I loop is unknown. However, DNAse I is a valuable tool to measure the G-actin content
of actin solutions. When DNAase I is bound by actin it no longer has the capacity to cleave DNA
(Lazarides and Lindberg, 1974; Hitchcock, 1980) and it can therefore be used to titrate actin.
Actin isoforms
The actins are classified into three groups according to their isoelectric point: α-, β- and γ-isoforms. In
mammals, there are at least six different actin isoforms, each encoded by a separate gene, and these
differ by <10% of the amino acid sequence (Vandekerckhove and Weber, 1978). In plants the number
of genes is higher; e.g. Petunia contains >l00 actin sequences in its genome (Baird and Meagher,
1987) and other plant species, such as soybean, tobacco, potato, rice, and lodgepole pine, also appear
to have dozens of actin genes (Meagher, 1991; Thangavelu et al., 1993; Meagher and Williamson,
1994).
The presence of an ancestral actin protein in bacteria was first suggested by Bork et al. (1992). This
protein, called MreB, was proposed to be the prokaryotic origin of the actin cytoskeleton (van den Ent
et al., 2001). Although the amino acid sequence homology of the MreB to eukaryotic actin is limited
to 15%, their overall size and shape are markedly similar (van den Ent et al., 2001). Furthermore,
MreB has similar actin-like cytoskeletal roles (Jones et al., 2001; Carballido-Lopez, 2006; Pradel et
al., 2006; Graumann, 2007; Vats and Rothfield, 2007).
11
Figure 2. Ribbon representation of the structure of uncomplexed actin in the ADP state (Otterbein et al., 2001).
Actin polymerization
Actin polymers assemble spontaneously through non-covalent interactions between monomeric
subunits and thus form highly dynamic structures with turnover at both ends. Actin monomers
polymerize under physiological conditions, i.e. high ionic strength (KCl concentrations >50 mM),
neutral or slightly acidic pH, high Mg2+ levels and elevated temperature (Asakura et al., 1960; Grazi
and Trombetta, 1985). The assembly of actin monomers (G-actin) into filaments (F-actin) occurs in
three steps: (1) a slow initial association into a dimer, (2) the formation of a more stable trimer that
represents the nucleus of polymerization, and (3) the elongation phase. Each actin monomer can bind
ATP. When an actin monomer is incorporated into the polymer the ATP is hydrolyzed to ADP
(Carlier, 1990, 1992; Carlier and Pantaloni, 1997; Romero et al., 2004; Zheng et al., 2007). The
elongation rate is directly proportional to the concentration of free actin and only ATP-actin
monomers are likely to participate in polymerization (Pollard, 1986). At the steady-state
concentration, the rate of actin assembly is the same as the rate of actin disassembly and the actin
filaments thus have a constant length. This phenomenon is known as treadmilling and it acts like a
motor for cell motility and pathogen locomotion (Disanza et al., 2005).
Several actin-binding proteins exist in vivo and these regulate different aspects of actin dynamics and
will be discussed in the next section.
12
1.2.2. Actin-binding proteins
Monomer availability for polymerization is regulated by actin monomer binding and actin filament
capping proteins (Weber et al., 1999; Pollard and Borisy, 2003). Capping proteins are F-actin-binding
proteins that interfere with the growth of an actin filament by blocking one of its ends. The activities
of these proteins are often regulated by signaling molecules and ions such as Ca2+ (Lehrer, 2002;
Oertner and Matus, 2005; Lange and Gartzke, 2006).
Actin-binding proteins can be grouped into three groups according to their function: (1) those that
participate in the formation of filaments from G-actin and the subsequent stability of these filaments,
(2) motor proteins that use F-actin for traction and (3) those that connect actin filaments to the cell
membrane or cross-link actin filaments to form different structures such as bundles, branching
filaments and three-dimensional networks. Examples of the latter group are all the members of the
spectrin superfamily.
1.2.3. Spectrin superfamily
The spectrin superfamily is composed of spectrin, α-actinin, dystrophin and utrophin. These proteins
are involved in the organization of the actin cytoskeleton. Proteins belonging to the spectrin
superfamily have three characteristic domains. They contain an N-terminal actin-binding domain
(ABD) and a C-terminal calcium-binding domain (CBD) linked by a rod domain. In spectrin, where
the basic unit is a heterodimer of α- and β-spectrin, the actin- and calcium-binding domains are both in
the N-terminal region of the dimer (Figure 3).
The actin-binding domain contains two calponin homology domains (CH1 and CH2 domains), both
with a low sequence similarity and functional diversity despite the predicted similarity in secondary
structure. Actin-binding studies on the isolated binding domain of α-actinin have shown that the two
domains have different roles in actin-binding (Way et al., 1992; McGough et al., 1994; Lorenzi and
Gimona, 2008). The CH1 domain by itself has a reduced affinity for F-actin. The CH2 does not have
any intrinsic actin-binding activity but contributes substantially to the interaction of the complete
actin-binding domain, perhaps by acting as a locator of low affinity docking sites on the actin filament
(Djinovic-Carugo et al., 1997; Bañuelos et al., 1998; Gimona et al., 2002). The fact that the single
calponin domain lacks actin-binding activity suggests the possibility that CH domains have additional
functions.
The calcium-binding domain is composed of EF-hand motifs located at the C-terminus (except for
spectrin, where is absent on the β-subunit). The EF-hand motif found in the spectrin superfamily
13
shares structural homology with calmodulin. For this reason, the calcium-binding domain has also
been named the calmodulin-like domain.
EF-hand regions are usually paired helix-loop-helix structures involved in the coordination of up to
two divalent cations, usually calcium but occasionally magnesium (Tufty and Kretsinger, 1975). The
binding of calcium to the EF-hands induces a conformational change that is implicated in the
regulation of the actin- binding activity of the protein (Lundberg et al., 1992; Trave et al., 1995).
However, divergent evolution has led in some members of the spectrin superfamily to a set of EFhands that no longer chelate calcium (Nakayama and Kretsinger, 1994).
α-actinin (100 kDa)
CH
CH
EF EF
CH-domain
Plekstrin Homology
EF-hand
Src3 Homology
Septrin repeat
β-spectrin (220 kDa)
WW domain
ZZ domain
Cys-rich domain
α-spectrin (240 kDa)
Dystrophin (420 kDa)
Utrophin (395 kDa)
Figure 3. Schematic representation of the different members of the spectrin superfamily (adapted from
Broderick and Winder, 2002).
The rod domain is composed of several repeats called spectrin repeats because they were initially
described in spectrin (Speicher and Marchesi, 1984). The folding of the spectrin repeats consists of
three α-helices in a coiled-coil assembly, where the three helices wrap around each other (Pascual et
al., 1996; Djinovic-Carugo et al., 1999, 2002; Broderick and Winder, 2005). The number of spectrin
repeats varies within the spectrin superfamily and ranges from 2–4 in α-actinin to 17–24 repeats in
spectrin, utrophin and dystrophin (Broderick and Winder, 2005). The repeat length has also been
14
found to differ between α-actinin, spectrin, and dystrophin, with 114–125, 106, and 109 residues,
respectively. This is mainly due to differences in the inter-helical loop length (Parry et al., 1992).
Spectrin superfamily evolution
Since gene codings for any member of the spectrin superfamily of proteins have not been found in the
bacterial and plant genomes available to date, it has been suggested that this superfamily appeared in a
primitive unicellular organism belonging to the animal kingdom (Virel and Backman, 2004). The
amino acid sequences and the structure analysis of spectrins, α-actinin and utrophin/dystrophin
proteins suggest that all three protein families arose from a single common ancestral protein that was
α-actinin-like (Byers et al., 1989 and 1992; Dubreuil, 1991).
Phylogenetic analysis indicated that the α-actinin-1 from the protozoan Entamoeba histolytica is the
earliest diverging α-actinin, followed by the α-actinin of Encephalitozoon cunculi (Virel and
Backman, 2004). These two proteins have the shortest rod domain, with only one spectrin repeat. The
presence of five repeats in the rod domain of Trichomonas vaginalis α-actinin has been suggested
(Addis et al., 1998; Bricheux et al., 1998), but only the first of these repeats shows some similarity
with those found in other α-actinins (Bricheux et al., 1998). An intragenic duplication gave rise to two
spectrin repeats (SR1 and SR4). This group of α-actinins is present in fungi and E. histolytica (αactinin-2). A subsequent, second, intragenic duplication added two more repeats (SR2 and SR3) (Virel
and Backman, 2004, 2007).
The most likely scenario for the evolution of the spectrin superfamily of proteins is that it began with
the introduction of seven repeats between repeat 2 and 3 of α-actinin, producing an elongated “αactinin”. At this point the dystrophin/utrophin lineage presumably diverged from the αβ-spectrin
lineage (Figure 4). The next step consisted of a duplication of the seven-repeat block in the elongated
“α-actinin” and the insertion of a repeat between the two seven-repeat blocks. The protein was split
into two parts at this inserted repeat (Figure 4A). Modern α- and β-spectrin evolved out of these two
different fragments. One α-helix (a0) of the cleaved repeat became the N-terminal end of what is now
known as α-spectrin and the two last α-helices (b17) became the end of what is now known as βspectrin (Figure 4A). A triple helix bundle, which is the characteristic structure of an entire repeat, is
completed when b17 binds to a0 to form the spectrin heterotetramer (Figure 4B) (Pascual et al., 1997;
Thomas et al., 1997; Viel, 1999).
The evolution of the spectrin superfamily can be divided into two phases. A first active phase is
characterized by intragenic duplication and concerted evolution. In this phase gene duplication
produced the α-actinin, dystrophin/utrophin and spectrin lineages. Just before arthropod/vertebrate
15
divergence the evolution of the spectrin superfamily entered a new, more stable phase (~500 MYA)
(Thomas et al., 1997).
A
B
Figure 4. A. Model for the evolution of the spectrin superfamily of proteins (adapted from Pascual et al., 1997;
Thomas et al., 1997; Virel and Backman, 2004). B. Spectrin heterotetramer (the functional unit of spectrin). All
the SR dark and light blue are similar.
16
The three lineages of the spectrin superfamily of proteins are present in all metazoans. In sequenced
bacterial and plant genomes, there is no evidence of genes coding for any member of the spectrin
superfamily. In protozoans, fungi and Oomycetes the superfamily is represented by the α-actinin
protein (Virel and Backman, 2004, 2007; Publications I and II). However, in plants, fungi and
Oomycetes the presence of 240–220 kDa spectrin-like proteins has been reported based on studies
using anti-spectrin antibodies (Michaud et al., 1991; Reuzeau et al., 1997; Kaminskyj and Heath,
1995; Holzinger et al., 1999; Degousée et al., 2000; Heath et al., 2003; Slaninová et al., 2003). This
discrepancy between the bioinformatics data and the immunological studies will be discussed in more
detail in Publications I and II.
The following section is focused on the basic principles of α-actinin structure and function. More
information about this protein is given in Publications III and IV, which represent the main part of the
thesis.
1.2.4. α-Actinin
α-Actinin is the smallest member of the spectrin superfamily. The functional unit is a homodimer
(Blanchard et al., 1989; Viel, 1999; Ylänne et al., 2001). There is antiparallel binding of two α-actinin
monomers, with the actin-binding domain (ABD) of one monomer facing the calcium-binding
domains (CBD) of the other (Figure 3). This organization gives α-actinin the ability to cross-link actin
filaments in a calcium-dependent manner (Tang et al., 2001). Another group of regulators,
phosphoinositides, are also able to regulate the interaction of α-actinin with actin filaments but these
can also regulate the association/dissociation of α-actinin with integrins, another class of α-actininbinding proteins (Fukami et al., 1992; 1996; Fraley et al., 2005). α-Actinin can also be phosphorylated
by kinases such as focal adhesion kinases. Focal adhesion plaques are an elaborate network of
interconnecting proteins linking actin stress fibers to the extracellular matrix. The phosphorylation of
α-actinin reduces its affinity for actin and prevents its localization to focal adhesion plaques (Izaguirre
et al., 2001; von Wichert et al., 2003). Moreover, an increase in the level of α-actinin phosphorylation
on tyrosine 12 of the ABD weakens the linkages formed between integrins and the cytoskeleton and
alters the focal adhesion dynamics (Rajfur et al., 2002; von Wichert et al., 2003). These observations
support the possibility that the phosphorylation of α-actinin may serve to modulate the
coupling/uncoupling of integrins to the cytoskeleton (Zhang and Gunst, 2006).
17
1.2.4.1. Functions
α-Actinin has many biological functions. In striated muscle it is the major thin filament cross-linking
protein in the muscle Z-disc (Suzuki et al., 1976; Fay et al., 1983; Luther, 2000), connecting actin
filaments of adjacent sarcomeres. In non-muscle cells, α-actinin is a major component of stress fibers,
a contractile structure analogous to the more organized units found in striated muscle cells (Otey and
Carpen, 2004).
α-Actinin is also found in adhesion sites close to the plasma membrane, where it cross-links cortical
actin to different adhesion and trans-membrane proteins, such as catenin (Knudsen et al., 1995) and
integrins (Otey and Carpen, 2004), and serves as a linker between trans-membrane receptors and the
cytoskeleton. In synapsis α-actinin-2 may play a role in the localization of the neurotransmitter
receptor NMDA and its modulation by Ca2+ (Wyszynski et al., 1997; Rycroft and Gibb, 2004; Franzot
et al., 2005).
It has been proposed that α-actinin participates in cytokinesis in fission yeast (Wu et al., 2001).
In addition, a number of important human diseases are caused by α-actinin dysfunction. Mutations in
α-actinin-4 cause a form of familial focal segmental glomerulosclerosis (FSGS) (Kaplan et al., 2000),
which is a common nonspecific renal lesion characterized by regions of sclerosis in some renal
glomeruli and often results in loss of kidney function. Different studies have found a relationship
between α-actinin dysfunction and different types of cancer, such as colorectal cancer (Honda et al.,
2005; Craig et al., 2007), lung cancer (Honda et al., 2004), breast cancer (Guvakova et al., 2002) and
neuroblastoma (Nikolopoulos et al., 2000).
1.2.4.2. Isoforms
Four isoforms of α-actinin exist in almost all vertebrate organisms: α-actinins-1, -2, -3 and -4
(MacArthur and North, 2004) classified on the basis of their Ca2+ affinity. α-Actinins-1 and -4 are
generally located in non-muscle cells and they have conserved EF-hand motifs that bind Ca2+. In
contrast, the muscle-isoforms α-actinins-2 and -3 have lost their Ca2+-binding ability. Furthermore,
alternative splicing generates additional isoforms in some vertebrates (Parr et al., 1992;
Kremerskothen et al., 2002; Honda et al., 2004). Only one gene coding for α-actinin has been found in
the invertebrate organisms studied to date. Therefore, the three isoforms described for Drosophyla are
probably due to the occurrence of alternative splicing (Roulier et al., 1992).
18
1.2.4.3. “Atypical” α-actinins
The rod domain of “atypical” α-actinins reported for some protozoa (Trichomonas vaginales and
Entamoeba histolytica), fungi and Oomycetes (Addis et al., 1998; Bricheux et al., 1998; Wu et al.,
2001; Virel and Backman, 2004, 2006, 2007; Virel et al., 2007; Publication I) is composed of one or
two spectrin repeats instead of the four observed in classical α-actinins (Virel and Backman, 2007)
(Figure 5). In these organisms there is generally only one α-actinin gene; the presence of different
isoforms due to alternative splicing has not been reported. However, in the protozoa Entamoeba
histolytica two proteins coded by two different genes have been characterized (Virel and Backman,
2006, Virel et al., 2007). “Atypical” α-actinin seems to represent the only member of the spectrin
superfamily in protozoa, fungi and Oomycetes (Virel and Backman, 2004; Publication I). It has been
proposed that this is the ancestor from which dystrophin, utrophin and spectrin evolved in two phases
(see section 1.2.3).
Figure 5. Diagrammatic representation of the “atypical” α-actinins. (CH: Calponin homology; SR: spectrin
repeat; CC: coiled coil region; CBD: Calcium-binding domain). The CC region from Phytophthora infestans is
shorter than that in T. vaginalis, corresponding to the accommodation of three and four putative SR, respectively.
The controversial reports of spectrin-like proteins in fungi and the relevance of the atypical fungal αactinins for the understanding of the evolutionary history of this superfamily make these organisms an
excellent model to obtain further insights into the evolution of this superfamily. The study of the
function of α-actinin in tip growth could reveal new roles for this actin-binding protein.
19
1.3. WHY STUDY FUNGI AS A MODEL OF TIP GROWTH?
Fungi represent the second largest group of organisms after insects, with about 1.5 million species –
most of which are filamentous fungi (Hawksworth, 2001). Fungi play an important role in the decay of
organic material and nutrient recycling.
The relationship between plants and fungi is sometimes positive, leading to the formation of symbiotic
structures inside plant roots. The mycorhizae form a group of organisms that are capable of such a
positive interaction. In other cases, fungi can be pathogenic agents and cause significant damage to
agricultural crops. Plant diseases caused by fungi include rusts, smuts, and leaf, root, and stem rots.
Fungi are also agents of animal diseases. From evolution point of view, fungi are more chemically and
genetically similar to animals than other organisms, making fungal diseases difficult to treat.
Fungi are also important in the pharmaceutical and food industries. They are used to produce enzymes
and secondary metabolites such as antibiotics and also participate in beer, champagne, cheese and
bread production (Hesseltine, 1965; Hersbach et al., 1984; Mapari et al., 2005; Schuller and Casal,
2005; Menacho-Márquez and Murguía, 2007; Wang and Lin, 2007).
The experimental tractability of fungi makes these organisms among the most important models in
fundamental research. Important knowledge in biochemistry, genetics and molecular biology has been
acquired from studies on fungi. Fungal cellular physiology and genetics share key components with
animal cells, including multicellularity, cytoskeletal structures, development and differentiation,
sexual reproduction, cell cycle, intercellular signaling, circadian rhythms, DNA methylation,
regulation of gene expression through modifications of the chromatin structure, and programmed cell
death (Colot and Rossignol, 1999; Borkovich et al., 2004; Dunlap and Loros, 2004; Galagan et al.,
2003; Galagan and Selker, 2004). The shared origins of the genes responsible for these fundamental
biological functions in humans and fungi continue to make the study of the fungal genes of vital
interest to human biology.
Hyphae of filamentous fungi belong to the most polarized cellular structures found in nature. The
study of fungal tip growth has been widely used to increase our understanding of the physiology of
other highly polarized cells, such as root hairs, pollen tubes and neurons. Common aspects of, as well
as differences in, cytoskeleton organization and function between these types of cells have provided
important insights into the relationship between the cytoskeleton and cell growth.
20
1.3.1. Filamentous fungi
In filamentous fungi polar extension is needed for vegetative growth and the development of complex
tissues. For several reasons, tip-growing cells represent an ideal system to study cell expansion. Fungi
concentrate their growth machinery at one cell surface site and only at this site does robust growth
occur. Localized growth implies that all material required for growth has to be present at, or delivered
to, the surface area where cell expansion takes place. Furthermore, the growth rate of the expanding
cell surface area in a tip growing cell is much higher than the growth rate of a cell that distributes the
growth machinery more or less evenly over its surface. This allows changes in growth rate and
direction to be observed more easily.
When the spores of filamentous fungi germinate, nuclear division is accompanied by a series of
ordered morphological events, including the switch from isometric to polar growth. As growth
continues, the hyphae become compartmentalized with the addition of more septa, and lateral branches
emerge from basal compartments.
Most of the studied filamentous fungi are of industrial interest, e.g. Aspergillus nidulans and Ashbya
gossypii (Steiner et al., 1995; Harris, 1997; Wendland et al., 1999; Momany and Taylor, 2000;
Kaminskyj, 2001; Goldman and Kafer, 2004; Guest et al., 2004; Oakley, 2004; Gattiker et al., 2007).
Other examples are plant pathogens such as Uromyces appendiculatus, Magnaporthe grisea, Botrytis
cinerea and Ustilago maydis (Barja et al., 1998; Dijksterhuis, 2003; Hamer and Talbot, 1998; Banuett
and Herskowitz, 2002; Silva et al., 2006; Ebbole, 2007; Klosterman et al., 2007). Examples of nonpathogenic fungi include the ascomycete N. crassa (Borkovich et al., 2004; Galagan and Selker, 2004;
Dunlap, 2006; Dunlap et al., 2007) and the chytridiomycete Allomyces arbuscula (Ojha, 1996; Ojha
and Barja, 2003).
The ascomycete N. crassa is the main experimental model used in this work, but other members of
this “phylum” have also been studied (M. grisea and B. cinerea). The complete sequence of these three
fungal genomes has been annotated by the Broad Institute (http://www.broad.mit.edu/annotation/fgi/).
N. crassa is an ascomycete that grows on semisolid media by forming colonies that spread. This
fungus was first described as an infectious agent in French bakeries and today is used in diverse
research programs.
The ascomycete Magnaporthe grisea is a plant pathogenic fungus that is responsible for an important
disease in rice, rice blast, but can also infect other agriculturally important cereals. This fungus has
been used as a model to understand plant-pathogen interactions.
Botrytis cinerea is another plant pathogen that affects many plant species, although its most notable
hosts are wine grapes. The fungus is usually referred to by the name of its asexual (anamorph) form
21
because the sexual phase is rarely observed. The sexual form (teleomorph) is called Botryotinia
cinerea.
In the work described here we also studied the chytridiomycete Allomyces arbuscula, which is a water
fungus that is distributed throughout the world and is particularly abundant in warm climates. The
filamentous cells of Allomyces divide in a characteristic dichotomous pattern. The life cycle can
alternate between two stages, gametophytic and sporophytic, reflecting the capacity for both sexual
and asexual reproduction. The reproductive structures are located at the end of the hyphae and are
separated by complete septa.
Phytophthora infestans, an oomycete in the kingdom of Stramenopila, has a filamentous phenotype
but is no longer considered as a fungus. In contrast to the fungi, Oomycetes are more closely related to
plants than to animals. Whereas fungal cell walls are made primarily of chitin, the cell walls of
Oomycetes are built mainly of cellulose. Ploidy levels and biochemical pathways are different
between these two kingdoms. P. infestans is an important potato pathogen that causes late blight and
has considerable economic impact.
The mode of growth of filamentous fungi is supported by the extreme polarization of the cytoskeleton
and endo-membrane network, which allows the long-range transport of vesicles containing precursors
required for apical extension to the tip region (Bourett and Howard, 1991; Roberson and Vargas,
1994).
1.3.2. Cytoskeleton in fungi
The principal components of the fungal cytoskeleton are microtubules and microfilaments. The
presence of intermediate filaments is more controversial and has only been reported in a few studies
(May and Hyams, 1998; McConnel and Yaffe, 1993; Geitmann and Emons, 2000). These cytoskeletal
elements are required for a variety of cellular processes including polarized growth, organelle
movement and positioning, secretion, endocytosis, cell division and chromosome segregation. As in
other eukaryotic cells, fungal microtubules are known to function in mitosis and chromosome
segregation (Morris and Enos, 1992; Thaler and Haimo, 1996; Jung et al., 1998). The importance of
microtubules for the establishment of polar growth and tip growth varies depending on the fungal
species (Oakley and Morris, 1980; Caesar-Ton That et al., 1988; Barja et al., 1993; Sawin and Nurse,
1998; Heath et al., 2000; Horio, 2007). Microtubules cooperate with microfilaments to control cell
shape, cell division and intracellular transport (Momany and Hamer., 1997; Schott et al., 2002;
Bretscher, 2005).
22
1.3.3. Actin cytoskeleton in fungi
1.3.3.1. Actin
Fungi contain either one or only a few actin genes (Tarkka et al., 2000; Helgason et al., 2003). In N.
crassa the presence of three isoforms, α- β- and γ-actins, has been described by Barja et al. (1991).
Nevertheless, as in other filamentous fungi and yeast there is only one bona fide actin gene, indicating
that the three isoforms may be products of post-transcriptional modifications. However, actin-related
proteins such as ARP 1, ARP 3 and RO 4 have recently been identified in Neurospora. Some of these
proteins share the same molecular weight and sequence similarity as actin (Robb et al., 1995; Tinsley
et al., 1998; Lee et al., 2001). Therefore, the reported three isoforms found in Neurospora could be the
result of a cross-reaction of anti-actin antibodies with these actin-related proteins.
