Nuclear centering in Spirogyra

Planta (1998) 204: 54±63
Nuclear centering in Spirogyra:
force integration by micro®laments along microtubules
Franz Grolig
Institut fuÈr Allgemeine Botanik und P¯anzenphysiologie, Justus-Liebig-UniversitaÈt, Senckenbergstr. 17, D-35390 Giessen, Germany
Received: 9 December 1996 / Accepted: 29 April 1997
Abstract. The contribution of microtubules and micro®laments to the cytomechanics of transverse nuclear
centering were investigated in the charophycean green
alga Spirogyra crassa (Zygnematales). Cytoplasmic
strands of enhanced rigidity and fasciate appearance
radiate from the rim of the lenticular nucleus through
the vacuole, frequently split once or twice and are
attached to the helical chloroplast bands in the peripheral cytoplasm. The nucleus is encased in tubulin and
a web of F-actin. Bundles of microtubules, emerging
from the nuclear rim, are organized into dividing
fascicles within the strands and reach to the inner
surface of the chloroplast envelope. Organelles are
translocated in both directions along similarly arranged
fascicles of micro®lament bundles which extend from the
nucleus to the peripheral actin cytoskeleton. Application
of microtubule- and/or micro®lament-depolymerizing
drugs a€ected the position of the nucleus only slowly,
but in distinct ways. The di€erential e€ects suggest that
nuclear centering depends on the tensional integrity of
the perinuclear sca€old, with micro®laments conveying
tension along stabilized microtubules and the actin
cytoskeleton integrating the translocation forces generated within the sca€old.
Key words: Cytomechanics ± Micro®lament ± Microtubule ± Nucleus (positioning) ± Phragmosome ±
Spirogyra
Abbreviations: CD=cytochalasin D; DAPI = 4¢-6-diamidino-2phenylindole; DIC = di€erential interference contrast; MF(s) =
micro®lament(s); MT(s) = microtubule(s); PNS = perinuclear
sca€old (sca€old with encased nucleus); RLP = rhodaminelabelled phalloidin
Correspondence to: F. Grolig, Biozentrum, Johann Wolfgang
Goethe UniversitaÈt, AK Kinematische Zellforschung, MarieCurie-Str. 9, D-60439 Frankfurt am Main;
E-mail: [email protected]; Fax: 49 (69) 79829607
Parts of this work have been presented at the meeting of the
Deutsche Gesellschaft fuÈr Zellbiologie, Hamburg, March 24±28,
1996, and at the 1st European Phycological Congress, Cologne,
August 11±16, 1996
Introduction
Nuclear movements represent the structurally most
diverse category of plant organelle movements (Williamson 1993; Menzel et al. 1996) and the mechanics of
most are not known. Premitotic nuclear positioning is a
major determinant of the plane of plant cytokinesis
(Gunning 1982; Lloyd 1991). In di€erentiated higher
plant cells, positioning is brought about by a cytoskeletal sca€old called the phragmosome (Sinnot and Bloch
1940), with nuclear translocation to the center of the
prospective division plane being functionally distinct
from ®nal anchorage (Wick 1991). The phylogenetic
origin of the higher plant phragmosome is obscure.
Following the new taxonomic system of the Chlorophyta (green algae) proposed by Pickett-Heaps and
Marchant (1972), the Charophyceae are now widely
accepted as being related to the ancestors of the land plant
lineage (Mattox and Stewart 1984; Graham et al. 