F-Actin appears in fungi in two principal forms: patches and cables (Heath et al., 2000; Walker and
Garrill, 2006). Patches are usually localized in growing regions of yeast or filamentous fungi. Cables
are described in yeast in a polarized manner from the bud along the mother cell. The organization
observed in yeast is less evident in filamentous fungi such as N. crassa or Aspergillus (Xiang and
Plamann, 2003), where the actin cytoskeleton appears principally as patches associated with the
hyphal tip and cell cortex (Barja et al., 1991, 1993; Heath et al., 2000). The absence of actin bundles
in some filamentous fungi has been explained in terms of the difficulty of preserving these structures
during the preparation of samples used for immunofluorescence microscopy (Heath, 1987; Harold and
Harold, 1992; Kaminskyj and Heath, 1994). However, in Aspergillus short actin cables were described
in the tip region of the hyphae (Pearson et al., 2004; Virag and Harris, 2006). In the basidiomycete
Ustilago maydis and the Oomycetes both structures, cables and patches, form the actin cytoskeleton
(Bachewich and Heath, 1998; Banuett and Herskowitz, 2002; Walker et al., 2006).
Actin is also localized in the cytoplasm and forms a diffuse network of microfilaments in the septa of
filamentous fungi (Harris et al., 1994; Capelli et al., 1997; Rasmussen and Glass, 2005, 2007) and in
the contractile actin rings of yeast (Pruyne and Bretscher, 2000; Schott et al., 2002; Kamasaki et al.,
2007).
The actin-binding proteins (ABP) form part of the actin cytoskeleton. These regulate its dynamics and
mediate the interaction with other proteins. The actin cytoskeleton also contains actin related proteins
(ARPs).
23
1.3.3.2. Actin-binding proteins
ABPs have been described principally in yeast. There are, e.g., twinfilin and cofilin/ADF, which sever
actin filaments (Moon et al., 1993; Okada et al., 2006; Moseley et al., 2006); profilin and formin,
which regulate the assembly of G-actin into filaments (Haarer et al., 1990; Sagot et al., 2002;
Evangelista et al., 2003; Kovar et al., 2005; Takaine and Mabuchi, 2007). In addition, there are
capping proteins, which regulate microfilament length (Amatruda and Cooper, 1992; Sizonenko et al.,
1996; Nakano and Mabuchi, 2006), fimbrin and α-actinin, which promote the bundling of
microfilaments (Adams et al., 1989; Wu et al., 2001; Goodman et al., 2003), and myosin, which is
involved in organelle movement (Watts et al., 1987; Matsui, 2003). Some ABPs were first described
in Saccharomyces as Aip1 (Amberg et al., 1995) and were shown to collaborate with cofilin, capping
the barbed ends of filaments severed by cofilin (Okada et al., 2006; Okreglak and Dubrin, 2007).
As far as filamentous fungi are concerned, there are some reports on proteins involved in the assembly
and stability of actin filaments such as formin. A. nidulans and N. crassa encode a single formin
(Xiang and Plamann, 2003). The A. nidulans formin, SepA, localizes to both septation sites and hyphal
tips, suggesting that filamentous fungi use site-specific regulatory mechanisms to control forminmediated actin polymerization (Harris et al., 1997; Sharpless and Harris, 2002). Genes encoding for
myosin proteins have been found in the genomes of N. crassa and A. nidulans (Xiang and Plamann,
2003; Steinberg, 2007) and has been characterized in these two organisms (van Tuinen et al., 1986;
McGoldrick et al., 1995; Osherov et al., 1998; Yamashita et al., 2000; Takeshita et al., 2002) and in
other filamentous fungi (Woo et al., 2003; Schuchardt et al., 2005; Weber et al., 2006). In N. crassa,
an actin-binding protein of 47 kDa with the same intracellular distribution as actin has been reported
(Capelli et al., 1997). This protein, which is named p47 and identified as EF1α (elongation factor
1α) (Taillefert, 1988), also binds to calmodulin (CaM). The actin-p47-CaM complex may participate
in the relationship between the actin cytoskeleton and protein synthesis machinery.
So far, only a relatively small number of ABPs has been identified in filamentous fungi. However, the
completion of their genome sequence will allow the identification of a large number of the ABPs in
these organisms. For example, most of the Saccharomyces genes coding for the actin cytoskeleton,
including the actin-binding proteins, have orthologs in N. crassa (Borkovich et al., 2004).
24
2. BACKGROUND AND AIMS OF THE STUDY
The cytoskeleton in fungi is not yet well understood. The functions of actin and tubulin have been
established to some extent by observing the effects of anti-cytoskeleton drugs on cell phenotype or on
the in situ localization of these proteins (Caesar-Ton That et al., 1988; Hoang-Van et al., 1989; Barja
et al., 1993; Riquelme et al., 1998; Torralba et al., 1998; Czymmek et al., 2005). Tools, such as
constructs of green fluorescence protein (GFP)-fusion proteins, improvements in mutant constructs
and bioinformatics have revealed more details on the composition and dynamics of the cytoskeleton
network. Nowadays, we try to understand the complexity of this structure through the interaction that
occurs between cytoskeletal proteins and other cellular structures, a process that is mediated by a
plethora of associated proteins.
We were interested in a group of actin-binding proteins, the spectrin superfamily, that participate in
the organization of the actin cytoskeleton and its connection with the plasma membrane in metazoans.
In the case of neurons, several studies report the essential role of spectrin in tip growth (Morris, 2001;
Spira et al., 2003). Initial studies on “spectrin-like” proteins in other highly polarized cells like root
hairs, pollen tubes and oomycetes and fungal hyphae assign “spectrin-like” roles in the process of
apical growth (Michaud et al., 1991; Kaminskyj and Heath, 1995; Bisikirska and Sikorski, 1997;
Reuzeau et al., 1997; De Ruijter et al., 1998, 2000; Holzinger et al., 1999; Degousée et al., 2000;
Braun, 2001; Heath et al., 2003; Slaninová et al., 2003; Toquin et al., 2006). In this thesis I provide
strong evidence for the absence of spectrin genes in any of the completed plant and fungal sequenced
genomes. Furthermore, the antibody used to report the presence of spectrin in these organisms crossreacts with proteins other than spectrin. α-Actinin is the only member of the spectrin superfamily in
fungi and this represents an early step in the present model for the evolution of this superfamily.
The goal of this thesis was to characterize the α-actinin protein in Neurospora crassa and to establish
the relationship between α-actinin and actin. This would allow a better understanding of the function
of the actin cytoskeleton in processes such as spore germination or growth of the mycelium tip. In
addition, elucidation of the structure and function of α-actinin in N. crassa will allow a comparison
with other better characterized metazoan models and lead to the possible discovery of new functions
for this protein in fungi. The first target of the study was to determine the veracity of the presence of a
“spectrin-like” protein in fungi and Oomycetes and to identify the peptide detected by the commercial
anti-spectrin antibody (commonly used so far in the scientific comunity) in these organisms.
25
For this purpose we planned to:
•
Look for the gene coding for a putative spectrin in the completed fungal genome
databases.
•
Perform mass spectrometric analysis on the peptide reacting with the anti-αβ-spectrin
antibody in N. crassa and P. infestans.
•
Use bioinformatics tools to study the features of the α-actinin protein in fungi and
Oomycetes.
•
Construct
a
GST
Neurospora
α-actinin
fusion
protein
for
biochemical
characterization.
•
Produce polyclonal anti-α-actinin using the GST-fusion protein as an immunogen for
further biochemical characterization of the fungal α-actinin.
•
Localize the protein in situ by immunodetection.
•
Construct a GFP-α-actinin fusion protein for localization of the protein in vivo.
•
Study of α-actinin knock-out Neurospora strain to provide an insight into protein
function.
In this thesis I provide strong evidence for the absence of spectrin genes in any of the completed plant
and fungal sequenced genomes. Furthermore, the antibody used to report the presence of spectrin in
these organisms cross-reacts with proteins other than spectrin. α-Actinin is the only member of the
spectrin superfamily in fungi and this represents an early step in the present model for the evolution of
this superfamily. Studies on this fungal protein may help to provide more details of the evolutionary
history of the spectrin superfamily as well as to give a greater insight into the function of the actin
cytoskeleton in fungi.
26
3. MATERIALS AND METHODS
Materials
Unless otherwise indicated, chemicals were obtained from Sigma-Aldrich/Fluka, Merck, Bio-Rad,
Amersham, and Roche Diagnostics (Mannheim). Restriction enzymes and buffers as well as other
DNA-modifying enzymes were purchased from New England Biolabs (Frankfurt am Main), Promega
and Roche. For the PCR-reactions the Primus 25 advanced PCRSystem (PeqLab) was used.
Antibodies were obtained from Sigma-Aldrich. Oligonucleotides were provided by Microsynth
(http://www.microsynth.ch/).
Fungal strains and cell culture
Neurospora crassa
Wild type N. crassa (FGSC 262, strain St. Lawrence STA 4) was obtained from the Fungal Genetics
Stock Center, School of Biological Sciences, Kansas City, MO. In order to produce large quantities of
macroconidia, the fungus was first grown on solid “Davis and De Serres” medium (Davis and De
Serres, 1970), for 3 days at 33°C in the dark and then at 25°C in artificial light for 4 days. Conidia
were harvested and inoculated at an inoculum density of 5x106 conidia/ml of Vogel’s minimal
medium (Vogel, 1956) enriched with 1.5% or 2% sucrose. The culture was incubated at 30°C for 0, 6,
12, 18 hours on a rotary shaker at 150 rpm.
“Davis and De Serres” medium with slight modification (1 liter)
5 g Na and K Tartrate, 3 g NaNO3, 0.5 g MgSO4.7H2O, 0.1 g CaCl2.2H2O, 3 g KH2PO4, 0.1 g
NaCl, 10 g Sucrose, 10 ml Glycerol, 0.1 ml Oligoelements*1, 0.1 ml FeCl3 (sol. 0.5%), 0.05
ml Biotine*2.
The pH value was adjusted to 5.6 with 1N KOH. Medium was dispensed in 150 ml
Erlenmeyer (20 ml/Erlemeyer) and solidified with 2% agar.
Vogel’s miminal medium (1 liter)
2 g NH4NO3, 2.5 g Citrate de Na.2H2O, 5 g KH2PO4, 0.2 g MgSO4.7H2O, 0.1 g CaCl2.2H2O,
0.1 ml Oligoelements*1, 50 μl Biotin*2 .
*1Solution of oligoelements (100 ml)
5.0 g Citric Acid, 5.0 g ZnSO4, 1,0 g Fe(NH4) 2 (SO4) 2.6 H2O, 5 mg H3BO3, 0.25 g CuSO4.
H2O, 0.05 g MnSO4.2H2O, 0,05 g Na2MoO4.H2O.
*2Solution of Biotin
Biotin 1 mg, in 10 ml 80% Ethanol (stock 4°C, during 6 months)
27
Magnaporthe grisea
M. grisea (wild type P1.2 strain) was kindly donated by Dr. M-H. Lebrun (Unité Mixte de Recherche,
Centre National de la Recherche Scientifique/ BayerCropScience, Lyon). M. grisea was grown on
solid rice medium with small pieces of filter for a few days until the filters were colonized and white
mycelium appeared.
For storage the colonized filters in solid rice medium containing M. grisea were put into a sterile
container and placed for at least 3 days in an oven at 37°C until they were dried, then stored at –20°C.
Mycelia for protein extraction were obtained from liquid culture in TNK medium containing 0.2%
Yeast extract and 1% Glucose (Ou, 1985). The cultures were allowed to grow for 48 hours in the dark
in a shaker (150 rpm). The average weight of mycelium collected per liter of liquid medium was 2.5 g.
Solid Rice Medium (200 ml)
4 g Rice powder, 0.4 g Yeast extract, 4 g Agar.
TNK Medium (1 liter)
2.0 g NaNO3, 2.0 g KH2PO4, 0.5 g MgSO4.7H2O, 0.1 g CaCl2.2H2O, 4 mg FeSO4.7H2O, 1 ml
Oligoelements*.
*Solution of Oligoelements (100 ml)
0.79 g ZnSO4.7H2O, 60 mg CuSO4.5H2O, 10 mg H3BO3, 20 mg MnSO4.2H2O, 14 mg
NaMoO4.2H2O.
Botrytis cinerea
B. cinerea (BO47) was kindly donated by Dr. R. Beffa (BayerCropScience, Lyon). Mycelia were
grown in solid potato dextrose agar medium (Difco) for one week on the bench with day periods of
light. Spores were then harvested by flooding the culture with water and separated from the mycelial
fragments by filtration. Spores obtained from a Petri dish (9 cm diameter) culture were used to
inoculate 200 ml of liquid potato dextrose broth medium (24 g/l, Difco). Mycelia were harvested after
24 hours of growth with agitation (150 rpm) at 25°C.
Allomyces arbuscula
A. arbuscula strain Burma LD was grown on filter pads placed on solid YpSs medium (Emerson,
1941). Zoospores were induced by shifting the filters in sterile Petri-plates containing 30-40 ml sterile
28
distilled water (water active liberation of zoospores) according to a procedure described by Ojha and
Turian (1981). Zoospores were inoculated in CGY medium (Turian, 1963) and grown for periods of 6,
12 and 18 hours with forced aeration.
Solid YpSs (1 liter) (Emerson, 1941)
7.5 g Soluble starch, 2 g Yeast extract, 0.5 g K2HPO4, 0.25 g MgSO4.7H2O, 15 g Agar.
GCY Medium (1 liter)
1.0 g K2HPO4 * , 0.2 g MgSO4.7H2O, 0.1 g NaCl, 0.1 g CaCl2.2H2O, 0.02 g FeCl3.6H2O, 3.0 g
Casein Hydrolysate, 5.0 g Sucrose, 0.1 g Yeast extract.
The pH was adjusted to 6.8 with 125 μl/l of 12 N HCl. *K2HPO4 was dissolved in water,
sterilised separately and added to medium before use.
Phytophthora infestans
P. infestans (PT78) isolate was kindly donated by Dr. R. Beffa (BayerCropScience, Lyon). Mycelium
was first grown on pea-agar (125 g of pea cooked and grinded in 1 liter of H2O, 2% agar). Sporangial
inoculum was prepared from a 8 to 12 days culture. The sporangia were detached from the mycelia by
flooding the culture with water and separated from the mycelial fragments by filtration. Sporangia
were inoculated in V8 liquid medium (50 ml tomato juice/liter of distilled water, the pH was adjusted
to 5 if necessary) at a final concentration of 105 sporangia/ml. Mycelia were harvested after 72 h of
growth in the dark at 20°C without agitation.
Protein extraction
To optimise the conditions for spectrin extraction, we tested different experimental conditions and
protocols for cell disruption including:
a)
several
detergents:
Triton-X-100,
Triton-X-114,
Empigen
1%,
Octyl
β-D
glucopyranoside,
b)
Sodium Dodecyl Sulfate (SDS) extraction. Dry fungal powder was resuspended in
buffer containing 4% SDS, 5% 2-β−mercaptoethanol, 5% sucrose, 10 mg of insoluble
polyvinil polypirrolidone (PVP) and boiled for 3 min before centrifugation (13000 x g;
20 min),
c)
Trichloroacetic Acid (TCA)-Acetone extraction (Granier, 1988). The dry powder was
resuspended in buffer containing 10% TCA, 0.07% 2-β−mercaptoethanol in cold
acetone and kept at -18°C for 1h. After 15 min centrifugation at 20000 x g the
supernatant was removed and the pellet was rinsed for 1 h at -18°C with cold acetone
29
containing 0.07% 2-β−mercaptoethanol. The rinsing solution was removed with caution
and the pellet vacuum-dried for 1 h. The pellet was resuspended in extraction buffer [5
mM Hepes-KOH pH 7.5; 2 mM EGTA, 2 mM dithiothereitol (DTT)],
d)
different concentrations of NaCl (0 to 100 mM) in 20 mM Tris-HCl pH 7.5,
e)
cells fixed in 3% paraformaldehyde before extraction,
f)
cell wall digestion with Lysing enzyme (Sigma 5 mg/ml) for 20 min before extraction,
g)
buffer with Vanadate, a phosphatase inhibitor, in order to reduce the susceptibility of
spectrins to cleavage by μ-calpain (Nedrelow et al., 2003),
h)
protein extraction from the cell wall-less mutant Neurospora crassa (FGSC 1118
fz;sg;os-1).
The standard procedure adopted to study the evolution of spectrin in growing mycelia was the
following: cells from different stages of development were harvested by filtration through Millipore
filters (pore size 0.5-1 μM; Millipore Corporation, Bedford, MA /USA), washed twice with distilled
water, frozen in liquid nitrogen and ground in a mortar kept at a low temperature using liquid nitrogen.
The frozen powder was suspended in cytoskeleton stabilizing buffer (2-4 ml per mg powder) of Abe
and Davies (1995) with slight modifications, containing 5 mM Hepes-KOH, pH 7.5, 250 mM sucrose,
15 mM Mg(OAc)2, 2 mM EGTA, 2 mM dithiothreitol (DTT), 25 mM K2O5S2, 10% glycerol and 0.5%
polyvinylpyrrolidone and the following protease inhibitors: 1 mM benzamidine, 2 mM
phenylmethylsulfonyl fluoride (PMSF), 2 μg/ml each of leupeptin, chymostatin, trasylol; or Complete
EDTA-free Protease Inhibitor Cocktail Tablets (Roche Applied Science). The homogenates were
incubated at 4°C with gentle agitation for 20 min and then centrifuged at 6000xg for 15 min at 4°C.
The supernatant was recovered for in vitro proteolysis, analysis by SDS-PAGE and immunoblotting.
The protein concentration in supernatants was measured according to Bradford (1976) with Bovine
Serum Albumine (BSA) as a standard.
One-dimensional gel electrophoresis
The proteins in the crude extract were separated on SDS-PAGE and native gels, according to
Chrambach and Rodbard (1971) and Laemmli (1970), respectively. The gels were stained with
Coomassie brilliant blue R-250. Samples of 50 μg or 10 μg of protein were loaded on each lane of a
15x10 cm gel or mini-gels respectively. Various molecular weight markers were utilized (for SDSPAGE gels: Precision Plus Protein Standards from BioRad, LMW-SDS Marker Kit from Amersham
Biosciences and Prestained Protein Marker IV from PeqLab. For non-denaturing gels: HMW Native
Marker Kit from Amersham Biosciences).
30
Two-dimensional electrophoresis
Proteins were first separated on isoelectrofocusing (IEF) gels according to O’Farrell (1975). The first
dimension gel was prepared in glass tubes and loaded with 300 μg of protein. The run condition used
was 400 V at room temperature for 19 hours and 1 hour at 500 V.
After equilibration, the gels were placed in a 6.5-10% SDS-PAGE gel according to Chrambach and
Rodbard (1971) in order to separate the proteins by their molecular weight. Gels could be stored for at
least one month at -20°C.
Gels where stained with Coomassie Blue or transferred to the appropriate membrane (nitrocellulose
for immunoblot assays or PVDF, ImmobilonP for EDMAN-sequencing).
Immunoblotting
The proteins were electrophoretically transferred to nitrocellulose as described by Towbin et al.
(1979). After blocking with TBS (10 mM Tris pH 7.5, 0.15 M NaCl) 0.5% Tween-20 and 5% BSA,
the nitrocellulose sheet was incubated with the primary antibody and then with goat peroxidaseconjugated secondary antibodies (dilutions of antibodies were prepared in TBS + 0.5% BSA + 0.5%
Tween-20). Washing solution was the same as blocking solution without BSA. The proteins were
revealed with 0.5 mg/ml of 3,3’-diaminobenzidine tetrahydrochloride (DAB) in 100 mM Tris-HCl, pH
7.5, containing 0.03% H2O2.
Mass spectrometric identification and EDMAN sequencing analysis of proteins
Protein spots corresponding to the immunoreacting peptides were excised from the 2D-gels and used
for further analysis. Proteins were subjected to trypsin digestion and subsequent identification by
MALDI-TOF MS, performed as a service by Alphalyse A/S (Odense, Denmark) and by the Section of
Pharmaceutical Sciences (University of Geneva). For N-terminal amino acid sequencing, also known
as EDMAN sequencing, the spots were transferred to a PVDF membrane. Analyses were performed
by the Analytical Research Services (University of Basel, Switzerland).
Immunofluorescence
Cells were fixed with 3% paraformaldehyde (v/v) in 50 mM phosphate buffer, pH 7.4, for 30 min to 1
hour at room temperature. Partial digestion of the cell wall was performed by incubation of the
31
mycelia for 5 to 10 minutes in a solution (5 mg/ml) of lysing enzyme (Sigma), in phosphate buffer, pH
6.5. The cells were rinsed three times with phosphate buffer and plasma membrane permeabilized with
0.1% Triton X-100 in the same buffer for 10 min. Triton was then removed by five washes in
phosphate buffer. All operations were performed at room temperature.
Permeabilized cells were blocked in phosphate buffer containing 3% BSA and then incubated 2 hours
at room temperature with the first antibody prepared in the same buffer. Samples were rinsed in
phosphate buffer and re-incubated for 1 h at RT with fluorescein-isothiocyanate (FITC) or crystalline
tetramethylrhodamine isothiocyanate (TRITC) goat anti-rabbit antibodies (Sigma F9887 and T5268,
respectively). After a final rinse in phosphate buffer, the cells were mounted in p-phenylenediamineglycerol (1 mg/ml p-phenylenediamine in 78% glycerol) containing 2.5 μg/ml DAPI for staining the
nuclei and examined with an epi-illumination microscope (Zeiss axioplan) or Leica SP2 Confocal
microscope. Both microscopes were equipped with selective filter combinations to visualize FITC and
TRITC-fluorescence patterns.
Cells treated for 4 hours with sordarin (50 μg/ml), a specific inhibitor of EF2, were also used in
immunofluroescence assays and compared with untreated hyphae.
Electron microscopy and immunogold
Cells were fixed at room temperature by the addition of a concentrated solution of fixative to obtain a
final concentration of 0.5% glutaraldehyde (v/v), 4% paraformaldehyde (v/v). This was done to
maintain the integrity of the culture and to minimize any alteration due to fixation. Samples were
collected by centrifugation, pre-embedded in 1.5% agar at 46°C, dehydrated respectively in 70% and
100% ethanol (for 30 min each), in 2:1 (v/v) ethanol : LR White resin (Polysciences), 1:1 (v/v) ethanol
: LR White, followed by 1:2 (v/v) ethanol : LR White for 1h and finally in 100% LR White for 24 h at
50°C for polymerization.
Ultrathin sections were made and taken on nickel grids, incubated for 2 h at room temperature in 50
mM phosphate buffer (K2HPO4/KH2PO4) pH 7.0 containing 2% BSA, 0.05% (v/v) Tween-20, and
then for a further 2 h with specific antibodies. The sections were rinsed with 0.05% Tween-20 in 50
mM phosphate buffer pH 7.0 and incubated with the secondary antibody conjugated to 20 nm gold
particles. After incubation, sections were successively washed with phosphate buffer, stained for 10
min in 2% uranyl acetate, 5 min in Reynold’s lead citrate and examined at 60 kV in a Philips M410
transmission electron microscope.
32
Proteolysis of cell-free extracts by the calcium-dependent protease CDP II
The cell-free extract, obtained as described earlier, was digested with 0.11 μg/μl CDP II (Ojha and
Wallace, 1988) (specific activity: 21 mM para-Nitroaniline (pNA) released μg-1 min-1) in 20 mM TrisHCl, pH 7.4, containing 4 mM EGTA, 1% β-mercaptoethanol and 10 mM free Ca2+. The reaction was
performed in a total volume of 75 μl, at enzyme: protein ratios of 1:100, 1:50 and 1:10. After
incubation at 37°C for 30 min, the reaction was stopped by the addition of 25 ml of 4X loading buffer
(Laemmli, 1970). The proteins were denatured in boiling water for 5 min, separated by SDS-PAGE,
transferred to nitrocellulose membranes and immunorevealed with spectrin polyclonal antibodies.
Reaction mixtures either without calcium or with leupeptine (10 mM), an inhibitor of CDP II activity,
were used as controls.
In situ proteolysis by CDP II
Hyphae from 12 h cultures were prepared as described earlier for immunofluorescence. Before
blocking, the cells were incubated for 1 hour at 37°C with CDP II in the enzyme reaction buffer
described above. The hyphae were then washed five times, for 5 min each, with phosphate buffer and
immunorevealed as described earlier (Ojha and Barja, 2003).
Construction of Neurospora GST-fusion proteins
Total RNA was isolated from hyphae after 18 hours of growth using TRIZOL reagent (Sigma)
according to the supplier’s protocol and treated with DNAse (Ambion, Cat. 1906). cDNA synthesis
was done using the Promega reverse transcription kit (A3802). The primers used for amplification of
the coding sequences of elongation factor 2 (ncu07700.4) and the full length and fragments of the αactinin (ncu06429.4) are shown in Figure 6A, where the restriction site added in the PCR product to
perform the cloning into the expression vector is also indicated. As the ncu06429.4 gene is 3084
nucleotides in length, we took advantage of an AatII site in the middle of the sequence to make the
construct in two steps. The PCR fragments were first cloned in the plasmid pGEMTeasy (Promega)
and then digested by BamHI and NotI to be introduced in the pGEX4T.1 plasmid (Amersham,
Biosciences) opened with these two enzymes.
33
A
B
Figure 6. Primers used for Neurospora GST-fusion protein constructs. A. Table with all the primers, indicating
the name of the target Neurospora gene, the name and sequence of the primer, and the restriction enzyme name
corresponding to the restriction site introduced with each primer. B. Chart of primer position in the gene coding
for α-actinin protein.
Bacterial transformation
pGEMTeasy constructs were transformed in Escherichia coli DH5α using the SEM-method (Inoue et
al., 1990). SEM-competent E. coli cells were thawed on ice. 100 µl of cell suspension were mixed
with 5 µl (10 ng) of the ligation (pGEMTeasy constructs). The bacteria were then incubated on ice for
30 min, at 42°C for 2 min and put immediately on ice for 5 min. The cells were then plated on LB 2%
agar plates containing 100 μg/ml Ampicillin (LB-Amp).
For electro-transformation of bacterial cells with the plasmid containing the expression constructs,
electrocompetent E. coli cells (BL.21) were thawed on ice. 45 µl of cell suspension was mixed with 5
µl of DNA (10 ng approximately) and placed in a pre-chilled sterile electroporation cuvette (BioRad,
distance between the electrodes: 2 mm). After a pulse of 2.5 kV and 25 µF, 950 µl of LB medium was
34
added immediately. The cells were gently shaken for 45 min at 37°C and then plated on LB-Amp agar
plates.
Fusion protein expression
After selection of clones expressing the fusion protein, growth conditions were evaluated for optimal
expression. In most cases, we adopted the following protocol: the bacteria were grown in LB-Amp
liquid medium at 37°C to an A600 of 0.4-0.5, and then protein induction was performed by adding 0.1
M Isopropyl-βD-thiogalactopyranoside (IPTG) at 20 °C for 10 hours to avoid inclusion body
formation. Proteins from induced and uninduced recombinant E. coli BL21 were extracted by
sonication in extraction buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, and 1 mM EDTA),
centrifuged, and separated on SDS-PAGE gel. The GST-α-actinin was purified by chromatography on
GSTrap FF affinity columns.
Immunization and preparation of anti-serum against Neurospora α-actinin
Antibody production was performed as described by Barnes et al. (1998). Prior to immunization, sera
of two rabbits were tested against N. crassa extracts for background exclusion or cross reactivity. The
rabbits were immunized for a period of one month, with 450 μg of GST-α-actinin protein. The sera
were tested weekly against the antigen preparation by Western blotting, starting from day 15 following
the first immunization.
Construction of Neurospora α-actinin-GFP fusion proteins
The α-actinin-egfp gene fusion was constructed by PCR amplification of the sequence encoding the αactinin gene (ncu06429.4), including the 930 bp of the N-terminal extension before the region coding
for the first CH-domain. The PCR was carried out using the oligonucleotides Actinin_forward (5’CGCCGCGGATCCTGGAGATGCTGGGGGTGGAG-3’)
and
Actinin_reverse
(5′-GT
CACGTTAATTAAATGATACCCATTCGGCTT -3′). This led to the insertion of BamHI and PacI
restriction sites (underlined) used to clone the gene in the egfp gene containing plasmid pMF272
constructed by Freitag et al (2004).
Transformation of Neurospora crassa
Transformation of N. crassa his-3 mutant (FGSC 9014; ridRIP1 mat A his-3) and heterokaryon
transformant selection were performed as described previously (Margolin et al., 1997; Freitag et al.,
2004).
Acquisition of live cell images
Cultures were prepared in liquid medium as describe above. For acquisition of images during long
times (12 hours), germinating spores were deposited on 8 chambered coverglass (Lab-Tek®, ref
35
155411) and covered with a small block of Vogel 1.5% agar medium in order to maintain the cells in
the same plane. GFP fluorescence was recorded with a Leica AF6000LX microscope. For the
acquisition of images during shorter times, 12 hour-old hyphae were deposited in similar culture
chambers as described above and observed with Leica SP2 microscope. Images were processed with
Adobe Photoshop 6.0.
Neurospora crassa heterokaryon knock-out (KO) strain
We asked the Neurospora Genome Project (Colot el al., 2006) to construct an α-actinin KO strain. It is
now available at the Fungal Genetic Stock Center (FGSC11835) as a heterokaryon because the
homokaryon KO strain was lethal.
Cell wall stain with calcofluor
Slides coated with Vogel’s medium containing 1.5% sucrose were inoculated with conidia and grown
at 25°C for 8 hours in a wet chamber. The mycelium was stained with 10 μM calcofluor (Fluorescent
Brightener 28) for 3 minutes and covered with a coverslip. An Orthoplan epi-illumination microscope
(Leica) equipped with fluotar optics and a selective filter combination was used to visualize calcofluor
fluorescence patterns. Fluorescent micrographs were obtained with a Leica-DFC490 camera and
processed with Adobe Photoshop 6.0.
36
4. RESULTS
4.1. Identity of anti-αβ-spectrin immunoreacting peptides in fungi and Oomycetes
Immunoblot
The spectrin immunoanalog proteins in fungi showed varying molecular weights under denaturing
conditions. In the ascomycetes N. crassa and M. grisea, a single band of 100 kDa was detected with
the anti-chicken αβ-spectrin (Fig. 1B in Publication I; Fig. 1A in Publication II), three bands
corresponding to 65, 105 and 230 kDa in the chitrydiomycete A. arbuscula (Figure 7), and a single
lower band corresponding to a relative mass of 67 kDa was observed in the oomycete P. infestans
(Fig. 1A in Publication II). Further studies in N. crassa suggested that this protein was present in the
same amount in all states of development during the vegetative life cycle (Fig. 1B in Publication I).
The specificity of the antibody used for the characterization of the spectrin immunoanalog protein in
fungi was demonstrated using human erythrocyte spectrin in a competition assay, showing progressive
diminution of the immuno-reacting band with increasing concentration of the competing human
spectrin (Fig. 1D in Publication I). However, the reaction of the antibody with the purified human
protein was weaker than with the fungal peptide (Fig. 1C in Publication I).
When crude extracts from Neurospora were immunoblotted in non-denaturing conditions two bands
were found (Figure 8, lane b). In an SDS-PAGE gel the lower band was confirmed to correspond to
the 100 kDa peptide (Figure 8, lane c) and the higher one corresponded to a 70 kDa peptide in
denaturing conditions (Figure 8, lane d), similar to a minor band that sometimes appeared in
immunoblotted crude extracts of Neurospora (Figure 8, lane e).
The Isoelectric point (pI) of this peptide was analyzed in a 2D gel; the predominant immuno-reacting
band had a molecular weight of 100 kDa and a pI in the range 6.5– 7.0 (Fig. 1E in Publication I; Fig.
2A in Publication II). The spot obtained in the 2D gels was subsequently identified by two different
techniques: (a) MALDI-TOF MS and (b) EDMAN sequencing. In both cases the results indicated that
the 100 kDa protein detected by the polyclonal anti-chicken αβ-spectrin corresponded to the
elongation factor 2 (EF2) in N. crassa (Fig. 2A and C in Publication II). The EF2 of Neurospora has a
predicted molecular weight of 95 kDa and a pI of 6.3.
The GST-elongation factor 2 fusion protein was also recognized by the commercial anti-spectrin
antibody used in this work (Fig. 1B in Publication II). An additional band of about 65 kDa, which
reacted strongly with the antibody, was co-purified with the fusion protein. We analyzed this peptide
to determine if it was a degradation product of the GST recombinant protein or a protein from the
bacterium. Analysis by MALDI-TOF MS revealed it to be GroEL from Escherichia coli.
37
The immunoreacting band in extracts from P. infestans differed considerably from the band obtained
in the analyzed Ascomycetes and did not correspond to the theoretical Mr and pI of EF2 in these
organisms. For this reason, we also attempted to identify this peptide and it was found to be heat shock
protein Hsp70 (Fig. 2B-C in Publication II).
38
Figure 7. Immunoblot of the Allomyces arbuscula protein revealed with anti-chicken αβspectrin antibody. Both lanes correspond to different crude extracts prepared under the same
conditions.
39
Figure 8. Relationship between the peptides from Neurospora revealed with the anti-chicken
αβ-spectrin antibody in non-denaturing (lanes a and b) and denaturing (lanes c-e) conditions.
(lane a) N. crassa crude extract on a native coomassie stained gel; (lane b) immunoblot; lanes c and
d correspond to the bottom and top bands from the native gel, respectively; (lane e) N. crassa crude
extract. Mr refers to the relative mass of the markers in the native PAGE gel.
41
Immunofluorescence
In all of the studied fungi the spectrin immunoanalog protein was localized at the peripheral region of
the cell and concentrated in the hyphal tip during the exponential phase of growth. A localization
study on this protein in N. crassa (Fig. 2A–E in Publication I) and A. arbuscula (Figure 9) during
development showed an iso-diametric signal in the germinating conidia. When the polarized germ tube
appeared, the fluorescence was concentrated in the peripheral region of the hyphae. In some cells we
observed that the fluorescence signal was concentrated as a cap in the hyphal tips and “branch
initials”. This concentrated signal may correlate with high growth activity. The same localization was
found in M. grisea (Figure 10) and P. infestans (Figure 11).
As they were growing, Neurospora hyphal cells were treated with sordarin – a drug that blocks the
interaction of EF2 with ribosomes – and the immunoreacting peptide was delocalized to the cytoplasm
(Fig. 3 in Publication II).
Immunogold
Immunogold labeling was performed in ultra-thin sections of N. crassa (Fig. 3 in Publication II) and
A. arbuscula (Figure 12). This study confirmed the association of the protein detected by the antichicken αβ-spectrin antibodies with the peripheral region of the cell. Control experiments with antispectrin antibody pre-absorbed with human spectrin or the use of only secondary antibodies showed
the absence of labeled grains, thus confirming the specificity of the primary antibody (data not
shown).
In vitro and in situ digestion with CDP II
Incubation of a crude extract from N. crassa with the protease CDP II under optimal conditions for
enzyme activity resulted in a diminution of the relative intensity of the band at low enzyme to protein
ratios (1:100, 1:50) and the complete absence of a band at a ratio of 1:10. The protein remains
unaffected in the absence of Ca2+ in the reaction mixture or in the presence of a specific protease
inhibitor (Leupeptin). Incubation of the extract without CDP II did not show any appreciable
proteolysis of the immuno-reacting protein, indicating that there was no other endogenous protease
targeting this protein under the test conditions (Fig. 4A in Publication I).
The digested protein in vitro appears as dots localized within the peripheral region of the cell instead
of localization along the plasma membrane and capped at the tip (Fig. 4B-C in Publication I).
43
The immunorevealed protein with anti-chicken αβ-spectrin antibody and the protease CDP II were colocalized in growing tips, as shown in Fig. 2F–I in Publication I. Outside of this region the
localization differs: the immunoreacting protein was predominantly associated with the plasma
membrane whereas CDPII showed diffuse cytoplasmic distribution.
44
Figure 9. Immunolocalization of the protein revealed with anti-chicken αβspectrin antibody in Allomyces arbuscula. A) Isodiametric distribution in
germinating zoospore with high fluorescence signal concentration along the plasma
membrane. B) Predominant antigen localization in growing rhizoid and tip of early
stages of germ tube formation. C) Distribution along the plasma membrane after 12
hours of growth. D) Predominant localization in hyphal tips after 18 hours of culture
growth (window: detail of rhizoid). Bar 10 μm
45
Figure 10. Immunolocalization of the protein revealed with anti-chicken αβ-spectrin
antibody in Magnaporthe grisea. Fluorescence signal distributed along the plasma membrane
(A-C) and in a branching tip (D). Bar 10 μm.
Figure 11. Immunolocalization of the protein revealed with anti-chicken αβ-spectrin antibody
in Phytophthora infestans. Fluorescence signal distributed along the plasma membrane in the
apical (A) and sub-apical region (B). Bar 10 μm.
47
Figure 12. Immunogold labeling of the protein revealed with anti-chicken αβ-spectrin
antibody in Allomyces arbuscula. A) General view of the apex showing predominance of the
antigen along the peripheral region of the cell, B) higher magnification of a portion of the tip. C)
Thin section of a rhizoid showing distribution of the signal along the plasma membrane. (cw: cell
wall; pm: plasma membrane; m: mitochondria; n: nucleus)
49
4.2. α-Actinin orthologs in fungi
Spectrin-like proteins have so far only been described in plants and fungi (Publication II, Table 1)
using the criteria of cross-reactivity with commercial anti-spectrin antibodies. We have demonstrated
here that the antibody predominantly used for these studies (anti-chicken αβ-spectrin) cross-reacts
with other proteins. A search in the fungi and oomycetes databases was initiated to verify the presence
of spectrin-related proteins in these organisms.
The first (unsuccessful) approach to search for spectrin-like proteins in fungi (M. grisea and N. crassa
genome sequences) and Oomycetes (Phytophthora sojae and Phytophthora ramorum genome
sequences, partial EST sequences of P. infestans) involved a homology search (BLAST tool) using the
entire spectrin amino acid sequences from human and chicken spectrins. The screening of the N.
crassa genomic database using the sequence of the domains defining the spectrin family (CH-domain,
spectrin repeats and EF-hand domain) gave a gene, ncu06420.4, coding for a hypothetical protein with
the three domains. This protein was closer in structure and sequence to an α-actinin, another member
of the spectrin superfamily. We used this protein to find its orthologs in other fungi and Oomycetes
(Publication III, Table 1).
This orthologous group is currently collected in the MIPS database and has been the object of two
recent publications concerning the bioinformatics approach (Virel and Backman, 2007; Publication
III).
51
4.3. Characterization of α-actinin from Neurospora crassa
Over-expression of α-actinin and α-actinin domains in a GST system and antibody production
The GST fusion proteins constructed were found to vary in their level of expression and solubility. In
general, both expression and solubility increased when the culture time was prolonged at low
temperature (25°C). The full length α-actinin-GST protein, containing the N-terminal extension and
characteristic domains (Figure 6B, Material and Methods), was used as an immunogen to produce
specific antibodies against N. crassa α-actinin. The resulting polyclonal antibody was named antiNeurospora α-actinin and has been used for several biochemical analyses described in Publications II
and IV and in this part of this work.
Immunochemical detection of proteins reacting with anti-Neurospora α-actinin antibody
The anti-Neurospora α-actinin antibody reacted against α-actinin from different ascomicetous fungi.
In N. crassa the antibody detected an 80 kDa peptide corresponding in molecular weight to the protein
containing the two N-terminal CH-domains, the rod domain and C-terminal EF-hand motifs (Figure
13; Fig. 1B, lane d in Publication II). In Magnapothe grisea the antibody detected a 72 kDa peptide
also corresponding to a protein with the specific α-actinin domains (Figure 13; Fig. 1B, lane e in
Publication II). The antibody was highly reactive against the 80 kDa α-actinin protein from a Botrytis
cinerea crude extract (Figure 13). In Phytophthora infestans an immunoreacting band with antiNeurospora α-actinin antibody was not obtained under the test conditions used (Fig. 1B, lane f in
Publication II).
We also tested the antibody against the three over-expressed fragments of the N. crassa α-actinin,
corresponding to the Actin-Binding Domain (ABD), the rod domain and the Ca2+-binding domain. The
antibody showed poor affinity for ABD (approaching the background signal). The principal epitopes
for the anti-Neurospora α-actinin antibody seem to be in the rod and Ca2+-binding domains (Figure
14).
Characterization of Neurospora α-actinin properties
The actin- and calcium-binding properties of Neurospora α-actinin have been described in detail in
Publication IV. The results show that calcium binds to α-actinin from N. crassa with the same affinity
as the α-actinin from chicken gizzard. Since we used recombinant GST-α-actinin for this binding
assay, the GST protein was used as a negative control and shows that this tag does not have affinity for
calcium (Fig. 4 in Publication IV). A co-sedimentation assay and electron microscopy were used to
analyze the actin-binding properties of Neurospora α-actinin. This protein binds actin in a calcium-
53
dependent manner (Fig. 2 in Publication IV) and cross-links the actin microfilaments to organize them
in parallel structures as bundles (Fig. 3B in Publication IV).
Localization of α-actinin in N. crassa
Neurospora α-actinin clearly localized within the septum (Fig. 5C-D and Fig. 7A-B in Publication IV;
Figure 15). Results obtained from the in vivo localization revealed that the α-actinin is only present at
this location during septum formation (Figure 15; Fig. 7 in Publication IV). Immunofluorescence and
GFP signal were found at the germination site in the conidia and in the tip of the emerging tube (Fig.
5A and Fig. 6A–C in Publication IV). α-Actinin immunolocalizes at the peripheral region of the cell in
growing hyphae (Fig. 5B in Publication IV). This localization was not confirmed by the α-actinin GFP
fluorescence.
Phenotype of the heterokaryon α-actinin knock-out strain of Neurospora
We approached the Neurospora Genome project (Colot et al., 2006) to create an α-actinin knock-out
strain. The heterokaryon knock-out strain was deposited in the FGSC. The homokaryon strain was
defined as lethal. The heterokaryon mutant in Davis and De Serres medium (1970) showed a
shortening of aerial hyphae (Fig. 8A in Publication IV) and a delay in colony expansion in Vogel 2%
saccharose plates in comparison to the wild type (Figure 16). The morphology of the hyphae was
different with respect to their branching pattern, showing a predominantly dichotomous phenotype
compared to sympodial branching in the wild type (Fig. 8B in Publication IV).
Immunochemical characterization of “α-actinin-GFP-expressing” and “heterokaryon α-actinin
knock-out” strains of N. crassa
We examined the crude extract of the α-actinin-GFP-expressing and wild type (wt) Neurospora strains
with the anti-Neurospora α-actinin antibody. This antibody reacted with the 80 kDa peptide both in
the wild type and the transformed strains (Figure 17, lanes a', b'), but additional bands appear at 110
and 140 kDa in the transformed strain (Figure 17, lanes b'). In an attempt to clarify the identity of
these two bands we used a commercial anti-GFP antibody, which reacted in this same crude extract
with the 110 and 140 kDa bands (Figure 17, lane d’). In order to confirm the identity of these two
bands as the recombinant GFP-fusion proteins proposed in Figure 17, it will be necessary to perform
sequence analysis.
In a crude extract from the heterokaryon α-actinin knock-out strain of N. crassa, the anti-Neurospora
α-actinin antibody gives a faint signal that could reflect its reduced gene copy number when compared
with wild type (Figure 17, lane c').
54
Figure 13. Immunoblot of α-actinin from different ascomycetous fungi. (Lane a) N. crassa;
(lane b) M. grisea; (lane c) B. cinerea.
Figure 14. Immunoreactivity of anti-Neurospora α-actinin antibody with the different
domains of α-actinin. (Lane a) full-length GST-α-actinin; (lane b) GST-Actin-binding domain;
(lane c) GST-rod domain; (lane d) GST-Calcium-binding domain. (Arrowhead shows the position
of the GST-Actin- binding domain).
55
Figure 15. Confocal images of germinating conidia expressing α-actininGFP showing its
localization during the septa formation. The GFP signal appears at the site of septum formation
and disappear following completion of the septum after 10 min. (Arrow: septum). Bar 5 μm.
57
Figure 16. Growth on solid medium of the Neurospora heterokaryon α-actinin knock-out
strain compared to the wild type.
59
kDa
170
116
140 kDa
GFP
110 kDa
GFP
80 kDa
76
a
Figure 17.
b
c
a’
b’
c’
d
d'
Anti-Neurospora α-actinin immunoreacting peptides in the heterokaryon α-
actinin knock-out and α-actinin-GFP containing strains compared to the wild type.
Immunoblot of the protein revealed with anti-Neurospora α-actinin (left panel) and anti-GFP ( right
panel, lane d’) antibodies. Lanes a-a’ corresponds to the wild type strain; lanes b-b’, to the αactinin-GFP containing strain and c-c’ to the heterokaryon α-actinin knock-out strain; lane d-d’ is
the same as b-b’.
61
Immunolocalization of α-actinin and actin in Botrytis cinerea
N. crassa was the principal model in this work for the characterization of α-actinin. The study of theis
protein in other fungi could give us more insights into the role of this actin-binding protein in fungi.
As the Western analysis of the whole crude extract proteins from B. cinerea resulted in a strong
reacting band with the anti-Neurospora α-actinin antibody, we have performed the in situ localization
of the α-actinin in this ascomycetous fungus. After 12 h of growth the cultures were in the exponential
growth phase. In this phase α-actinin was localized throughout the cytoplasm and concentrated in the
septum and tip region (Figure 18A–D). Actin principally localized as cortical patches and occasionally
an immuno-fluorescence signal was detected in the septum (Figure 18E–F).
63
Figure 18. Immunolocalization of Botrytis cinerea α-actinin (A-B) and actin (C-D). α-Actinin
localization in the tip region (A) and in the septum of the hyphae (B). Actin was mostly
concentrated in the septum and as dots in the apical region (C-D). Bar 5 μm.
65
5. DISCUSSION, CONCLUSIONS AND PERSPECTIVES
Discussion
Identity of the spectrin-like protein in fungi and an Oomycete
Our experimental models are filamentous fungi, mainly Neurospora crassa and Magnaporthe grisea.
In these organisms, the existence of a fibrous network of peripheral proteins known as membrane
skeleton has been proposed by Degousée et al. (2000) and Torralba and Heath (2001). According to
these authors, this membrane cytoskeleton is likely to be composed mainly of actin, spectrin and
integrin. In support of this hypothesis studies have been published describing the presence of high
molecular weight spectrin in plants, fungi and Oomycetes that reacts with anti-spectrin antibodies
(Table 1 in Publication II). Our results using the same primary antibody that was used to report the
presence of spectrin-like proteins in these organisms, indicated that the major immunoreacting peptide
is a 100 kDa protein in N. crassa and M. grisea and a 60 kDa protein in the oomycete P. infestans
(Publications I and II). Despite a great deal of effort, we were unable to confirm the presence of a high
molecular weight spectrin-like protein as described by the authors cited above. Furthermore, in the
available sequenced genome database for fungi and Oomycetes, a putative gene coding for high
molecular weight spectrins was not found. This contradicts previous reports of a spectrin-like gene
product of 240–220 kDa in fungi (Kaminskyj and Heath, 1995; Degousée et al., 2000; Heath et al.,
2003; Slaninová et al., 2003; Toquin et al., 2006). The peptides reacting with the anti-αβ-spectrin
antibody in N. crassa and the oomycete P. infestans are not related to the spectrin superfamily of
proteins but to two different proteins: EF2 in N. crassa and Hsp70 in P. infestans (Publication II). The
three proteins; spectrin, EF2 and Hsp70, were found to share one characteristic: a predicted chaperone
activity due to a hydrophobic region. Further, another anti-β-spectrin antibody has been reported to
cross-react with the E. coli chaperone, GroEL (Czogalla et al., 2003).
The localization of N. crassa EF2 and P. infestans Hsp70 in the peripheral region of the cell is difficult
to interpret, especially for EF2. Heat shock proteins have previously been found to localize in the cell
walls of fungi (López-Ribot and Chaffin, 1996; López-Ribot et al., 1996; Purin and Rillig, 2008)
where they participate in processes associated with fungal cell wall biosynthesis. However, the
localization of EF2 to the peripheral region is not consistent with its function in protein elongation,
associated with ribosomes. Localization of elongation factors to the plasma membrane has been found
in bacteria (March and Inouye, 1985). All living cells utilize conserved systems responsible for
membrane protein biogenesis. In eukaryotic cells, integral membrane proteins, as well as many
secreted proteins, are targeted to the endoplasmic reticulum. Bacteria utilize a similar pathway,
however in these organisms ribosome target to the cytoplasmic membrane (Herskovits and Bibi, 2000,
2002 and reference cited therein). In fungi, as in the other eukaryotic cells, the relationship between
protein synthesis and the plasma membrane is less probable and has not been probed.
67
The existence of other unknown functions for EF2 that may explain this unexpected localization
cannot be ruled out. EF2, as other elongation factors (the EF1α and EF1β) is an actin-binding protein
(Yang et al., 1990; Bektaş et al., 1994; Condeelis, 1995; Gross and Kinzy, 2005). The EF1α has also
other functions besides its role in the elongation phase of protein synthesis and its ability to bind actin
(Yang et al., 1990). It has also been described as an activator of the phosphatidylinositol-4-kinase
(Yang et al., 1993).
The explanation for the results of the anti-spectrin antibody cross-reactivity is discussed in Publication
II.
Features of α-actinin in fungi and Oomycetes
The absence of spectrins in fungi does not exclude the possibility that other members of the spectrin
superfamily may be involved in actin cytoskeleton organization and function. In 2001 Wu et al.
reported the presence of a gene coding for an α-actinin-related protein (Ainp1) in
Schizosaccharomyces pombe but observed that its ortholog is absent in the budding yeast
Saccharomyces cerevisiae. Complete genome sequences of filamentous fungi were not available at the
time until the publication of the N. crassa genome in 2003 (Galagan et al., 2003). In recent years other
fungal genomes have been completely sequenced and this enabled us to compare orthologs in these
fungi and Oomycetes. In a recent study on the evolution of α-actinins, Virel and Backman (2004)
provided a direct evidence for the presence of α-actinin orthologs in two fungi: N. crassa and S.
pombe. While our work on the evolution of α-actinin in fungi using CH-domain sequences was in
progress, a paper by the same authors appeared documenting actinin orthologs in fungi using the rod
domain criteria (Virel and Backman, 2007). We extended these observations by including a
representative from the Chytridiomycota and carried out a detailed analysis of α-actinin in
Saccharomycotina. In this sub-class, which includes very diverse organisms (Dujon, 2006), we found
only one species, Yarrowia lipolitica, that contains a gene coding for α-actinin. There is no gene
coding for a bona fide α-actinin in S. cerevisiae (Wu et al., 2001; Virel and Backman, 2004) or in the
more recently sequenced genomes of Kluyveromyces lactis, Candida glabrata and Ashbia gossypii. In
other yeasts, we found hypothetical proteins with one conserved N-terminal CH-domain in C. albicans
and two in Pichia stipitis, Debaryomyces hansenii and Candida sp. (Candida guilleimondi and
Candida tropicalis). Spectrin repeats or C-terminal EF-hand motifs were not found. The estimated
molecular weights (Mw) of these proteins are between 70–80 kDa, similar to the estimated Mw of
fungal α-actinins. However, this does not confirm their identity as actinin-related proteins. It is clear
that there are other proteins having CH-domains besides spectrin superfamily members, e.g., calmin,
enaptin and nuance. These proteins have been described as having two N-terminal CH-domains and a
C-terminal trans-membrane domain(s) (Gimona et al., 2002 and references cited therein). However,
these proteins generally have higher molecular weight than expected for fungal α-actinin. Other
68
proteins with two N-terminal CH-domains, not mentioned in the review of Gimona et al. (2002), are
the cortexillins I and II, present in the cellular slime mold Dictyostelium discoideum (Faix et al.,
1996). We used the blast tools of NCBI (National Center of Biotechnology Information,
http://www.ncbi.nlm.nih.gov/) and also found these proteins in another cellular slime mold
Polysphondylium pallidum as well as in an amoeba Entamoeba histolytica. The sequences found,
however, code for a protein of lower molecular wight, about 40 kDa and are implicated in cytokinesis
(Faix et al., 1996) and mechanical properties of the cell cortex (Simson et al., 1998). We did not find
any orthologs of these proteins, containing N-terminal CH-domains, in the fungal species examined.
The existence of “atypical” α-actinins with only one or two conserved N-terminal CH-domain(s) can
be explained by evolutionary pressure where genes become highly diverged, modified beyond
recognition or completely lost. Gene loss during evolution in several lineages is not a rare event and
has been shown to occur for many genes (Aravind et al., 2000; Roelofs and Van Haastert, 2001;
Krylov et al., 2003).
Despite the fact that the relationship between these proteins in the Saccharomycotina and other fungi
is not clear, we can hypothesize that they have evolved from a common ancestor through two different
pathways (as shown in Fig. 1 in Publication III). One pathway led to the loss of the α-actinin gene, as
in S. cerevisiae, or severely modified, as in Candida albicans and the yeasts mentioned above.
However, other yeasts such as Y. lipolytica followed another pathway in which the gene was
preserved. This hypothesis is supported by the fact that even though we cannot recognize any
functional domain in the middle and C-terminal regions of these hypothetical proteins, the gene is still
present with a relatively large ORF. The absence of a “classical” fungal α-actinin in nearly all
Saccharomycotina may be due to a complementation in function by other proteins, possibly fimbrin, as
happens in S. pombe (Wu et al., 2001) or other actin-binding proteins (Rivero et al., 1999). Fimbrin is
interesting from this point of view since it contains two conserved domains that are also found in αactinin but with a reverse orientation, i.e., N-terminal Ca2+-binding EF-hand motifs and C-terminal
actin-binding CH-domains. α-Actinin is present in all fungi examined, except most members of the
Saccharomycotina, where α-actinin was found only in Y. lipolytica.
In other fungi, α-actinin seems to be highly conserved and display a significant sequence homology to
chicken and human α-actinins. The estimated molecular weight for fungal α-actinin varies in the range
70–85 kDa. Some ascomycetes such as N. crassa, M. grisea, Sclerotinia sclerotia and Fusarium
graminearum have N-terminal extensions of varying length and do not have sequence homology to
any known protein. Considering the size of these N-terminal extensions, the expected protein should
vary between 80 to 110 kDa. The α-actinins of these four species contain a second putative ATG-start
codon located immediately upstream of the first CH-domain. The molecular weight of the protein
resulting from transcription starting at the second start codon fits perfectly with the molecular weight
69
of other fungal α-actinins. The anti-Neurospora α-actinin antibody reveals bands of 80 and 72 kDa,
respectively, in N. crassa and M. grisea, suggesting that the expressed protein should have initiated at
the second ATG-start codon under the conditions used in this work. The N-terminal extension could
be a regulatory element in the mRNA, an alternate use of the two ATG start codons or a pro-domain in
the protein, cleaved immediately after protein synthesis. The latter hypothesis is supported by the
analysis of crude extracts N. crassa recombinant α-actinin-GFP expressed in E.coli. In these extracts
three bands with relative masses of 80, 110 and 140 kDa were revealed with the anti-Neurospora αactinin antibody. These peptides could correspond to the native short α-actinin, the short α-actininGFP and full-length α-actinin-GFP, respectively (the presence of the GFP tag in these polypeptides
was confirmed using anti-GFP antibody). The presence of full-length and short α-actinin-GFP
suggests that the cell possesses the enzymatic machinery necessary to cleave the pro-domain of the
protein. These results do not rule out the possibility of an alternate use of the two in-frame methionine
start codons. Indeed, such a phenomenon has been described for another protein, frequency (FREQ),
implicated in the regulation of the circadian clock in Neurospora (Diernfellner et al., 2005). In this
protein, the alternate use of the start codons of FREQ is regulated by one intron and the UTR-region.
Biochemical characterization of N. crassa α-actinin
The interaction of α-actinin with actin was calcium dependent, as demonstrated by co-sedimentation
assays. It was shown that α-actinin has the ability to cross-link F-actins and to organize
microfilaments in a parallel way. Actin filaments cross-linked by α-actinin were 15–25 nm apart. The
distance between the filaments was shorter than those observed in other actin-α-actinin complexes
(30–40 nm) (Taylor and Taylor, 1993; Tang et al., 2001; Hampton et al., 2007), but agrees with the
difference in size between α-actinin of higher eukaryotic cells and the shorter fungal α-actinins, which
contain only half the number of spectrin repeats (Virel and Backman, 2007). The calcium dependence
of Neurospora α-actinin in actin-binding assays and the insensitivity of vertebrate α-actinin support
the idea that the calcium-dependent regulation of actin-binding was lost during the invertebrate–
vertebrate divergence (Dixson et al., 2003; Virel and Backman, 2004).
Localization of α-actinin in N. crassa
The localization of proteins can provide information on their function. Two approaches were used to
localize α-actinin in N. crassa: in situ detection, using specific antibodies, and in vivo GFP
fluorescence localization. α-Actinin was found to immunolocalize to some septa. In a manner
consistent with the immunofluorescence results, the GFP signal was also transiently found in the septa
during their formation. Other proteins required for septation in filamentous fungi, such as formin SepA
(Sharpless and Harris, 2002) and actin (Momany and Hamer, 1997; Rasmussen and Glass, 2005),
show the same pattern. α-Actinin also localizes in emerging tubes and the apical region of the hyphae,
70
suggesting a role for the protein during germination and tip growth. The peripheral localization in the
hyphae suggests additional roles in cellular physiology required for optimal growth. One can speculate
that these functions are related to the lethal phenotype of the homokaryon knock-out mutant.
However, this localization was not observed in vivo, probably because the GFP signal was too weak to
be detected or that the GFP tag partially modified the localization of the fusion protein (Ikonen et al.,
1995; Schneider et al., 2000).
Function of N. crassa α-actinin
The knock-out phenotype was lethal. It is difficult to assume that the lethal phenotype is due only to a
supposed role in septum formation. The aseptate mutants in Neurospora (Rasmussen and Glass, 2005;
2007) and other filamentous fungi (Ayad-Durieux et al., 2000; Wendland and Philippsen, 2002; Kim
et al., 2006) are viable.
Although the exact function of α-actinin in Neurospora is still not clear, we can predict that a
collaboration with actin plays a major role. The results from protein localization suggest a role in
cytokinesis, which would agree with the function of its orthologs in S. pombe (Wu et al., 2001).
However, differences can be expected because cytokinesis in filamentous fungi and yeast are two
principally different mechanisms (Walther and Wendland, 2003). Moreover, α-actinin function in
yeast and also in D. discoideum can be complemented by other actin-binding proteins, notably fimbrin
(Wu et al., 2001; Rivero et al., 1999), resulting in non-lethal phenotypes for α-actinin knock-out
mutants in these organisms.
The dichotomous phenotype of the α-actinin heterokaryon knock-out strain may be a consequence of
splitting at the tip, also called “dichotomous branching”. This process has been observed in several
other filamentous fungal mutants in which polarity maintenance proteins were targeted (Sharpless and
Harris, 2002; Geissenhöner et al., 2001; Han and Prade, 2002). A similar phenotype has been observed
in Neurospora treated with Cytochalasin A (Riquelme et al., 1998) and Neurospora actin mutants
(Virag and Griffiths, 2004), suggesting that α-actinin together with actin may play a role in branching.
However, other putative roles can be expected.
The list of newly identified proteins and molecules interacting with α-actinin has increased over the
last several years, confirming that α-actinin is not merely an actin-binding protein but a scaffold for
other protein-protein interactions
connecting the cytoskeleton to diverse signaling pathways
(Critchley and Flood, 1999; Otey and Carpen, 2004).
71
Conclusions
The conclusions drawn from this work are as follows:
•
There are no genes coding for spectrin in fungi and Oomycetes.
•
The anti-spectrin antibody that has been used to demonstrate the presence of spectrin-like
proteins in fungi and Oomycetes cross-reacts with proteins other than spectrin.
•
In fungi, there is evidence for a gene coding for a spectrin superfamily protein. This protein
has been identified as α-actinin.
•
α-Actinin gene is not present in all fungi. In some saccharomycotina yeasts it has been lost.
•
N. crassa α-actinin has a molecular weight of 80 kDa and pI of 5.9.
•
N. crassa α-actinin has the ability to bind actin in a calcium-regulated manner.
•
In N. crassa α-actinin localizes in situ to: (1) the site of the initiation of germ tubes in
germinating spores, (2) within the peripheral region of growing hyphae, and (3) in septa
during their formation.
•
Localization in situ was confirmed in vivo, with the exception of the plasma membrane
localization.
•
Deletion of the α-actinin gene in N. crassa is lethal suggesting that at least in this organism its
function cannot be complemented by other actin-binding proteins.
•
Although the role of α-actinin in N. crassa has not been precisely determined, localization and
characterization of the knock-out heterokaryon suggests that the protein participates together
with actin in the coordination of cellular activities such as germination, septum formation and
branching.
72
Perspectives
Our results represent an important step towards understanding the role of actin cytoskeleton in the
spatial organization of hyphae during cell life cycle. Future experiments will be aimed at determining
the function of the α-actinin N-terminal extension. First, it will be necessary to determine if this
extension present in the mRNA is a UTR-region, a coding sequence with an alternative start-codon
usage or a pro-domain of the protein. To achieve this, 5' RACE (Rapid amplification of 5'
complementary DNA ends) experiments will allow us to determine the start-codon of α-actinin
mRNA. Further, point mutations in the first putative start-codon will confirm the location of the
translation initiation and answer whether the N-terminal extension is a pro-domain of the protein.
Studies into the effects of actin inhibitors on in situ and in vivo α-actinin localization will provide a
greater insight into the cooperation between these two proteins, α-actinin and actin. Other GFPconstructs, such as N-terminal GFP tagging of the “full-” and “short-length” α-actinins, will help us to
understand why α-actinin localization to the peripheral region has not been found in vivo. Comparison
of the three transformed strains can provide information about the role of the N-terminal α-actinin
extension.
Considering that α-actinin deletion is lethal in N. crassa, future experiments using siRNA may help to
elucidate the function of this protein.
The evolutionarily related α-actinins characterized in N. crassa and S. pombe seem to differ in their
cellular localization, function and indispensability. Studies of these proteins in other fungi may help to
better understand their role as components of the actin cytoskeleton in these organisms.
73
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94
ANNEXE
95
CURRENT MICROBIOLOGY Vol. 53 (2006), pp. 311–316
DOI: 10.1007/s00284-006-0057-y
Current
Microbiology
An International Journal
ª Springer Science+Business Media, Inc. 2006
Proteolytic Cleavage of a Spectrin-Related Protein
by Calcium-Dependent Protease in Neurospora crassa
M. Cotado-Sampayo,1 M. Ojha,2 R. Ortega-Prez,1 M.-L. Chappuis,1 F. Barja1
1
2
Laboratory of Bioenergetics and Microbiology, University of Geneva, 10 Chemin des Embrouchis, CH-1254 Jussy-Geneva, Switzerland
Department of Biochemistry, University of Geneva, 30 Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland
Received: 3 February 2006 / Accepted: 18 May 2006
Abstract. To investigate the functional significance of a cytoskeletal spectrin-like protein, we studied
its localization pattern in Neurospora crassa and sought the answer to whether it is a substrate for
another apically localized protein, the calcium-dependent protease (CDP II). Immunoblots of crude
extracts from exponentially growing mycelia, separated by one- and two-dimensional sodium dodecyl
sulfate–polyacrylamide gel electrophoresis using antichicken a/b-spectrin antibodies, revealed a single
band of approximately relative mass (Mr) 100 kDa with an isoeletric point (pI) in the range of 6.5 to 7.0.
Despite rigorous efforts, we could not confirm the presence of an Mr 240- to 220-kDa spectrin-like
protein in N. crassa. The immunofluorescence- and immunogold-labeling Mr 100-kDa protein showed
its predominance along the plasma membrane of the conidia during the swelling phase of germination.
In contrast, in the germ tubes and the growing hyphae, the localization was polarized and concentrated
mainly in the apical region. The in vitro proteolysis experiments showed that indeed this protein is a
preferred substrate of CDP II which is, as mentioned previously, also localized in the apical regions of
the hyphae. These results indicate a putative functional relationship between these two proteins
(spectrin-like protein and CDP II) in the dynamics of tip growth.
The filamentous fungi represent a large group of
organisms that grow and colonize substrates by
branching and apical elongation. Cytoskeletal elements
are considered to be involved in this process and have
been the subject of intensive investigation. Ultrastructural and immunologic studies of the apical zone, for
example, have shown that this region is rich in cytoskeletal elements, such as actin [1–4], tubulins [5–11],
integrins, and spectrins [12–15]. These proteins are
considered to provide stability to the growing hyphal
tubes by the linkage among themselves and to the
plasma membrane. For the hyphae to grow, this linkage
must be reorganized.
Another important factor implicated in regulation of
polarized growth and branch formation is calcium signaling [16–19]. We have reported the presence of two
calcium-dependent proteases (CDPs I and II) in the
Correspondence to: F. Barja; email: [email protected]
apical region of Allomyces, an aquatic fungus, and shown
that their presence or absence is related to growth [4, 20–
24]. An immunoanalogue of CDP II was also discovered
and shown to be exclusively localized in the apical
regions of the hyphae of an ascomycete fungus, Neurospora crassa, and a phytopathogenic basidiomycete,
Uromyces appendiculatus [25]. Therefore, this colocalization of cytoskeletal proteins and CDP might have
some functional significance. Indeed, we have shown
selective proteolysis of a-tubulin by CDP II in the apical
region of the growing hyphae and cell-free extracts [24].
We sought to determine whether other apically
localized cytoskeletal proteins are also targets of this
protease. Because a spectrin-like protein has been
shown to be localized in the apex of the growing hyphae
of N. crassa using antichicken a/b spectrin antibodies
[14], we re-examined the spatial localization of this
protein and CDP II during early development of
N. crassa and studied the proteolysis of the former
in situ as well as in cell-free extracts by this protease.
312
Materials and Methods
Organism and culture conditions. A wild-type strain of N. crassa
(FGSC 262, strain St. Lawrence STA4) was used in this study.
Production of conidial inocula and culture growth conditions were as
described previously [26].
Protein extraction and analysis. Crude extract was prepared
according to Abe and Davies [27] with slight modifications. Briefly,
the buffer contained 5 mM HEPES-KOH at pH 7.5, 250 mM sucrose, 15
mM Mg(OAc)2, 2 mM EGTA, 2 mM dithiothreitol, 25 mM K2O5S2, 10%
glycerol, 0.5% polyvinylpyrrolidone, and protease inhibitors (1 mM
benzamidine, 2 mM phenylmethylsulfonyl fluoride, and 2 lg/ml each
for leupeptin, chymostatin, and trasylol). Protein concentration was
determined according to Bradford [28]. Sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional
analysis were done according to Laemmli [29] and OFarrell [30],
respectively.
Immunoblotting. Proteins
from
unstained
monodimensional
and bidimensional gels were electrophoretically transferred to
nitrocellulose membranes (BA85, pore size 0.45 lm), using the
transfer buffer described by Burnette [31], and immunoblotted as
described earlier [24]. Antichicken a/b spectrin (Sigma S1390) and
antirabbit IgG antibodies coupled to horseradish peroxidase were used
at dilutions of 1:800 and 1:2000 in Tris-buffered saline, respectively.
Immunofluorescence microscopy. Conidia were suspended in
Vogels growth medium, and a sample was taken immediately, which
represented the start point (0 hours). Further samples were taken at 6,
12, and 18 hours of culture growth and fixed instantaneously by the
addition of paraformaldehyde in the culture medium to a final
concentration of 3%.
Primary antichicken spectrin antibodies and secondary goat antirabbit fluorescein isothiocyanate (green, Sigma F9887) or crystalline
tetramethylrhodamine isothiocyanate (red, Sigma T5268) antibodies
were used at dilutions of 1:50 and 1:100, respectively, in phosphate
buffer [24]. These two secondary antibodies were used to differentiate
the distribution of spectrin-like protein and CDP II in the colocalization experiments. The immunolabeled cells were examined with a
microscope (Zeiss axioplan) equipped for epi-illumination with fluotar
optics and selective filter combinations. Spectrin-like protein was revealed using Sigma antichicken a/b spectrin and anti-CDP II using
laboratory stock of anti-CDP II prepared from Allomyces arbuscula.
Colocalization was done using respective primary antibodies. The
images were taken with a Hamamatsu color, chilled 3 CCD camera,
developed by Raster Optics video captor and treated by the program
Adobe PhotoShop 7.
Electron microscopy and immunogold labeling. Spores (0 hours)
and 6-hour germinated conidia were used for immunogold labeling of the
spectrin-like protein using antichicken spectrin antibodies diluted to 1:50
in phosphate buffer and secondary goat antirabbit antibodies conjugated
to 20 nm gold particles and diluted to 1:30 in the same buffer. The
experimental procedure used has been described in detail elsewhere [24].
Sections were examined at 60 kV using a Philips M400 transmission
electron microscope.
Proteolysis of cell-free extract by CDP II. The cell-free extract,
obtained as described in the section on protein extraction and analysis,
was digested with CDP II (laboratory stock purified from A. arbuscula
as described in Ojha and Wallace [20], specific activity 21 lM
paranitroaniline released lg–1min–1) in a reaction mixture containing
20 mM Tris-HCl at pH 7.4, 4 mM EGTA, 3 mM MgCl2, and 1% bmercaptoethanol. 6 mM CaCl2 was added to obtain 5 mM free Ca2+ to
CURRENT MICROBIOLOGY Vol. 53 (2006)
initiate the proteolytic activity. The reaction was performed in a total
volume of 75 ll at enzyme-to-protein ratios of 1:100, 1:50, and 1:10.
After incubation at 37C for 30 minutes, the reaction was stopped by
the addition of 25 ll 4 · loading buffer [29]. The proteins were
denatured in boiling water for 5 minutes and separated by SDS-PAGE,
transferred to nitrocellulose membranes, and immunorevealed with
spectrin polyclonal antibodies. Reaction mixtures either without
calcium or with leupeptin (10 lM), an inhibitor of CDP II activity,
were used as controls.
In situ proteolysis by CDP II. Hyphae from 12-hour cultures were
prepared as described previously for immunofluorescence. Before
blocking, the cells were incubated for 1 hour at 37C with CDP II in
the enzyme reaction buffer described previously, and washed with
phosphate buffer five times for 5 minutes each, and then
immunorevealed as described earlier [24].
Results
Proteins from the cell-free extract, separated in nondenaturing PAGE and immunoblotted with chicken
antispectrin polyclonal antibodies, showed two immunoreacting bands (Fig. 1A). The lower band (arrow) from
nondenaturing gel was cut and migrated in SDS-PAGE; a
single strong band of Mr 100 kDa appeared (data not
shown). In direct SDS-PAGE of the cell-free extract, a
single heavily stained band of Mr 100 kDa was also observed. The intensity of this band did not change significantly in preparations obtained from cells either during
germination or exponential growth (Fig. 1B). Human
erythrocyte spectrin, used as a positive control, revealed
two bands of Mr 240 and 220 kDa each (Fig. 1C).
The specificity of immunoreaction was demonstrated using human erythrocyte spectrin in a competition assay. The results showed a progressive diminution
of the immunoreacting band with increasing concentration of the competing human spectrin (Fig. 1D). In twodimensional gels, the predominant immunoreacting
band was Mr 100 kDa, with a pI in the range of 6.5 to
7.0, in addition to a faint 80-kDa band with similar pI,
representing probably a degradation product of the major immunoreacting protein (Fig. 1E).
Immunofluorescence of the protein revealed with
polyclonal antispectrin antibodies was intense all around
the peripheral regions of the isodiametric germinating
conidia, representing the swelling phase of germination
(earliest visible phase) (Fig. 2A). With the appearance
of germ tube outgrowth representing the polarized
growth phase, much of the fluorescence was concentrated in the plasma membrane of the germ tubes
(Fig. 2B). In the cells from the 12- and 18-hour cultures,
representing filamentous hyphal growth and corresponding to exponential growth phase, the fluorescence
was also polarized and concentrated as a cap in the
hyphal tips and branch initials (Figs. 2C through 2E).
M. Cotado-Sampayo et al.: Spectrin-Related Protein As Target of Calcium-Dependent Protease
Fig. 1. Immunoblot of a N. crassa spectrin-like protein revealed with
anti–a/b-spectrin antibodies. (A) Native PAGE of crude extract proteins; arrow indicates the band that gives the 100-kDa peptide in the
SDS-PAGE gel. (B) SDS-PAGE of crude extract proteins from 6, 12,
and 18 hours of culture development. (C) Human erythrocyte spectrin
as control (1 lg). (D) Specificity assay of spectrin polyclonal antibodies in N. crassa with antibodies preadsorbed to human erythrocyte
spectrin: (a) human erythrocyte spectrin revealed with unadsorbed
antibodies; (b) crude extract with unadsorbed antibodies; (c and d)
antibodies preincubated with human erythrocyte spectrin at ratios of
1:0.8 and 1:2, respectively. Preincubation of antibodies was performed
for 30 minutes at room temperature before using them in immunoblot
reactions. (E) Two-dimensional analysis of a N. crassa spectrin-like
protein revealed with anti a/b-spectrin antibodies. The pI range of the
protein is 6.5 to 7.0.
In the colocalization experiments shown in Figs. 2F
through 2I, both spectrin-like protein (green) and CDP II
(red) in 6-hour germinating conidia and branch initials
of the hyphae, were abundantly present in the same region of the cell, i.e., the growing tip. There was, however, clearly one difference: the predominance of
spectrin-like protein along the plasma membrane and a
more diffuse distribution of CDP II in the cytoplasm as
evident in the merge of the localization of the two
proteins (Figs. 2H through 2I).
Immunogold labeling the protein in ultrathin sections of conidia in the isodiametric and polarized phases
313
Fig. 2. (A through E) Immunolocalization of a N. crassa spectrin-like
protein revealed with polyclonal antichicken a/b-spectrin in cells of
different stages of growth. (A) Conidia. (B) Germinating conidia with
germ tube. (C and D) Growing hyphae. (E) Tip of branch initial.
Bar = 10 lm. (F through I) Colocalization of CDP II and spectrin-like
protein in N. crassa hyphae. (F) Localization of spectrin-like protein.
(G) Localization of CDP II. (H) Merge of two proteins in the tip of the
germ tube. (I) Merge of two proteins in the branch initials.
Bar = 10 lm.
of germination showed uniform distribution in the cortical region and along the plasma membrane (Fig. 3).
Control experiments with antichicken antibody preadsorbed with human spectrin, or the use of only secondary antibodies, showed an absence of labeled grains
indicating the specificity of the primary antibodies (data
not shown).
Proteolysis of 100-kDa protein by CDP II in the
presence of Ca2+ was evident by the diminution of the
relative intensity of the band at low enzyme-to-protein
ratios (1:100 and 1:50) and complete absence at the ratio
of 1:10 (Fig. 4A, c through e). Under similar experimental conditions, but in the absence of Ca2+ or in the
presence of leupeptin, this protein remained unaffected
(Fig. 4A, f through g). Incubation of the extract without
CDP II did not show any appreciable proteolysis of the
immunoreacting protein, indicating there was no other
endogenous protease targeting this protein under the test
condition used. An examination of the relative intensity
of Ponceau-stained bands after transfer from SDS-PAGE
314
CURRENT MICROBIOLOGY Vol. 53 (2006)
Fig. 3. Immunogold labeling of a N. crassa
spectrin-like protein revealed with anti–a/bspectrin antibodies during development.
(A) Ultrathin section of conidia showing
isodiametric distribution along the plasma
membrane. (B) Distribution along the plasma
membrane shown at a higher magnification
of a portion (A). (C) Labeling along the
plasma membrane in a section of conidia
after 6 hours of germination. (D) Higher
magnification of a portion of (C).
m = mitochondria; n = nuclei; pm = plasma
membrane; v = vacuole; cw = cell wall.
to nitrocellulose membrane did not show any massive
general proteolysis.
Fixed and permeabilized cells incubated with CDP
II in the enzyme reaction mixture and processed for
immunofluorescence using antispectrin antibodies
showed partial digestion of this protein where it appeared as faint fluorescent dots (Fig. 4C) instead of
continuous fluorescence along the plasma membrane
and capped localization at the tip observed in untreated
cells (Fig. 4B). This pattern was similar to the one noted
in our previous work with tubulins, in which b-tubulin,
which is not affected by CDP II, had no modification in
its hyphal distribution, whereas a-tubulin appeared as
faintly dispersed fluorescent dots in the cytoplasm.
Discussion
A search in the genomic database of N. crassa for the
spectrin superfamily proteins yielded two genes, the
NCU06429.2 and NCU03992.2. The gene NCU06429.2
codes for a hypothetical protein corresponding to
a–actinin, i.e., theoretical Mr 110 kDa. This protein has a
C-terminal prodomain, two calponin homology domains,
a rod domain composed of two spectrin repeats, and a
N-terminal EF band domain. The gene NCU03992.2
codes for a protein (i.e., theoretical Mr 72 kDa) corresponding to fimbrin and comprised of four calponin
homology domains and no spectrin repeat units. Western
blot analysis of the cell-free extracts of N. crassa—using
the same primary antibody that was previously used to
recognize spectrin epitopes in plants [32–36], i.e., the
oomycetes Saprolegnia ferax [12] and green algae
[37]—yielded evidence for the occurrence of only one
related protein of Mr 100 kDa. This protein could correspond to the gene product NCU06429.2. Contrary to
published results [14] using the same antibodies and the
same organism (N. crassa), we did not find the presence
of an Mr 240- to 220-kDa protein.
The spatial localization of the Mr 100-kDa protein
by immunofluorescence showed that it is localized in the
apical zone of the fungal hyphae (especially along the
plasma membrane) confirming the published results
[14]. The plasma membrane localization of the spectrinlike protein was more evident in the images obtained
M. Cotado-Sampayo et al.: Spectrin-Related Protein As Target of Calcium-Dependent Protease
315
from the immunogold-labeling experiments. This
localization may confer stability to plasma membrane by
anchoring it to other cytoskeletal proteins. It is to be
expected that proteolysis is necessary to weaken the
attachment of cytoskeletal proteins to plasma membrane
or other underlying hyphal tube structures and permit
the expansion of the growing hyphal apex. Proteolysis of
this plasma membrane–associated protein and possible
weakening of the membrane skeleton may also facilitate
the access of intracellular vesicles to the plasma membrane and allow its expansion. This is of importance
because the tip of the hyphae has been shown to be a site
of membrane insertion by exocytosis processes [38, 39].
The question arises, what is the identity of the protease
responsible for this proteolysis? The apical localization
of CDP II, as shown in Fig. 2H, essentially confirmed
our previous finding reported earlier [25]. The colocalization of the spectrin-like protein and CDP II in the N.
crassa hyphal apices (Fig. 2I) suggests that this spectrinlike protein at some point in development is proteolyzed
by CDP II and regulated in its function.
Considering the in vitro and in situ proteolysis of
this spectrin-like protein and its colocalization with CDP
II, we suggest that it is a likely candidate for one of the
specific substrates in vivo.
ACKNOWLEDGMENTS
We gratefully acknowledge financial support from the Academic
Society of Geneva (F. B.) and National Science Foundation Grant No.
3100-056786.99 (M. O.). Thanks are due to R. Strasser for interest in
the project and encouragement, A. Cattaneo for technical assistance,
and A. Fehr for secretarial assistance.
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Fig. 4. (A) Proteolysis of a spectrin-like protein in the crude extract of
N. crassa (50 lg/slot) with CDP II revealed by immunoblotting. The
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was revealed with antibodies against a/b-spectrin. Lane a = Extract
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In situ digestion of a spectrin-like protein with CDP II. (B) Localization of spectrin-like protein along the plasma membrane. (C) After
digestion with CDP II protease. (D) 4;6-Diamidino-2-phenylindoleÆ2HCl (DAPI)-staining nuclei. Bar = 10 lm.
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Available online at www.sciencedirect.com
Fungal Genetics and Biology 45 (2008) 1008–1015
www.elsevier.com/locate/yfgbi
Specificity of commercial anti-spectrin antibody in the study of fungal
and Oomycete spectrin: Cross-reaction with proteins other than spectrin
Marta Cotado-Sampayo a, Pilar Okenve Ramos a, Rubén Ortega Perez a,
Mukti Ojha b, Francßois Barja a,*
a
Laboratory of Bioenergetics and Microbiology, University of Geneva, ch. des Embrouchis 10, CH 1254 Jussy-Geneva, Switzerland
b
Department of Biochemistry, University of Geneva, Quai Ernest Ansermet 30, CH 1211 Geneva4, Switzerland
Received 18 December 2007; accepted 13 February 2008
Available online 21 February 2008
Abstract
Spectrin was first described in erythrocytes where it forms a filamentous network in the cytoplasmic face of the plasma membrane and
participates in the membrane’s structural integrity in addition to controlling the lateral mobility of integral membrane proteins. In fungi,
spectrin-like proteins have been described in the plasma membrane, concentrated mainly in the region of maximum apical expansion.
This localization led to the idea of a spectrin based membrane skeleton in fungi participating in mechanical integrity of the plasma membrane, generating and maintaining cell polarity. The occurrence of spectrin-like proteins in filamentous fungi, yeasts and Oomycetes,
however, is questionable since the presence of such proteins has only been demonstrated with immunochemical methods using antibodies
whose specificity is unclear. There is no evidence of a gene coding for the high molecular weight ab-spectrin in the genome of these organisms. Mass spectrometric analysis of the anti ab-spectrin immunoreacting peptides from Neurospora crassa and Phytophthora infestans
identified them as elongation factor 2 (NCU07700.4) and Hsp70 (PITG_13237.1), respectively. An attempt was made to correlate the
reactivity of anti-spectrin antibody to a common feature of these three proteins i.e., spectrin, elongation factor 2 and heat shock protein
70, in that they all have a hydrophobic region implicated in chaperon activity.
Ó 2008 Elsevier Inc. All rights reserved.
Keywords: Spectrin; Neurospora crassa; Elongation factor 2; Heat shock protein 70; Polyclonal antibody
1. Introduction
Spectrin is a major constituent protein of the metazoans
cell membrane, first purified to homogeneity from red
blood cells. It is an elongated heterodimer composed of
non-identical a and b subunits (about 30% identity) with
molecular weight of 240 and 220 kDa estimated, respectively, from electrophoretic mobility and shown to localize
at the cytoplasmic face of the plasma membrane. It also
interacts with a wide variety of proteins creating a cellular
network (Bennett and Gilligan, 1993; Winkelmann and
Forget, 1993). These interactions with cytosolic and membrane proteins control the elasticity of the lipid bilayer and
*
Corresponding author. Fax: +41 22 37 93756.
E-mail address: [email protected] (F. Barja).
1087-1845/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.fgb.2008.02.003
therefore the cell shape. (Steck, 1989; Lee et al., 1993; Viel
and Branton, 1996; Bennett and Baines, 2001).
The presence of spectrin in non-animal cells has been
demonstrated mainly through studies employing immunochemical techniques (Western blot, immunofluorescence
and/or immunogold) using commercial anti-spectrin antibodies (Table 1). The proteins recognized have been
reported as spectrin-like proteins in protists (Hemphill
et al., 1991; Ghazali et al., 1995; Holzinger et al., 1999),
plants (Michaud et al., 1991; Faraday and Spanswick,
1993; Bisikirska and Sikorski, 1997; De Ruijter et al.,
1998, 2000), Oomycetes (Kaminskyj and Heath, 1995)
and fungi (Degousée et al., 2000; Slaninová et al., 2003;
Cotado-Sampayo et al., 2006). However, our search for
the bona fide spectrin in fungal-specific and general databases (Broad Institute Database, MIPS and NCBI) using
M. Cotado-Sampayo et al. / Fungal Genetics and Biology 45 (2008) 1008–1015
1009
Table 1
Reports of spectrin-like proteins in fungi, Oomycetes, plants and low eukaryotes organisms
Organism
Antibodies
Neurospora crassa
Saccharomyces
cerevisiae
Anti ab-Spectrin
S1390
Anti ab-Spectrin
S1390
Anti ab-Spectrin
S1390
Anti ab-Spectrin
S1390
Sigma
220, 240, 60, several low
molecular weight bands
Schizosaccharomyces
japonicus
Anti ab-Spectrin Sigma
S1390
220, 240, 60, several low
molecular weight bands
Saprolegnia ferax
Anti ab-Spectrin Sigma
S1390
Anti-Spectrin (ICN, St.
Laurent)
Anti ab-Spectrin Sigma
S1390
Anti b-Spectrin
Anti ab-Spectrin Sigma
S1390
Anti-Spectrin Sigma S1515
Anti ab-Spectrin (Lorenz
et al., 1995)
Anti ab-Spectrin S3396
Anti ab-Spectrin S1515
246, several low molecular
weight bands
Geotrichum candidum
Phytophtora infestans
Tomato plants
Vivia sativa
Pisum sativum
Onion cells
Green algae,
Desmidiaceae
Chara globularis
Molecular weight (kDa)
Localization
Reference
Sigma
220, 240, 100
Degousée et al. (2000)
Sigma
100
Plasma membrane
Apex
Plasma membrane
Apex
Plasma membrane
Patches in the apex
Plasma membrane
Vacuolar membrane
Cytoplasmic
Plasma membrane
Cytoplasmic
Septum
Plasma membrane
Sigma
Anti ab-Spectrin Sigma
S1390
Anti-Spectrin Sigma S1515
Anti-Spectrin Sigma S3396
Anti ab-Spectrin Sigma
S1390
Anti-Spectrin Sigma S1515
Cotado-Sampayo
et al. (2006)
Heath et al. (2003)
Slaninová et al. (2003)
Slaninová et al. (2003)
Kaminskyj and Heath
(1995)
240, 220, 100, 70, 50, 30
Plasma membrane
Toquin et al. (2006)
240, 220
Plasma membrane
Root hair tips
Michaud et al. (1991)
De Ruijter et al.
(1998)
Native: 800, 280, 170, 110,
70
Native: 210/230 zone, 580,
380 and smaller bands 100,
130, 170
220, 120, 70
195, 170
Associated to plasma membrane.
Endoplasmic reticulum and
endomembrane system
Plasma membrane
Endoplasmatic reticulum
Vesicles
Endoplasmatic reticulum
(ER) agregates
Tip of rhizoids
Bisikirska and
Sikorski (1997)
Reuzeau et al. (1997)
Holzinger et al. (1999)
Braun (2001)
195, 170, 110
conserved sequence of CH-homology domain did not show
any gene coding for spectrin. A protein with features of the
spectrin superfamily has been proposed to exist in the
Ascomycete Neurospora crassa (NCU06429.4) and the
Oomycete Phytophtora infestans (PITG_13237.1), since an
immunoreacting peptide was revealed with anti-spectrin
antibodies (Degousée et al., 2000; Cotado-Sampayo
et al., 2006; Toquin et al., 2006). This protein, however,
is smaller in molecular weight than the fungal spectrins
reported earlier (Kaminskyj and Heath, 1995; Degousée
et al., 2000). In more recent studies this protein has been
considered to be an a-actinin related protein (Cotado-Sampayo et al., 2006; Virel and Backman, 2004 and in MIPS
and Broad Institute data bases).
In this report, using the filamentous fungi N. crassa and
Magnaporthe grisea and the Oomycete, P. infestans, we
demonstrate that the polyclonal anti-chicken ab-spectrin
antibody does not react with a-actinin (NCU06429.4),
the spectrin superfamily protein present in these organisms.
Further, we clarify the identity of the protein that reacts
with anti- chicken spectrin antibody and demonstrate that
the reacting protein is EF2 in Neurospora and Hsp70 in
Phytophthora.
2. Materials and methods
2.1. Strains and culture conditions
Wild-type strain N. crassa (FGSC 262, strain St. Lawrence STA4) was used in this study. Production of conidial
inocula and culture conditions were as described previously
(Ortega Perez et al., 1994). M. grisea was kindly given by
Dr. M.-H. Lebrun (Unité Mixte de Recherche, Centre
National de la Recherche Scientifique/BayerCropScience,
Lyon). The fungus was inoculated on solid rice medium
and allowed to grow in the dark at 27 °C for a few days
until white mycelia appeared. In sterile conditions, small
pieces were cut and inoculated in 200 ml of Tanaka minimal medium (Ou, 1985) with 0.2% yeast extract and 1%
sucrose. The culture was grown for 48 h with agitation
(150 rpm) in the dark. Mycelia were collected by filtration
and weighed.
The P. infestans strain was kindly provided by Dr. R.
Beffa (Unité Mixte de Recherche, Centre National de la
Recherche Scientifique/BayerCropScience, Lyon) and
grown on pea-agar medium. Sporangial inoculum was prepared from a 8–12 days culture, detached from the mycelia
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M. Cotado-Sampayo et al. / Fungal Genetics and Biology 45 (2008) 1008–1015
by flooding the culture with water and separated from the
mycelial fragments by filtration. Sporangia were inoculated
in V8 liquid medium (50 ml tomato juice/liter of distilled
water, pH adjusted to 5 if necessary) at a final concentration of 105 sporangia/ml. Mycelia were harvested after
72 h of growth in the dark at 20 °C without agitation.
2.2. Protein extraction and analysis
To optimize the preservation of proteins against proteolysis during the harvest of mycelia, grinding and suspension of the mycelial powder in the extraction buffers,
different protocols were followed. These included rinsing
of mycelia with protease inhibitors before freezing in liquid
nitrogen as described by Kaminskyj and Heath (1995),
quick freezing, use of different detergents and TCA–acetone extraction (Granier, 1988); in all representing some
10 experimental conditions. Mycelia were always ground
in liquid nitrogen. The standard procedure finally adopted
was essentially as described earlier (Cotado-Sampayo et al.,
2006).
Protein concentration was determined according to
Bradford (1976). SDS–PAGE and 2-D analysis were done
according to Laemmli (1970) and O’Farrell (1975),
respectively.
2.3. MALDI-TOF MS analysis of anti-spectrin antibody
reacting proteins
Protein identification was performed on several occasions in different laboratory services (collaboration with
Lyon-Bayer Crop. Sciences, Lyon, France; Section of
Pharmaceutical Sciences, University of Geneva and Alphalyse A/S, Odense, Denmark). In brief, cross-reacting protein bands were cut and subjected to matrix-assisted laser
desorption ionization-time of flight mass spectrometry
(MALDI-TOF MS) peptide mass fingerprinting and identified by matches across the peptide sequences in the
National Center for Biotechnology Information database
(Shevchenko et al., 1996). Corresponding entries were
searched in the Broad Institute Database (http://
www.broad.mit.edu/annotation/fgi/). As control, known
EF2-GST fusion protein and GST were also sequenced
to confirm the accuracy of the analysis. Alphalyse A/S
analysis facility uses internal controls in each identification
experiments.
The identity of the immunoreacting protein with antispectrin antibody (S1390) from N. crassa was also confirmed by EDMAN sequence analysis at Analytical
Research and Services, University of Bern.
2.4. Expression of N. crassa elongation factor 2 and a-actinin
Elongation factor 2 and a-actinin in N. crassa are coded
by the genes ncu07700.4 and ncu06429.4, respectively. To
prepare cDNA total RNA was isolated from hyphae after
18 h growth using TRIZOL reagent (Sigma) according to
the supplier’s protocol and treated with DNAse (Ambion,
Cat. 1906). cDNA was synthesized using Promega reverse
transcription kit (A3802). Amplifying primers for the coding sequence of the elongation factor 2 were: forward 50 GCGCGCGGATCCATGGTCAACTTCACGATTGAC
G-30 and reverse 50 -ATAAGAATGCGGCCGCTTAGA
GCTTGTCGTAGT AG-30 . Amplification of a-actinin
was performed in two steps, taking advantage of an internal AatII site. Primers used for the amplification were: forward, 50 -CGCCGCGGATCCATGGAGATGCTGGGG
GTGGAG-30 and reverse, 50 -TCACGGACGTCCACTTC
GTCATAGCACG-30 and, forward 50 -AAGTGGACGTC
CGTGATTTCAGCGGCAG-30 and reverse 50 -ATAAGA
ATGCGGCCGCCTAATGATACCCATTCGGCTTG-30 .
The BamHI, NotI and AatII digestion sites are respectively
underlined, in bold and in italics. Amplification program
was: denaturation at 94 °C for 3 min, 30 cycles at 94 °C
for 30 s, 55 °C for 50 s and 72 °C for 2 min, and a final
elongation step of 72 °C for 4 min. PCR fragments were
digested by BamHI and NotI, and cloned into pGEX4T1 (Amersham Biosciences). This recombinant plasmid
was used to transform competent Escherichia coli BL21
strain. To study the expression of the cloned gene, the
transformed cells were grown in LB medium containing
100 lg/ml ampicillin and induced with 100 lM Isopropylb-D-thiogalactopyranoside (IPTG). Proteins from induced
and uninduced E. coli BL21 strain were extracted by sonication in extraction buffer (140 mM NaCl, 2.7 mM KCl,
10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3, and 0.4 mg/
ml of lysozyme), centrifuged and analyzed on SDS–PAGE
gel. Partial digestion was performed with thrombin (5 l/mg
of the GST fusion protein) at 25 °C for 20 min in a final
volume of 20 ll.
2.5. Anti-N. crassa a-actinin antibody production
Purified GST-NCU06429.4 recombinant protein, verified by MALDI-TOF MS (Alphalyse A/S) was used to
immunize two rabbits according to Barnes et al. (1998).
Sera from immunized rabbits were tested 20 days after
immunization and pre-immune serum was used as control
for background or cross-reactivity.
2.6. Immunoblotting
Proteins from unstained mono- and bi-dimensional gels
were electrophoretically transferred to nitrocellulose membranes (BA85, pore size 0.45 lm) using the transfer buffer
described by Burnette (1981) and immunoblotted as
described earlier (Ojha and Barja, 2003). The membrane
was blocked overnight at 4 °C with 5% BSA in TBS-Tween,
incubated for 2 h at room temperature with polyclonal
anti-chicken ab-spectrin (Sigma S1390) or anti-N. crassa
a-actinin antibody at 1:800 and 1:500 dilution in blocking
buffer. Subsequently, the primary antibody was replaced
with anti-rabbit IgG antibodies coupled to horseradish peroxidase (Sigma A8275) diluted at 1:2000 and reincubated
M. Cotado-Sampayo et al. / Fungal Genetics and Biology 45 (2008) 1008–1015
1011
for 1 h. Following four washes in TBS, the membranes
were treated with DAB-H2O2 to develop peroxidase activity, as described earlier (Huber and Ojha, 1994).
Although the anti-chicken ab-spectrin antibody (S1390),
which has been extensively used by others and in our hands
gave better resolution, was used in most experiments
described here, we also tested monoclonal anti-spectrin
antibody (Sigma S3396). The reactivity of this antibody
was very poor (also reported by Slaninová et al., 2003)
and therefore not continued.
2.7. Immunofluorescence microscopy
Mycelia from 10 h culture were treated for 2 h with sordarin (Sigma S1442) at 50 lg/ml concentration; the control
was a 12 h culture without treatment. Samples were fixed
by the addition of paraformaldehyde to the culture medium to a final concentration of 3% (v/v).
Primary anti-chicken spectrin-antibody and secondary
goat anti-rabbit fluorescein-isothiocyanate (FITC, green,
Sigma F9887) antibodies were used at dilutions of 1:50
and 1:100 in phosphate buffer (Ojha and Barja, 2003).
The fluorescent signal was analysed with a Leica TCS
SP2 AOBS confocal microscope. Images were treated with
the program Adobe PhotoShop 7.
3. Results
3.1. Immunochemical detection of proteins reacting with
anti-chicken ab-spectrin and anti-N. crassa a-actinin
antibodies
Neurospora crassa, M. grisea and P. infestans protein
extracts, separated by SDS–PAGE, transferred to nitrocellulose membrane and immunoblotted with anti-chicken abspectrin antibody showed a single band of about Mr
100 kDa both in N. crassa and M. grisea and a band of
68 kDa in P. infestans (Fig. 1A, lanes a–c). The same protein extracts blotted with anti-N. crassa a-actinin antibody
gave a reaction with a protein of 80 kDa in N. crassa,
72 kDa in M. grisea and a band of 140 kDa in the extract
of recombinant over-expressed GST-a-actinins which agree
with the relative mass deduced from the protein sequences
(Fig. 1B, lanes d, e and g). In P. infestans no immunoreacting band with the anti-N. crassa a-actinin antibody was
obtained (Fig. 1B, lane f).
The monoclonal anti-spectrin antibody did not reveal in
crude extract from N. crassa any protein of 240, 220 or
100 kDa (results not shown).
3.2. Anti-chicken ab-spectrin cross-reacting proteins of N.
crassa and P. infestans
Neurospora crassa and P. infestans protein extracts separated in 2D-gels and immunoblotted with anti-chicken abspectrin antibody revealed a predominant polypeptide of
100 kDa with a pI between 6.5 and 6.8 in N. crassa
Fig. 1. Western blot analysis of crude extract proteins. (Lanes a and d)
Neurospora crassa, (lanes b and e) Magnaporthe grisea, (lanes c and f)
Phytophtora infestans, (lane g) over-expressed cloned GST-tagged aactinin in E. coli BL21. Proteins were immunoblotted with anti-chicken
ab-spectrin (A) anti-N. crassa a-actinin (B) antibodies.
(Fig. 2A) corresponding in the gel to three spots of 6.5,
6.6 and 6.8 (described earlier by Cotado-Sampayo et al.,
2006) and 64 kDa with a pI of 5.0 in P. infestans
(Fig. 2B). The different spots of 100 kDa protein, from N.
crassa in 2-D gels, were analyzed by MALDI-TOF MS, first
as one sample, to discard the possibility that the results did
not include a contaminant protein of 100 kDa, due to its relatively ‘‘broad range” of pI (6.4–6.8), each spot were separately analyzed and all showed to be EF2. MALDI-TOF
MS analysis of the 64 kDa peptide from P. infestans
resulted to be the heat shock protein 70 (Hsp70) (Fig. 2C).
3.3. Neurospora crassa elongation factor 2(EF2) overexpression and its reactivity to anti-chicken ab-spectrin
antibody
The elongation factor 2 gene of N. crassa was cloned
and expressed in E. coli BL21 as described in Section 2.
Fig. 3A (lanes a–c) shows over-expression of the fusion
protein demonstrating the induction of a protein of
126 kDa corresponding to 26 kDa glutathione S-transferase (GST) and 100 kDa N. crassa elongation factor 2.
Immunoblotting with anti-chicken spectrin antibody recognized the recombinant protein GST-EF2 (Fig. 3A lanes a0 –
d0 and 3B lane b), with the same intensity as the immunoreacting band from Neurospora protein extracts (Fig. 3A
lanes d0 –e0 ). However, the antibody recognized an additional protein of about 63 kDa of the host cell (even better
than the 126 kDa polypeptide, Fig. 3A lanes a0 –d0 ).
MALDI-TOF MS analysis revealed this protein to be the
GroEL chaperone.
Neurospora crassa elongation factor 2 was cleaved from
GST-fusion protein by partial digestion with the site-specific protease, thrombin, and analyzed by Western blot
with anti-chicken ab-spectrin antibody. As shown in
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M. Cotado-Sampayo et al. / Fungal Genetics and Biology 45 (2008) 1008–1015
Fig. 3. Western blot analysis using anti-chicken ab-spectrin antibodies.
(A) Coomasie blue staining of total proteins in a 10% SDS–PAGE gel
(lanes a–e), corresponding immunoblot (lanes a0 –e0 ). Lanes a, b and c
corresponds to different amount of partially purified GST EF2 protein
(0.2 lg, 0.5 lg and 1 lg, respectively). Lane d corresponds to a mixture of
crude extract of Neurospora crassa (10 lg) and 0.2 lg partially purified
GST EF2 protein and lane e corresponds to a crude extract from N. crassa
(10 lg). (B) Immonoblot of crude extract from N. crassa (lane a), purified
GST EF2 (lane b) and purified GST EF2 partially digested with thrombin
(digestion was performed to remove the GST component from the
recombinant protein).
a cap (Fig. 4B). Incubation of the hyphae with sordarin (an
antifungal compound that interacts specifically with the
elongation factor 2 blocking its binding to the ribosome;
Domı´nguez et al., 1999) caused a clear dislocation of protein from the peripheral region and hyphal tips towards
the cytoplasm (Fig. 4C and D).
4. Discussion
Fig. 2. Sequence analysis of anti-chicken ab-spectrin immunoreacting
peptides. Two-dimensionalgel analyses of Neurospora crassa (A) and
Phytophtora infestans (B) protein extracts with anti-chicken ab-spectrin
antibodies. Arrows indicate the analyzed peptides. Table with results of
sequence analysis (C), each peptide is designated with the organism (Nc,
Neurospora crassa; Phi, Phytophthora infestans), and their pI, ‘‘Broad
number” refers to Broad Institute Accession number of the protein.
Fig. 3B lane c the protein band of 100 kDa corresponding
to EF2 was recognized by the antibody and had the same
molecular weight as the immunoreaction peptide from N.
crassa protein extracts (Fig. 3B lane a).
3.4. Indirect immunofluorescence
The localization of EF2 in the hyphae of N. crassa was
examined by indirect immunofluorescence microscopy with
anti-chicken spectrin antibody as primary antibody and
goat anti-rabbit conjugated to FITC as secondary antibody. The antibody revealed continuous fluorescence in
the peripheral region (Fig. 4A) in the sub-apical region
but concentrated in the entire region of the hyphal tips as
The presence of a spectrin related protein in fungi using
polyclonal anti-chicken ab-spectrin antibody has been
reported by many authors (Degousée et al., 2000; Heath
et al., 2003; Cotado-Sampayo et al., 2006). However, in
spite of the efforts to identify a spectrin sequence in the
sequenced genomes of N. crassa, M. grisea or P. infestans
no gene coding for the Mr 240–220 spectrin, as described
for higher eukaryotic cells, was found. An a-actinin related
protein seems to be the only member of the spectrin superfamily protein present in fungi and Oomycetes, but it is not
recognized by the anti-spectrin antibody.
In N. crassa and M. grisea a protein with relative
mass of 100 kDa shows strong reactivity to anti-chicken
ab-spectrin antibody. The MALDI-TOF MS analysis of
the Neurospora peptide reacting with the antibody corresponds to elongation factor 2, a protein with apparently
no similarity in function, structure or sequence with any
protein belonging to the spectrin superfamily of proteins. These results were reproducible. Immunoblotting
of cloned, expressed and purified elongation factor 2
confirmed the identity of the immunoreacting peptide.
In P. infestans, however, the results of mass spectra
M. Cotado-Sampayo et al. / Fungal Genetics and Biology 45 (2008) 1008–1015
Fig. 4. Effect of sordarin treatment in the localization of the protein
revealed with anti-chicken ab-spectrin antibodies in N. crassa. Nontreated (A and B) and treated (C and D) cells. Bar = 10 lm.
analyses of the immunoreacting band matched the heat
shock protein 70 (Hsp70). The relationship between
spectrins, the Neurospora EF2 and Hsp70 is difficult
to interpret.
It has been reported that GroEl from E. coli, a chaperonin belonging to the Hsp60 class of proteins, reacts with
anti-b-spectrin antibodies (Czogalla et al., 2003). The commercial anti-chicken ab-spectin antibody used in the present study and the work of others reporting the presence
of spectrin in plants and fungi, clearly shows a strong reactivity to GroEl of E. coli (Czogalla et al., 2003). The evidence that this commercial antibody reacts with GroEl
and Hsp70 and the fact that both proteins have a chaperone function (Ellis, 1987; Craig, 2003) suggest that some
anti-spectrin antibodies could react with an eptitope from
chaperones. Indeed, EF-G, EF-Tu (the prokaryotic counterpart of EF2 and EF1a, respectively) and initiation factor
2 (IF2), have a chaperone-like activity (Kudlicki et al.,
1997; Caldas et al., 1998, 2000; Hotokezaka et al., 2002;
Malki et al., 2002).
Spectrin has also been reported to have a chaperone-like
activity (Chakrabarti et al., 2001; Bhattacharyya et al.,
2004). It has the hydrophobic binding sites for lipophilic
molecules through which it can interact with the denatured
proteins. These regions in a- and b-spectrins have been
found to have sequence homology with known chaperone
proteins (about >50% similarity and 30% identity). This
indicates that there could be a putative chaperone-like
domain in spectrin, located near the N-terminus of a-spectrin and the C-terminus of b-spectrin (Bhattacharyya et al.,
2004).
1013
Considering that elongation factors, spectrin, heat shock
proteins and GroEL all have a chaperone activity, we suspect that the anti-chicken ab-spectrin reacts with an epitope in a domain implicated in this activity, maybe
located in a hydrophobic region.
The localization of the immunoreacting protein with
anti-spectrin antibody, identified now as EF2, was found
along the peripheral part of the sub-apical region and as
a cap in the tips during exponential phase of growth in
Neurospora (Cotado-Sampayo et al., 2006). This pattern
was also found in many fungi and other organisms (Table
1). The elongation factors have been described to be associated with the plasma membrane in bacteria. The LepA
protein, proposed recently as a new elongation factor,
EF4, (Qin et al., 2006), is localized in the plasma membrane
of E. coli (March and Inouye, 1985).
The association of DnaK from E. coli (Hsp70) to the
plasma membrane has also been described (El Yaagoubi
et al., 1994). About 47 kDa fragment of Hsp70 in Candida
albicans (Mathews et al., 1998) and the 80 kDa homologue
of Hsp70 in Histoplasma capsulatum (Gomez et al., 1992)
have been shown to be localized in the cell wall. The presence of the heat shock proteins in the cell wall of fungi has
been also described (López-Ribot and Chaffin, 1996;
López-Ribot et al., 1996).
The commercial antibody that we have employed is
polyclonal and produced using as antigen sequences from
two proteins, the a- and b-spectrin, that are similar in
structure but not in sequence (30% identity). It has been
shown that polyclonal antibodies can cross-react with several proteins sharing simple peptide sequences (Michaud
et al., 2003). Therefore, the results obtained with this polyclonal antibody must be interpreted with caution.
Acknowledgments
We gratefully acknowledge financial support from the
Academic Society of Geneva (F.B.). Thanks are due to
R. Strasser for interest in the project and encouragement,
M.-L. Chappuis for technical assistance, A. Fehr for secretarial assistance and B. Peck for reading this manuscript.
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1
Features of α-Actinin in Fungi and Oomycetes
Marta Cotado-Sampayo
Laboratory of Bioenergetics and Microbiology, University of Geneva, 10, chemin des
Embrouchis, CH-1254 Jussy-Geneva, Switzerland
Tel. +41 22 379 37 50, FAX: +41 22 379 37 50,
E-mail: [email protected]
Spectrin superfamily proteins are characterized by their domain structure: N-terminal
calponin homology domains (CH) and C-terminal EF-hand motifs both linked together by a rod
domain. Corresponding to these features, α-actinin, spectrin, utrophin and dystrophins are
considered as closely related members (Blanchard et al. 1989; Pascual et al. 1997). These
proteins are well known in metazoans where they frequently interact with actin and plasma
membrane, and considered to play a role in cell-cell interactions and intercellular signaling (Otey
and Carpen 2004). α-Actinin is a smaller member of the superfamily and its functional unity is a
rod shaped homodimer with monomers assembling in opposite polarity to form the dimer
(Wallraff et al. 1986; Amos and Amos 1991).
In a recent study on the evolution of α-actinins, Virel and Backman (2004) gave a direct
evidence for the presence of its orthologs in two fungi, Neurospora crassa and
Schizosaccharomyces pombe. Whilst our work was in progress on the orthologs and evolution of
the α-actinin in fungi using CH-domain structure a paper by the same authors has appeared
documenting actinin orthologs in fungi using the criterium of rod domain (Virel and Backman
2007). We have extended these observations by including a representative from Chytridiomycota
(Table 1) and a detailed analysis of α-actinin in Saccharomycotina. In this sub-class (Table 1 and
2
fig. 1), which includes very diverse organisms (Dujon 2006), we have found only one species,
Yarrowia lipolitica containing a gene coding for α-actinin. There is no gene coding for a bona
fide α−actinin in Saccharomyces cerevisiae (Wu et al. 2001; Virel and Backman 2004) or in
more recently sequenced genomes of Kluyveromyces lactis, Candida glabrata and Ashbya
gossypii. In other yeasts, we have found hypothetical proteins with one conserved N-terminal αactinin-like actin binding domain (CH-domain) in C. albicans and two in Pichia stipitis,
Debaryomyces hansenii and Candida sp (Candida guilleimondi and Candida tropicalis) but no
spectrin repeats or C-terminal EF-hand motifs (Table 1). The estimated molecular weight (Mw)
of these proteins is between 70-80 kDa, similar to the estimated Mw of the fungal α-actinins.
However, this can not be taken for sure as a criterion for actinin related protein. Obviously, there
are other proteins having CH-domains besides spectrin superfamily members, like Calmin,
Enaptin and Nuance, described as having two N-terminal CH-domains and C-terminal transmembrane domain(s) (Gimona et al. 2002 and references cited therein), but these proteins are
generally of higher molecular weight than expected for fungal α-actinin. Other proteins with two
N-terminal CH-domains, not mentioned in the revision of Gimona et al. (2002), are two
cortexillins, I and II, present in the cellular slime mold Dictyostelium discoideum (Faix et al.
1996). Using blast tools of NCBI, we have also found these proteins in another cellular slime
mold Polysphondylium pallidum and in the ameba Entamoeba histolytica and, these are low
molecular weight proteins of about 40 kDa and considered to be implicated in cytokinesis (Faix
et al. 1996) and mechanical properties of the cell cortex (Simson et al. 1998). We have not found
orthologs of any of these proteins, either high or low molecular weight, containing N-terminal
CH-domain, in the fungal species examined.
3
The “atypical” α-actinins with only conserved N-terminal CH-domain(s) can be explained as
a process in evolution where genes are highly diverged or lost (or have modified beyond
recognition). Gene losses during evolution in several lineages is not a rare event and has been
shown to occur for many genes (Aravind et al. 2000; Roelofs and Van Hasteert 2001; Krylov et
al. 2003).
Even though we are not certain of the relationship between these proteins in the
Saccharomycotina and other fungal α-actinins, we can hypothesize that they have evolved from a
common ancestor through two different pathways. In one, the α-actinin seems to have been lost
like in S. cerevisiae, or severely modified during evolution as in Candida albicans and the yeasts
mentioned above but preserved in other, for example Y. lipolytica (fig. 1).
This hypothesis is supported by the fact that, even though we can not recognize any
functional domain in the middle and C-terminal region of these hypothetical proteins, the gene is
still present with a relative large ORF. Anyway, the absence of a “classical” fungal α-actinin in
nearly all of Saccharomycotina might be due to a complementation in function with other
proteins, maybe fimbrin as in S. pombe (Wu et al. 2001) or other actin binding proteins (Rivero et
al. 1999). Fimbrin is interesting from this point of view since it has two conserved domains of αactinin but with reverse polarity, i.e., N-terminal Ca2+ binding EF-hand motifs and C-terminal
actin binding CH-domains.
We have also analyzed the α-actinin gene of other fungi and an Oomycete from the available
genome database. As shown in Table 1 and figure 2, the sequence of α-actinin is organized into
the three distinct domains: two N-terminal calponin homology domains (blue boxes), a rod
domain (yellow boxes) and the C-terminal Ca2+-binding domain. The CH-domain is the best
conserved region in the protein and contains three actin-binding sites (red boxes) able to bind one
4
actin monomer (Keep et al. 1999; Gimona et al. 2002). In most fungi rod domain, composed of
two spectrin repeats, is the least conserved region. In Oomycetes we can expect the presence of
four, given the size of the rod domain but only the first is relatively conserved and the other three
are difficult to define. The spectrin repeats in fungi and Oomycetes loose some features such as
tryptophan in position 17. However, spectrin repeats are defined more by their length and their
3D-structure than the sequence. In fungi they are of 101-115 amino acids in length and the
structural prediction indicates that each domain contains three segments of α helix in the form of
a three stranded coiled-coil.
The C-terminal calcium binding domain is composed of three EF-hand motifs (green boxes).
The EF-hand motif consists of a twelve residue loop flanked on both sides by a twelve residue αhelical domain. The bioinformatic tools predicted only one putative calcium binding EF-hand
domain. So far, occasionally a second putative EF-hand calcium insensitive domain has been
detected with a very low score but is considered as a match primarily because EF-hand domain is
known to be repeated and not likely to occur as a single copy in a protein. Using the pattern of
EF-hand motifs, PS00018 (http://us.expasy.org), a putative third C-terminal EF-hand domain
was found, manually, about 70 nucleotides downstream from the first one. This domain, present
only in Ascomycota, is probably calcium insensitive, given that position three, five and seven in
the loop do not exactly correspond to the pattern of the canonical EF-hand domain.
Among the fungi examined here what is remarkable about Ascomycetes (N. crassa, M.
grisea, S. sclerotia and F. graminearum) is the presence of an N-terminal extension upstream of
the first CH-domain that has a low sequence similarity. It is variable in length and does not have
any conserved domain motif (InterPro). This N-terminal region has not been considered in the
annotation for Neurospora α-actinin in MIPS database, nevertheless we have demonstrated that it
5
is present in the mRNA of N. crassa (Cotado-Sampayo et al. 2006 and article in preparation).
The presence of N-terminal extension in some fungi could be attributed to a: UTR-region in the
mRNA, a pro-domain of the protein or an alternate open reading frames (ORF). As an UTRregion, it will be too long (with 930 nucleotides length) and if one considers it to be translated as
a pro-domain and cleaved afterwards, we do not know any putative enzyme cutting just upstream
of the first calponin homology domain. In case of an alternative use of ATG-start, one can
conclude that the full-length protein will be translated in special conditions since we have found
only the short form of the protein in the crude extract from standard cultures of N. crassa (80
kDa) or M. grisea (72 kDa) (Cotado-Sampayo et al. 2008).
Our detailed analyses of the features of fungal α-actinin highlight the target for future
experimental studies i.e. their biochemical and functional characterization in order to understand
why they are absent or highly modified in most Hemiascomycetous yeast.
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Cotado-Sampayo M, Ojha M, Ortega Perez R, Chappuis M-L, Seum C, Barja F. Spectrin family
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6
Conference on Fungal Genetics. Vienna University of Technology, Book of abstracts. 2006.
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commercial anti-spectrin antibody in the study of fungi and oomycetes: cross-reaction with
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7
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redundant roles in the growth and development of Dictyostelium. J Cell Sci. 1999; 112:27372751.
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and adhesion energy of wild-type and mutant cells of Dictyostelium lacking talin or
cortexillins. Biophys J. 1998; 74:514-522.
Virel A, Backman L. Molecular evolution and structure of α-actinin. Mol Biol Evol. 2004;
21:1024-1031.
Virel A, Backman L. A comparative and phylogenetic analysis of the α-actinin rod domain. Mol
Biol Evol. 2007; 10:2254-2265.
Wu JQ, Bahler J, Pringle JR. Roles of a fimbrin and an α-actinin-like protein in fission yeast cell
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LEGENDS
Table 1.
Accession Number, Domain Structure and Taxonomic position of organisms used in this study.
The Accession number for: Batrachochytrium dendrobatidis, Rhizopus oryzae, Neurospora
8
crassa, Magnaporthe grisea, Fusarium graminearum, Sclerotinia sclerotiorum, Botrytis cinerea,
Aspergillus nidulans, Candida sp., Cryptococcus neoformans, Coprinus cinereus, Chaetomium
globosum and Phytophthora sp., sequences were obtained from the Broad Institute database
(http://www.broad.mit.edu/annotation/fgi/).
Sequences
for
Yarrowia
lipolitica
and
Debaryomyces hansenii were obtained from the yeast specific database Genolevures
(http://cbi.labri.fr/Genolevures/). The sequences for Entamaeba cuniculi, Uromyces maydis,
Aspergillus fumigatus, Aspergillus oryzae, Schizosaccharomyces pombe and Pichia stipitis were
derived from Swiss-prot protein database (http://ca.expasy.org/sprot/). No putative α-actinin
gene was found in completed genomes of Saccharomyces cerevisiae, Candida glabatra,
Kluyveromyces lactis and Ashbya gossypii.
Structure domain is represented with rectangles for the CH-domain, ovals for spectrin repeats and
squares for EF-hands.
FIG. 1.- The α-actinin protein evolution in hemiascomycetous yeast. The arrows indicate the
major events in evolution.
FIG. 2.- Alignment of amino acid sequence of α-actinins from Ascomycetes. Domains are
indicated in color boxes, CH-domain (blue), spectrin repeat (yellow) and EF-hand motif (green).
Acting Binding Sites (ABS) are indicated with black underlined red boxes.
Table 1
Taxon
MICROSPORIDIA
CHYTRIDIOMYCOTA
Organism
Accesion no
Entamoeba cuniculi
Q8STW7
Batrachochytrium dendrobatidis
BDEG_05746
Rhizopus. oryzae
RO3G_05027.1
Phycomyces blakesleeanus
Phybl1_37239
Saccharomyces cerevisiae
--
Candida glabatra
--
Kluyveromyces lactis
--
Ashbya gossypii
--
Candida albicans
CAWG_04250.1
Candida tropicalis
CTRG_01638.3
Candida guilleimondi
PGUG_05348.1
Debaryomyces hanseni
DEHA0C07491g
Pichia stipitis
A3GG62
Yarrowia lipolitica
YALI0F07601g
Schizosaccharomyces pombe
O13729
Chaetomium globosum
CHGG_05684.1
Neurospora crassa
NCU06429.4
Magnaporthe grisea
MG06475.4
Fusarium graminarum
FG07284.1
Sclerotinia sclerotiorum
SS1G_00266.1
Botrytis. cinerea
BC1G00894.1
Aspergillus nidulans
AN7707.2
Aspergillus fumigatus
Q4WUF8
Aspergillus. oryzae
Q2U7P8
Uromyces maydis
Q4P6F1
Cryptococcus neoformans
CNAG_02992.1
Coprinus cinereus
CC1G_03395.1
Phytophthora sojae
PSOJAE 137006
Phytophthora ramorum
PRAMORUM 85609
Phytophthora infestans
PITG13237
Domain structure
CH
SR
CH
EF
CH
CH
SR
SR
EF
CH
CH
SR
SR
EF
ZYGOMYCOTA
CH
HEMIASCOMYCOTA
CH
ARCHIASCOMYCOTA
BASYDIOMYCOTA
OOMYCOTA
CH
CH
CH
CH
CH
SR
CH
CH
CH: Calponin homology domain; SR: Spectrin repeat; EF: EF-hand motif.
SR
SR
SR
SR
EF
EF
EF
EF
EF
EF
Fig. 1
S. cerevisiae
Loss of α-actinin
gene
Loss of rod and
calcium binding
domains in α-actinin
C. glabatra
K. lactis
A. gossypii
D. hansenii
P. stipitis
C. tropicalis
C. guilleimondi
C. albicans
Y. lipolytica
Fig. 2
10
20
30
40
50
60
70
80
90
100
110
120
----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:Afumigatus/1-645
-----------------------------------------------------------------------------------------------------------------------------Aoryzae/1-645
-----------------------------------------------------------------------------------------------------------------------------Bcienrea/1-660
-----------------------------------------------------------------------------------------------------------------------------Ssclerotiorum/1-931 MFSALKKSLRIEPKVEAPKDVTSNTVAKEENVENVGIEDTIVVKDLDATRKEAEKEKVEDVREEDVL-VIKDLDAKWEERPDMRAEEEARDMEKGEDVK-GEEDTLVVKDLDVRVESEGVKEEDVE
Cglobosum/1-643
-----------------------------------------------------------------------------------------------------------------------------Fgraminearum/1-889 ------------MDAVRNSVQAFQGML-------------PL-----------------QPGNLDGN-EKFPDFCLHSEALCSNDSHEDLEREQEQQHDKG-----------------LHNKQSKQ
Mgrisea/1-788
----------------------MDHQY-------------PH-----------------DAGSVPSNREDWPLF------------------TNENSYHPG-----------------GHTDNSLN
Ncrassa/1-1027
-----MEMLGVEIGEPGPSVQSGQPAA-------------ALPDPATAPARAAAQSPTQSTNSTTST-RSGPASLARSRSSTAGASTTCSSRSWGETHSGGPAQVEKFPDLPTHTTSPEHQQQPLH
Ylipolytica/1-616
-----------------------------------------------------------------------------------------------------------------------------Spombe/1-621
-----------------------------------------------------------------------------------------------------------------------------130
140
150
160
170
180
190
200
210
220
230
240
250
---|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|-Afumigatus/1-645
-----------------------------------------------------------------------------------------------------------------------------Aoryzae/1-645
-----------------------------------------------------------------------------------------------------------------------------Bcienrea/1-660
-----------------------------------------------------------------------------------------------------------------------------Ssclerotiorum/1-931 MEVEYVN-EEVGEEEEEEEEGKREREPTITSNSTNYIGIPSPPSSEIGDESDDVI----------GDEESEGEQINSTPGNDRCLVGSSSVTAANPDSEVNKNEETSISTTPRHSRAISPTPIENR
Cglobosum/1-643
-----------------------------------------------------------------------------------------------------------------------------Fgraminearum/1-889 RHPHFHYPRPHSPSASISESSRL--SCISTA------------SVSASSSTSSRVSSASFD--SFSTPI-----------------------SPTLDSSLHSPRSSLASNT--CSSPVSEFIAR-Mgrisea/1-788
RPP------------------SL--SLSSTT------------SSYD---------------------------------------------FSDKDSVFDSQRAS--------------FIST-Ncrassa/1-1027
HHPHPHH-RHHHHHQPASEAVRRDGTCAHTAE-----GTPTPPAAPAAPATPTPVPRLNTDHTELASPGPPPAASCPAIGRPPSLSIHSTASSSDHSSSLSDGDLVFDSRR--SS-----VVTS-Ylipolytica/1-616
-----------------------------------------------------------------------------------------------------------------------------Spombe/1-621
-----------------------------------------------------------------------------------------------------------------------------260
270
280
290
300
310
320
330
340
350
360
370
--:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:--Afumigatus/1-645
--------------------------------------------------------------------------------------------------------------------MLTVEKSWVN
Aoryzae/1-645
--------------------------------------------------------------------------------------------------------------------MLTVEKSWVN
Bcienrea/1-660
-------------------------------------------------------------------------------------------------------------------MAFAEQQQWIT
Ssclerotiorum/1-931 SHTQNQEKRISSSTADFEINAPSTPPATPPRLVRRERSASQREREILRSRQFSE---TARLQRSAEKPEKTDENLGLGKRRKRYQHRHKSK-GSNAGSGEALKAVEPLHEHQLGKMAFAEQQQWIT
Cglobosum/1-643
-------------------------------------------------------------------------------------------------------------------MAFAEQQRWIT
Fgraminearum/1-889 ----------NRSLTGASLSS-------VS--------SSPPSSPTPRNRWDKPQPYAGRAQRSGSTTTNVSSPVGVHKQEQSSSPR--SF-RSNPIPADALAAVES-HDTKLIKMAFAEQQRWVT
Mgrisea/1-788
----------SSSIDSCSIAC---------------------GSPLPRSRSNAKQLALARLQRSGSNPVGLSSKKADSPSR-----K--RF-YSSGSAQEALAATEP-AGGGLTKM----EEDWVS
Ncrassa/1-1027
----------ASTASAYNYPLS--PSCHQP--------DSSPEHPLTRTRRDAKQLHAARLERSGSISLALASTKREAPSPALRNPT--RFQRASSEPTRAPKLVTE-QPAGFTKMAFAEQQQWIT
Ylipolytica/1-616
----------------------------------------------------------------------------------------------------------------------MSLPQWIA
Spombe/1-621
----------------------------------------------------------------------------------------------------------------------MQANQWQS
380
390
400
410
420
430
440
450
460
470
480
490
500
-|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|---Afumigatus/1-645
VQQKTFTKWLNDKIKVRGI-LIDDLVTDLSDGVILIHLLEILGGESLGRYASKPKLRVQKFENVNKSLDFIRGRRIQMTNIGAEDIVDGNRKIILGLIWTLILRFTISDINAEGMTAKEGLLLWCQ
Aoryzae/1-645
VQQKTFTKWLNDKLKVRRL-FIEDLVSDLSNGIILIHLLEILGGEPLGKYASNPRLRVQKFENVNKSLDTIKGRGIQMTNIGAEDVVDGNRKIILGLIWTLILRFTISDINEEGMTAKEGLLLWCQ
Bcienrea/1-660
VQQKTFTKWLNTKIAHRKL-EVIDLVKDLSDGVILIHLLECLSNESLGRYAAKPKLRVQRFENANLSLDFIKSRGIQMTNIGAEDVVDGNRKIILGLIWTLILRFTISDINLEGMTAKEGLLLWCQ
Ssclerotiorum/1-931 VQQKTFTKWLNTKIAHRKL-EVVDLVKDLSDGVILIHLLECLSNESLGRYAAKPKLRVQRFENANLSLDFIKSRGIQMTNIGAEDVVDGNRKIILGLIWTLILRFTISDINLEGMTAKEGLLLWCQ
Cglobosum/1-643
VQQKTFTKWLNTKVEARGL-EVKDLVQDLSDGVMLIHLLECLSSESLGRYAAKPKLRVQRFENANRALDFIKSRGIQMTNIGAEDVVDGNRKIILGLIWTLILRFTINDINEEGMTAKEGLLLWCQ
Fgraminearum/1-889 VQQKTFTKWLNTKIEARNL-EVKDLVKDLSDGVMLIHLLECLSHESLGRYASKPKLRVQKFENANTALDFVKSRGIQMTNIGAEDVVDGNQKIVLGLIWTLILRFTISDINEEGMSAKEGLLLWCQ
Mgrisea/1-788
TQQKTFQKWANSKLAERSL-ETKNLVEDLKDGVLLIHLLECLASESLGRFASKPKLPVQQYENANTALGFIQSRGIRLTNCGAEDIVKGNRKIVLGLIWTLILRFTISDINEEGLTAKEGLLLWCQ
Ncrassa/1-1027
VQQKTFTKWLNTKIEVRGL-EVKDLVKDLSDAVMLIHLLECLSGDSLGRYAAKPKLRVQRFENANLALNFIKSRGIQMTNIGAEDIVDGNRKIILGLIWTLILRFTINDINEEGMTAKEGLLLWCQ
Ylipolytica/1-616
TQHKAFLRWANTYLEANQIGTMVSLETDFCDGVRLCQLIEIIGKESLGRYSGQPRMRFQMIENVNTALAFIRHRGVQLHNIGAEDICDGNLKLILGLLWILILRFTIEDISEEGLSAKEGLLLWCQ
Spombe/1-621
VQNRTFTKWFNTKLSSRDLPSVFDLRKDLSDGILLIQLLEIIGDENLGRYNRNPRMRVHRLENVNKALEYIKSKGMPLTNIGPADIVDGNLKLILGLIWTLILRFTIADINEEGLTAKEGLLLWCQ
510
Afumigatus/1-645
Aoryzae/1-645
Bcienrea/1-660
Ssclerotiorum/1-931
Cglobosum/1-643
Fgraminearum/1-889
Mgrisea/1-788
Ncrassa/1-1027
Ylipolytica/1-616
Spombe/1-621
520
530
540
550
560
570
580
590
600
610
620
630
:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|
RKTACYE-GVEVRDFSTSWNDGLAFCALLDIHRPDLIDFDALDKKDHRGNMKLAFDIATNEIGIPDLLDVDDVCDVAKPDERSLMTYIAYWFHAFSQLERVENAGRRVEKFINNMHGAWEMQNSYE
RKTACYE-EVEVRDFSTSWNDGLAFCALLDIHRPDLIDFDALDKKDHRGNMKLAFEIAANEIGIPDLLDVDDVCDVPRPDERSLMTYIAYWFHAFSQLERVENAGRRVEKFINNMHGAWEMQNSYE
RKTACYE-EVDVRNFTDSWNDGLAFCALLDIHRPDLIDYDTLDKDDHRGNMQLAFDIATKEIGIPALLDVEDVCDVAKPDERSLMTYIAYWFHAFSQMEKVENAGRRVEKFVNNMQGAWEMQSAYE
RKTACYE-EVDVRNFTDSWNDGLAFCALLDIHRPDLIDYDTLDKDDHRGNMQLAFDIATKEIGIPALLDVEDVCDVAKPDERSLMTYIAYWFHAFSQMEKVENAGRRVEKFVNNMQGAWEMQSAYE
RKTACYD-EVDVRDFSASWNDGLAFCALLDIHRPDLIDYDALDKSDHRGNMQMAFDIAHKEIGIPKLLDVEDVCDVAKPDERSLMTYIAYWFHAFSQMEKVENAGRRVEKFVNNMRGAWDMQSAYE
RKTACYE-EVEVRDFSGSWNDGLAFCALLDIHRPDLIDYDALDKADHRGNMQLAFDIAHKEIGIPKLLDVEDVCDVAKPDERSLMTYIAYWFHAFSQMEKVENAGRRVEKFVNNMQGAWEMQSAYE
RKTACYE-ECDVRDFSASWNDGLAFCALLDIHRPDLIDYDALDKTDHKGNMQMAFDIAHKEIGIPKLLDVEDVCDVAKPDERSLMTYIAYWFHAFSQMEKVENAGRRVEKFVNNMQGAWEMQSAYE
RKTACYD-EVDVRDFSGSWNDGLAFCALLDIHRPDLIDYDALDKSDHRGNMQLAFDIAHAEIGIPKLLDVEDVCDVAKPDERSLMTYIAYWFHAFSQMEKVENAGRRVEKFFNNMQGAWEMQSAYE
RKTAGYK-GVAVKDFSGSWSDGLAFCALLDKHRPDLIDFAQLDPTKPRENMELAISIATEQIGIPQILDVEDICGVAKPDERSVMTYVAYWFHAFSALDMIENAGRRLEKFVEMTSSAYAMQSGYE
RKTANYHPEVDVQDFTRSWTNGLAFCALIHQHRPDLLDYNKLDKKNHRANMQLAFDIAQKSIGIPRLIEVEDVCDVDRPDERSIMTYVAEYFHAFSTLDKVETAARRVERFTEVLMSTHDMKIDYE
640
650
660
670
680
690
700
710
720
730
740
750
----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:Afumigatus/1-645
RRMKELLRLIRAQREAWKNASFE--GTYKDAKEQARQFSLYKRNEKRRWVAEKSDLAALLGNIKTKLSTYRLRPYDPPEELRLEVCDNEWELLTRDEHERSQLINETIRDIKNALRRSFADKANDF
Aoryzae/1-645
RRMKELLRLIRAQREEWKNASFE--GTYKDAKDQAFQFSLYKKKQKRQWVAEKSDLAALLGNIKTKLSTYRLRPYDPPAELSLEVCDQEWECLTRDEHERSQLINETIRDIKNALRRSFADKANDF
Bcienrea/1-660
KRMRALLKSIKEQVITWQDATFE--GTYADAKKQATHFSSYKRGQKREWVAEKSDLAALLGNIKTKLSTYRLRAYDPPPELRLSVLDEEWAKLMKGEMARGQLINETIRDIKNALRRSFADKANDF
Ssclerotiorum/1-931 KRMRALLKNIKEQVITWQDATFE--GTYVDAKKQASHFSSYKRGQKREWVAEKSDLAALLGNIKTKLSTYRLRAYDPPPELRLSVLDEEWALLMKGEMARGQLINETIRDIKNALRRSFADKANDF
Cglobosum/1-643
RRMRELLKVIREQMESWQLAKFE--GTYTDAKAQAADFAAYKRGLKREWVAEKSELATLLGNIKTKLGTYRLRPYDPPAELRLEVLDQEWANLTKAEMARGQLINETIRDIKNALRKSFADKANDF
Fgraminearum/1-889 RRMRALLKAIQERIEVWKEATFE--GTYTDAKAQLNQFFDYKRGKKREWVAEKSDLATLLGNIKTKLGTYRLRPYDPPAELSLDALEQRWAELASNEMRRAQLINETIRDIKNALRKSFADKANDF
Mgrisea/1-788
RRMAALLQAIRAQVESWQQAKFE--GSYTDAKAQATDFASYKRGKKREWVAEKSELATLLGNIKTKLGTYRLRPYEPPAELRLDVLDGEWANLAAAEMKRGQLINETIRDIKNALRKSFADKANDF
Ncrassa/1-1027
RRMAALLKAIREQVVSWKGSTFD--GTYADAKAQAFQFASYKKGKKREWVAEKSDLATLLGNIKTKLATYRLRPYDPPAHLRMEVLDDEWGNLSKAEMSRGQLINETIRDIKDALRKSFADKANDF
Ylipolytica/1-616
ERMKALLKAIATQKEKWEQAADPEHLAYVEVKQQTAEHATFKIKTKREWTREKASLASLLGNIRTKLATYNLKEYSPPVGLRSADVEAAWKELHMGEINRSKLLNQSMRRLKESLRKRFADAANEF
Spombe/1-621
SRMKRLLGSIARMQEYWHTVQFE--NNYTDVKSHSNNFAKFKATEKREWVKEKIDLESLLGTIQTNLKTYQLRKYEPPAGLKIVDLERQWKDFLSEEANQSKLINTHMREIKESMRIAFADRANSF
760
770
780
790
800
810
820
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840
850
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870
880
---|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|-Afumigatus/1-645
ALTLKTLSLAISGLDGDVEDQLEHVKRLNDNLPPLDAFLETIAELDEQCVEANIEENDFTTYTLDELSYELSLVKSSISKKLAFLENQLVARNMTNLTPIQLEEFESVFRHFDRDSSNTLHELEFS
Aoryzae/1-645
ALTLKTLSLAISGLDGDVEDQLAHVKRLNDNLPPLDAFLDTIAEIDEQCEEANIEENDYTTYTLDELSYELSLVKSSISKKLAFLDNQLVARNMTNLTPIQLEEFESVFRHFDRDSSNTLHELEFS
Bcienrea/1-660
ATTLNTMQLAISGLEGDVEDQLVHVRRLHDNLPPLNQFLDQIEAIDKKCEEANIEENDFTTYTYDELCYEMSLVKNSVAKKLAFLDNQVVARNMTNLTPIQLEEFESVFRHFDRDATNSLQELEFS
Ssclerotiorum/1-931 ATTLNTMQLAISGLEGDVEDQLVHVRRLHDNLPPLNQFLDQIEAIDKKCEEANIEENDFTTYTYDELCYEMSLVKNSVAKKLAFLDNQVVARNMTNLTPIQLEEFESVFRHFDRDASNSLQELEFS
Cglobosum/1-643
ALALNTIQLAISGLEGDVEDQLHHVRKLSENLPPLDAFLKTIAAVDAKCQEANIEENDFTTYTYDELCYELSLVKSSVSKKLAFLENQMVARNMTNLTPIQLEEFESVFRHFDRDDTNCLSELEFS
Fgraminearum/1-889 AMALNTMQLALSGLDGDVEDQLHHVRKLSESLSPLDQYLDKISELDQKCQEANIEENDFTTYTYDELSYELGLVKTSVQKKLAFLENQMVARSMTNLTPIQLEEFESVFRHFDRDDTNSLQELEFS
Mgrisea/1-788
ALALNTIQLAISGLEGDVEDQLHHVRKLSENLPPLDEYLKTIGAVDAKCQEANIEENDFTTYTYDELCYELGLVKSSVAKKLAFLDNQMVARNMTNLTPIQLEEFESVFRHFDRDDSNSLAELEFG
Ncrassa/1-1027
ALALNTMQLAISGLEGDVEDQLHHVRKLSENLPPLDAYLKTIEAVDLKCQEANIEENDFTTYSYDELCYELSLVKSSVSKKLAFLENQMVARNMTNLTPIQLEEFESVFRHFDRDDTNSLSELEFS
Ylipolytica/1-616
SDRLSVLSTAISQMDGPLEDQLEEIADISEKLRPLTEKIRLLKELDTSCVEANVEENDYTVYSYDELEYDLGLAKESVKKKLAFIENQIVARNMTNLTPIQLEEFESVFRHFDKSQHNALLESEFS
Spombe/1-621
SKMLSTISNEITNLQGDWRDQLDHVEFLQEHLGPLEVELASVKVLYDNCFQAGIEENDYTMFSYEDLEHEFGITANIIANKIKYLENELLEREKRTLSKQELDGITKVFRHFEKKKSNMLNEVEFY
890
900
910
920
930
940
950
960
970
980
990
1000
--:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:----|----:--Afumigatus/1-645
AALASLGLVYDEEEMHQVYVETCGPARLAQNAGVSFEQFIHFMVSVTEDQHTAEQVFQSFREVADGKPYVTELDLRHSLIPDEVIEHLVQTMPLHQGPDLLEDRDLPKYDYISFMEKMM
Aoryzae/1-645
AALASLGLVYDEDEMHEVYVETCGPARLAQNAGVSFEQFIRFMVSVTEDQNTAEQVLQSFREVADGKPYVTELDLRHSLIPDEVIDHLVQTMPRHEVFDRGEDQNEPKYDYYSFMQKMM
Bcienrea/1-660
AALASLGLVFSEDEMHDYFLDTSNGKDY-----VTFEEFIRFMVDVTEDQNTAEQVFQSFREVADGKPYVTEMDLRHSLVPDEVIEKLTQFIPIHKGPDLQEDRGMPQYDYISFMDKLL
Ssclerotiorum/1-931 AALASLGLVFSEEEMHHYFLDTSNGKDY-----VTFEEFIRFMVDVTEDQNTAEQVFQSFREVADGKPYALDRWI-------------------------------------------Cglobosum/1-643
AALASLGLIFSEDEMHDYFLDTSGGLDR-----VTFEQFIRFMVDVTEDQNTAEQVFQSFREVADGKPYVTEMDLRHSLVPDDVIEKLVEIMPGHSGPDMQSDRGQPQFDYIAFMDKMI
Fgraminearum/1-889 AALASLGLVFSEDEMHDYFHATSGGRDY-----VTFEQFIRFMVDVTEDQNTAEQVYQSFREVADGKPYVTEMDLRHSLVPDEVIDQLVEIMPAHSGPDMSEDRGMPQYDYISFMEKLI
Mgrisea/1-788
AALASLGVVFSEGEMHEYFVETAKGRDR-----ITFEQFIRFMVEVTEDQNTAEQVYQSFREVADGKPYVTEMDLRHSLVPDEVIDKLIEIIPAHNGPDTAQDRGMPQYDYIAFMDKFI
Ncrassa/1-1027
AALASLGLVFSEDEMHEYFLSTSNGRDR-----VTFEQFIRFMVDVTEDQNTAEQVFQSFREVADGKPYVTEMDLRHSLVPDEVIEKLIEIIPKHTGPDMQSDRGMEQYDYIAFMEKLI
Ylipolytica/1-616
GALASLGLVYSETEMHEVFQAASEGQVS-----VSFEQFITFMVEVTEDQLSAEQVLQSFAEVADGKMYVTELDLQNSLIPEPMIDQLKDTMP----------KTADGFDYIAYMERLT
Spombe/1-621
AALASLGLVYDTEEGTALFHRAANSEEG-----VTYERFTEIVMEELEDRDSARQVLYAFCDVADGKSYVTSDDLLRSQVRPNIVKFLECNMNKHS----------EGLDYLTWIKQLL
1
Characterization of Neurospora crassa α-actinin
Marta Cotado-Sampayoa, Ruben Ortega Pereza, Mukti Ojhab, Carol Seumc,
François Barjaa*
a
Department of Botany and Plant Biology, University of Geneva, ch. des Embrouchis
10, CH 1254 Jussy-Geneva, Switzerland
b
Department of Biochemistry, University of Geneva, Quai Ernest-Ansermet 30, CH
1211 Geneva 4, Switzerland
c
Department of Zoology and Animal Biology, University of Geneva, Quai ErnestAnsermet 30, CH 1211 Geneva 4, Switzerland
* Corresponding author. Fax: +41 22 379 3756.
E-Mail address: [email protected]
2
Abstract
α-Actinin, an actin binding protein belonging to the spectrin superfamily is
present in most eukaryotes except plants. It is composed of three domains: Nterminal CH-domains, a C-terminal calcium binding domain (with EF-hand motifs)
and a central rod domain. α-Actinin from Neurospora crassa has been cloned and
expressed as GST fusion protein for biochemical characterization and as GFP fusion
protein for in vivo localization. In this report we show that the α-actinin from N.
crassa has the ability to cross-link actin filaments in a calcium regulated manner just
as some α-actinins from higher eukaryotes. The localization in situ and in vivo of αactinin led us to propose an interaction of this protein with the actin cytoskeleton in
the process of germination, hyphal elongation and septum formation. The deletion of
the α-actinin gene in N. crassa has a lethal effect suggesting that its function is not
complemented by other actin binding proteins as observed for other organisms such
as Schizosaccharomyces pombe or Dictyostelium discoideum. These organisms can
survive the knock-out of the α-actinin gene.
Keywords: α-actinin; Neurospora crassa; actin binding protein
1. Introduction
In Neurospora a wide variety of cell types is found during its cell cycle. For its
vegetative and sexual phases 28 morphologically distinct cell types have been
described (Britis et al., 2003). This complexity must be supported by a proper
coordination between skeletal structures (cell wall and cytoskeleton) and signaling
pathways. The actin cytoskeleton which participates in this coordination has a major
3
role in the establishment of hyphal tips and the maintenance of polarized growth
(Barja et al., 1993; Heath, 1999; Heath et al., 2000; Virag and Griffiths, 2004). The
dynamics of the actin cytoskeleton is regulated by a large number of actin-binding
proteins (ABPs).
Taking advantage of the existence of complete genome sequences of several
fungal genomes, many of these actin binding proteins were annotated by the Munich
Information Center for Protein Sequences (MIPS) database but their biochemical and
functional characterization is not so well advanced. One of these proteins is α-actinin
which has recently been presented in a study on its evolution, by Virel and Backman
(2004, 2007), and in our own work on the analysis of a putative spectrin-superfamily
protein in fungi (Cotado-Sampayo et al., 2006, 2008).
α-Actinin belongs to the spectrin superfamily of which other members are
spectrin, dystrophin and utrophin. All these proteins contain three functional and
structural domains: two calponin homology (CH) domains at the N-terminal, a
central rod domain containing spectrin-repeats and a C-terminal domain with EFhand motifs. The functional unit of α-actinin is a homodimer of two anti-parallel
monomers (Critchley and Flood, 1999; Djinovic-Carugo et al., 1999). As a
consequence, α-actinin cross-links F-actin by binding with its each N-terminal end to
an actin filament (Podlubnaya et al., 1975). The actin-binding ability is regulated by
calcium but in skeletal muscle isoforms (α-actinin-1 and α-actinin-4) this regulation
is calcium independent since their EF-hand motifs are non-functional (Blanchard et
al., 1989).
α-Actinins has been described in most eukaryotic cells (Fryberg et al. 1990;
Barstead et al., 1991; Beggs et al., 1992; Mills et al., 2001; Virel and Backman,
4
2006; Virel et al., 2007), in the protozoa Trichomonas vaginalis and Dictyostelium
discoideum (Brier et al., 1983; Addis et al., 1998; Bricheux et al., 1998; Rivero et al.,
1999) and in the fission yeast, Schizosaccharomyces pombe (Wu et al., 2001).
Although its function is generally based on its ability to bind actin filaments, αactinin is not merely a cross-linking protein. Recent studies have revealed an
expanded number of α-actinin binding proteins, such as paladin, syndecan 4,
CaMKII, fesselin, and others that suggest variety of roles in the cell (Otey and
Carpen, 2004 and references cited therein; Pham and Chalovich, 2006; Bozulic et al.,
2007). Basically, α-actinin links the cytoskeleton with plasma membrane proteins,
thus providing structural stability (Belkin and Koteliansky, 1987; Otey et al., 1990;
Knudsen et al., 1995; Rajfur et al., 2002; Dandapani et al., 2007). Some of these
membrane proteins are receptors and α-actinin constitute a piece in the scaffold to
integrate signaling pathways (Wyszynski et al., 1997; Dunah et al., 2000; Otey and
Carpen, 2004). In S. pombe α-actinin (Ainp1) seems to have a different function,
playing a role in cytokinesis, but the interaction between this protein and the plasma
membrane has not been established (Wu et al., 2001).
There is evidence for the presence in filamentous fungi of genes coding for αactinins sharing high sequence similarity with Ainp1 of fission yeast (Virel and
Backman, 2004, 2007; Cotado-Sampayo et al, 2006, 2008). However, the
biochemical and functional characteristics of these proteins are still unknown.
The N. crassa genome contains one open reading frame, potentially coding for an
α-actinin protein with an atypical N-terminal extension preceding the conserved CHdomain. In this report, we present a study on the α-actinin from N. crassa and
5
provide insight in its domain structure, biochemical properties, localization and
expression during development.
2. Materials and methods
2.1. Strains and culture conditions
Wild type N. crassa (FGSC 262, strain St. Lawrence STA4) and a heterokaryon
α-actinin knockout strain (FGSC11835, Colot et al., 2006) were obtained from the
Fungal Genetics Stock Center, School of Biological Sciences, Kansas City, MO. The
procedure for the preparation of the conidial inoculum has been described earlier
(Cotado-Sampayo et al., 2006). Conidia were inoculated at a density of 5x106
conidia/ml in Vogel’s liquid medium (Vogel, 1956) containing 2% sucrose. The
cultures were grown on a rotary shaker (150 rpm) at 30 °C for 6, 12 and 18 h.
2.2. Immunochemical characterization
Cells from different stages of development were harvested by filtration, washed
twice with distilled water, frozen and ground in a mortar kept cool with liquid
nitrogen. The frozen mycelial powder was suspended in cytoskeleton stabilizing
buffer (Abe and Davies, 1995) with slight modifications as described earlier
(Cotado-Sampayo et al., 2006). The homogenate was incubated for 20 min at 4 °C
while gently agitated and centrifuged at 6.000xg for 15 min at the same temperature.
Protein concentration in the supernatant was measured according to Bradford (1976)
using bovine serum albumin (BSA) as standard. 2-Dimensional gel electrophoresis
was carried out according to O’Farrel (1975); proteins were transferred to
nitrocellulose membrane in a semidry system and stained with 0.05% (w/v) Ponceau
S in 3% (w/v) trichloroacetic acid (TCA). The membrane was blocked overnight at 4
6
°C with 5% BSA in TBS-Tween, incubated for 2 hours at room temperature with
polyclonal anti-Neurospora α-actinin antibody (Cotado-Sampayo et al., 2008) at
1:500 dilution, washed and re-incubated for 1 hour with anti-rabbit IgG antibodies
coupled to horseradish peroxidase (Sigma A8275) diluted at 1:2000. Following 4
washes in TBS, peroxidase activity was assayed with 3,3’-diaminobenzidin
tetrahydrochloride (DAB) and H2O2 as described earlier (Cotado-Sampayo et al.,
2006).
2.3. Construction of recombinant plasmids
Recombinant plasmids were constructed, A) to express and purify the fusion
protein for biochemical analysis and B) to study in vivo expression:
A. Construction and expression of GST-α-actinin fusion protein for biochemical
analysis
The α-actinin entry in the Neurospora data base (NCU06429.4, Broad Institute)
is a protein with two putative methionine start codons, giving two products with
different predicted molecular weights (100 kDa and 80 kDa). The coding sequence of
GST-fusion proteins of both full- (100 kDa) and short-length (80 kDa) were
amplified
using
the
forward
CGCCGCGGATCCATGGAGATGCTGGGGGTGGAG-3’
primers
for
full-
5’and
5’-
CGCCGCGGATCCATGGCTTTCGCAGAGCAACAAC-3’ for short- length αactinin.
The
reverse
primer
5’-
ATAAGAATGCGGCCGCCTAATGATACCCATTCGGCTTG-3’ was common for
both constructions. The PCR product was cloned in pGEX4T.1 at BamHI and NotI
restriction sites (underlined) and this construct was used to transform competent
Escherichia coli BL21 cells. For the expression of fusion proteins, transformed cells
7
were inoculated in 0.5 to 1 liter LB medium containing 100 μg/ml ampicillin and
incubated at 37 °C till an OD600 of 0.4–0.6 was reached. Expression of the protein
was induced by the addition of IPTG (final conc. 100 mM) and the cultures were
grown for a further period of 16 h at 23 °C, harvested by centrifugation, suspended in
lysis buffer (50 mM Tris-HCl, pH 8.0, 2 mM EDTA, 100 mM NaCl) and lysed by
sonication. The soluble proteins were recovered by centrifugation (16.000xg, 15 min)
and purified on GST-sepharose column. The construction of “short” GST-α-actinin,
without the N-terminal extension, was used to characterize the actin- and calcium
binding properties of the protein.
B. Construction of α-actinin-egfp gene containing plasmid and transformation
The α-actinin-egfp gene fusion was constructed by PCR amplification of the
sequence encoding the α-actinin gene (ncu06429.4), including the 930 bp of the Nterminal extension, the region upstream of the sequence coding for the first CHdomain.
PCR
was
carried
out
using
forward
primer
5’-
CGCCGCGGATCCATGGAGATGCTGGGGGTGGAG-3’ and reverse primer 5′GT CACGTTAATTAAATGATACCCATTCGGCTT-3′. This led to the insertion of
BamHI and PacI restriction sites (underlined) and used to clone the gene in the egfp
gene containing plasmid pMF272 constructed by Freitag et al. (2004).
Transformation of the N. crassa his-3 mutant (FGSC 9014; ridRIP1 mat A his-3)
and heterokaryon transformant selection were performed as described previously by
Margolin et al. (1997) and Freitag et al. (2004).
2.4. Biochemical analysis of α-actinin
Actin binding assays
8
To monitor the co-sedimentation of Neurospora α-actinin with actin, the
concentration of actin was maintained constant at 3.5 μM but the concentration of αactinin was varied. G-Actin from rabbit muscle (Molecular Probes, Eugene, OR,
USA) and GST-α-actinin were mixed in actin binding buffer (5 mM Tris-HCl, pH
8.0, 0.2 mM ATP, 0.2 mM CaCl2, 0.5 mM β-mercaptoethanol) and polymerization of
actin was initiated by the addition of KCl and MgCl2 to a final concentration of 100
mM and 1 mM, respectively. Samples were incubated at room temperature for 20
min and centrifuged for 30 min at 13,000 rpm in an Eppendorf centrifuge. The effect
of calcium on the regulation of actin-binding capacity of α-actinin was tested by
addition of 0.5 mM EGTA in all samples except for the one containing 3 mM CaCl2.
After centrifugation the supernatants and pellets were collected and electrophoresed
in 10% SDS-PAGE and immunobloted with anti-actin antibody (Sigma A2668).
Cross-linking assay and electron microscopy visualization
For cross-linking assays, the “GST” tag from the recombinant protein, was
removed with thrombin (5 units/mg protein for 30 min at 25 °C). The completion of
the digestion was confirmed by gel SDS-PAGE (10%). The resulting α-actinin (2
mM) was mixed with 2 mM G-Actin in the actin binding buffer supplemented with
actin polymerization solution (KCl and MgCl2, final concentrations of 100 mM and 3
mM, respectively). After incubation for 1 hour at room temperature, the mixture was
stained with 0.5% sodium phosphotungstate (PTA) at pH 7.0 and deposited on a
carbon-coated grid, and visualized at 60 kV using a Philips EM410 transmission
electron microscope. A control without α-actinin was prepared under the same
conditions.
9
Calcium binding assays
The extent of calcium binding was determined using a
45
Ca2+ overlay assay
(Maruyama et al., 1984). Briefly, 10 μg of GST-α-actinin was slot-blotted onto a
nitrocellulose membrane. The membrane was equilibrated in imidazol buffer (10 mM
imidazol, 5 mM MgCl2 and 30 mM KCl, pH 8.0) and then incubated with
45
Ca2+
(13 μCi/ml 45Ca2+) as CaCl2. 10 μg of calmodulin and α-actinin from chicken gizzard
(Sigma A9776) were used as positive controls and GST (10 µg) as negative control.
2.5. Localization of α-actinin in situ
Samples for immuno-localization assays were prepared as described earlier
(Cotado-Sampayo et al., 2006). Anti-Neurospora α-actinin was used at a 1:20
dilution and FITC conjugated anti-rabbit secondary antibody at a dilution of 1:80.
2.6. In vivo expression of α-actinin-GFPand acquisition of live cell images
Cultures were prepared in liquid medium as described above. For long time
image acquisition (12 hours), germinating spores were deposited in 8 chambered
coverglass (Lab-Tek®,ref 155411) and covered with a small block of Vogel 1.5%
agar medium in order to maintain the cells in the same plane. The GFP expression
was recorded with a Leica AF6000LX microscope. For shorter time image
acquisition, 12 hours hyphae were deposited in culture chambers as described above
and followed with a Leica SP2 microscope. Images were processed with Adobe
Photoshop 6.0.
2.7. Cell wall staining with calcofluor
10
Slides coated with Vogel’s medium containing 1.5% sucrose were inoculated
with conidia and grown at 25 °C for 8 hours in a wet chamber. The mycelia were
stained with 10 μM calcofluor (Fluorescent Brightener 28) for 3 minutes and covered
with a coverslip. An Orthoplan epiillumination microscope (Leica) equipped with
fluotar optics and a selective filter combination was used to visualize calcofluor
fluorescence patterns. Fluorescent micrographs were obtained with a Leica-DFC490
camera and processed with Adobe Photoshop 6.0.
3. Results
3.1. Immunoblot
A Western blot of N. crassa crude extract and partial thrombin digested full
length GST-α-actinin recombinant protein separated in 2D-gels showed three bands
with different relative molecular weights and Isoelectric points (pI): the undigested
(140 kDa, pI 6.0), the thrombin cleaved recombinant protein (110 kDa, pI 5.9) and
the “native” (80 kDa, pI 5.6) Neurospora α-actinin (Fig. 1A). These features
correspond to the theoretical estimated values based on the sequences. The 140 kDa
band agrees with the molecular weight of the fusion protein (110 kDa full length αactinin plus 30 kDa of the GST), 110 kDa represents the full length α-actinin and the
80 kDa protein corresponds to the conserved region containing the characteristic
domains of the α-actinins (Fig. 1B).
3.2. Actin-binding property
Our results show that actin sedimentation is modified by the presence of αactinin and Ca2+. Low speed centrifugation sedimented neither G-Actin, nor F-Actin,
11
nor fusion GST-α-actinin (Fig. 2a, b and c, respectively). Under the sedimentation
conditions used the amount of the sedimented actin depended on the amount of the
GST-α-actinin when calcium was chelated with EDTA (Fig. 2d and e). In the
presence of excess calcium, α-actinin failed to co-sediment with actin (Fig. 2f).
3.3. Cross-linking assay and electron microscopic visualization
Electron micrographs of negatively stained cross-linked samples also showed the
affinity of α-actinin for actin filaments. α-Actinin cross-linked actin filaments and
organized them in parallel structures as bundles (Fig. 3B). Bundle formation was
observed when the ratio between actin and α-actinin was 1:1. High concentrations of
actin relative to α-actinin resulted in a complete disorganization of the actin
filaments (Fig. 3C). The inter cross-linker space between actin filaments was difficult
to determine with precision but it was approximately between 15 and 25 nm.
3.4. Calcium-binding assay
45
Ca overlay experiments showed that Ca2+ binds to α-actinin. For the same
amount of protein the signal was higher for GST-actinin (Fig. 4b) than for
commercial chicken gizzard α-actinin (Fig. 4a). Calmodulin was used as positive
(Fig. 4c) and purified GST as negative control (Fig. 4d).
3.5. α-Actinin localization
Immunofluorescence microscopy was used to detect the spatial localization of αactinin in the germ tubes and growing hyphae of wild-type strain. The results showed
that the protein was localized in the tip region (Fig. 5A), along the plasma membrane
(Fig. 5B) and the septa (Fig. 5C, D). This localization was confirmed by in vivo
12
expressed α-actinin-GFP. GFP fluorescence was observed at the site of germ tube
tips and the emerging tubes, (Fig. 6A, B, and C). In the growing hyphae α-actininGFP was also located in the zone of hyphal fusions (Fig. 6F and Fig. 7C) and the
septa (Fig. 7A, B). The localization in septa and the hyphal fusion zone seemed to
disappear after completion of these structures. The plasma membrane localization
was not observed in vivo in the α-actinin-GFP transformed strain (Fig. 6 and Fig.7).
3.6. Heterokaryon α-actinin knockout strain phenotype
The α-actinin knockout strain was deposited in the FGSC as heterokaryon (Colot
et al., 2006), considering that the the homokaryon has a lethal phenotype because of
poor or inexistent germination of the first generation of ascospores. Compared to the
wild type, the heterokaryon showed shortened aerial hyphae (Fig. 8A) in Davis and
De Serres (1970) medium. The morphology of the hyphae was different with respect
to their branching pattern. The mutant showed a predominantly dichotomous
phenotype compared to sympodial branching in the wild type (Fig. 8B).
4. Discussion
The antibody prepared against α-actinin from Neurospora crassa reacted
specifically with a 80 kDa protein in the Western blot of the crude extract. This
relative molecular weight (Mr) agrees with the estimated molecular weight of the
Neurospora α-actinin annotated in the MIPS database (as an α-actinin related
protein) based on the initiator methionine immediately upstream of the coding
sequence for the first CH-domain. All close homologs in other fungi confirm that this
methionine is the initiator and the resulting protein contains all the domains defining
13
α-actinins. However, there are two start codons present in the ncu06429.4 gene
annotation of the Broad Institute database and we have cloned the gene from the
cDNA which included the first start codon indicating that this N-terminal extension
is present in α-actinin mRNA and could be an UTR region or pro-domain of the
protein. Alternatively, another interpretation could be that the translation of mRNA is
initiated at both methionine codons located in the ORF. The UTR region hypothesis
is not very likely for several reasons. The first reason is its length of 930 nucleotides.
5’UTR- are generally shorter than 3’UTR- regions and the average length is roughly
200 nucleotides (Pesole et al., 2000), although there are reports of unusually long
UTR regions in some genes (e.g. the cyclin CLN3 from S. cerevisiae (864
nucleotides) (Polymenis and Schmidt, 1997; Vilela and McCarthy, 2003) or frq
(frequency) from N. crassa with ~1.5 Kb (Diernfellner et al., 2005). The second
reason is that the first 5’AUG could be a good initiator codon according to the
predicted Kozak sequence of filamentous fungi (Bruchez, 1993). The putative fulllength peptide would be of 310 amino acids longer than the short one. Alternative
initiation of translation has been reported in N. crassa (Garceau et al., 1997;
Diernfellner et al., 2005) and could explain the presence of the two in frame ATG in
a situation where the full-length α-actinin was translated under conditions other than
those used in our analysis. The hypothesis of a pro-domain in the protein is more
plausible even if we were not able to detect the product in the crude extracts. It might
be that it was very unstable and quickly degraded.
The interaction of α-actinin with actin was negatively regulated by calcium, as
demonstrated by the sediment assays. It was shown that the α-actinin has the ability
to cross-link F-actin and organize the microfilaments in a parallel way. Cross-linked
14
actin filaments with α-actinin are 15-25 nm apart. The distance between the
filaments was shorter than observed in other actin-α-actinin complex (30-40 nm)
(Taylor and Taylor, 1993; Tang et al., 2001; Hampton et al., 2007), but agrees with
the difference in size between α-actinin from higher eukaryotic cells and the shorter
fungal α-actinins which contains only half the number of spectrin repeats (Virel and
Backman, 2007).
The calcium dependence of the Neurospora α-actinin and insensitivity of
vertebrate α-actinin in actin binding assays, supports the idea that calcium dependent
regulation of actin binding was lost in the vertebrate-invertebrate divergence (Dixson
et al., 2003; Virel and Backman, 2004).
Higher levels of α-actinin were observed in germinating conidia at the site of
hyphal tube emergence and were maintained in the tip of the emerging tubes
suggesting that α-actinin plays a role in germination and branching. A similar
localization has been observed for actin which was shown to be essential for germ
tube initiation (Barja et al., 1993). α-Actinin was also localized along the plasma
membrane in the tip region of the hyphae suggesting additional roles in cellular
physiology. α-Actinin facilitates the stabilization of focal adhesion plaques in animal
cells (Xu et al., 2000). The homological structures in fungi, are the actin plaques or
dots (Torralba and Heath, 2001) where F-actin binding to the plasma membrane
occurs (Hoch and Staples, 1983; Adams and Pringle, 1984; Kaminskyj and Heath,
1996). The connection of the α-actinin with the plasma membrane is mediated by
other proteins such as β1,β3-integrin, vinculin, α-catenin (Otey and Carpen, 2004).
None of these proteins have homologs in the Neurospora genome but we can not
exclude the presence of other unknown α-actinin partners (Otey and Carpen, 2004).
15
α-Actinin-GFP was not observed in the plasma membrane region. Is possible that the
signals were too weak to detect or alternatively the GFP tag may have partially
modified the localization of the fusion protein, a phenomenon that has been
described before (Ikonen et al., 1995; Schneider et al., 2000). The localization of αactinin in the septa was observed both in situ and in vivo. The disappearance of αactinin from the septa once their formation is complete, has also been described for
other proteins required for septation in filamentous fungi, such as formin SEPA
(Sharpless and Harris, 2002) and actin (Momany and Hamer, 1997; Rasmussen and
Glass, 2005, 2007).
The knock-out phenotype was lethal. It is unlikely that the lethal phenotype is
based only on a role in septum formation. The aseptate mutants in Neurospora
(Rasmussen and Glass, 2005, 2007) and other filamentous fungi (Ayad-Durieux et
al., 2000; Wendland and Philippsen, 2000; Kim et al., 2006) are viable.
The exact function of α-actinin in Neurospora is still not clear. We can assume that a
major role is played by a collaboration between actin and α-actinin. The results on
protein localization suggest a role in cytokinesis. This would agree with the function
of its orthologs in S. pombe (Wu et al., 2001). However, differences can be expected
since cytokinesis in filamentous fungi and yeast are two principally different
mechanisms (Walther and Wendland, 2003). Moreover, the function of α-actinin in
yeast and also in D. discoideum can be complemented by other actin-binding
proteins, notably fimbrin (Rivero et al., 1999; Wu et al., 2001). This result in nonlethal phenotypes for α-actinin mutants in these organisms.
The dichotomous phenotype of the α-actinin heterokaryon knock-out strain is a
consequence of a splitting at the tips. This is also called “dichotomous branching”,
16
and has been observed in several other filamentous fungi in which polarity
maintenance proteins were mutated (Geissenhöner et al., 2001; Han and Prade, 2002;
Sharpes and Harris, 2002). A similar phenotype has been observed for Cytochalasin
A treated Neurospora (Riquelme et al., 1998) and Neurospora actin mutants (Virag
and Griffiths, 2004) suggesting that α-actinin may also collaborate with actin in the
formation of branching patterns.
However, other roles may be expected. The list of newly identified proteins and
molecules interacting with α-actinin has increased over the last years, demonstrating
that α-actinin is not merely an actin-binding protein but a scaffold for other proteinprotein interactions and to connect the cytoskeleton to diverse signaling
pathways (Critchley and Flood, 1999; Otey and Carpen, 2004).
Acknowledgment
We gratefully acknowledge financial support from the Academic Society of
Geneva (F. B.). Thanks are due to R. Strasser for interest in the project and
encouragement, M-L. Chappuis for technical assistance, A. Fehr for secretarial
assistance.
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Fig. 1. 2D-PAGE (10%) analysis of the N. crassa α-actinin and the GST-α-actinin
constructs. A. A mixture of N. crassa crude extract and partially digested GST-α-actinin
fusion protein immunoblotted with anti-Neurospora α-actinin antibody. B. Diagrammatic
representation of GST-α-actinin construction, bars underline the region of the protein
reacting with the anti-Neurospora α-actinin antibody shown in A. (GST, means
gluthathione S-transferase; CH, Calponin Homology domain; SR, spectrin repeat and EF,
refers to EF-hand motif).
Fig. 2. Analysis of α-actinin-actin interactions. SDS-PAGE (10 %) coomassie blue
stained gel (upper panel) of supernatants (sn) and pellets (p) of α-actinin-actin mixtures
and the corresponding immunoblot with the anti-actin antibody (lower panel). Lanes a, b
and c are 3.5 μM of G-actin, F-actin and GST-α-actinin respectively. Lanes d and e
represent reaction mixtures containing 3.5 μM actin and 2 μM (lane d) and 3.5 μM (lane
e) α-actinin. Lane f is the same as lane e but with excess calcium.
Fig. 3. Electron micrographs of α-actinin-F-actin cross-reaction products. A. 2 μM
actin; B. α-actinin to actin molar ration 1:1 (2 μM); C. α-actinin to actin molar ratio 1:2
(1 μM:2 μM). Bar 100 nm.
Fig. 4. Calcium-binding of N. crassa α-actinin probed by calcium overlay using
45
Ca. a, 10 μg of GST-α-actinin; b, α-actinin from chicken gizzard; c, calmodulin and d,
GST protein were slot-blotted onto a nitrocellulose membrane.
Fig. 5. Immunolocalization of N. crassa α-actinin during different stages of growth.
Germinating conidia, emerging germ tubes and growing hyphae corresponding to 1 hour
(A) and 12 h (B-D) growth. Tip region of the hyphae with a branch initial (B).
Localization of α-actinin in the septum (C-D). Bar 5 μm.
Fig. 6. Time lapsed images of live cells for a duration of 5 hours. Laser scanning
confocal microscopy images of Neurospora hyphae expressing α-actinin-GFP. Images
correspond to intervals of approximately 1 hour. After one hour of growth the
fluorescence concentrated at the site of germination (A), this concentration was
maintained until germ tube formation during the first 3 hours of growth (B-C), during
hyphal growth no specific localization was found (D-E). The GFP signal accumulated at
the fusion site of two hyphae (F). Bar 10 μm.
Fig. 7. In vivo localization of α-actinin in N. crassa. A-B. Transmission and GFP
signal is shown for each acquisition image; complete septum formation took less than 40
min. A. Time series corresponding to the first step of septum formation, the septum is not
evident in the transmission images made during the first 20 min. B. Stack serie of the
hyphae at 30 minutes, the fusion pore was visualized in the medial acquisition image. C.
confocal image showing a hyphal fusion using the transmission image (a), GFP signal (b)
and calcofluor stain (c).
Fig. 8. Phenotype of the α-actinin heterokaryon knock-out strain. A. Wild type (wt)
hyphae (left) and α-actinin heterokaryon knock-out strain (right). B. Calcofluor stained
hyphae of N. crassa wt (a-b) and α-actinin heterokaryon knock-out strain (c-d). b and d
are images at higher magnification of a and b. Bar 10 μm.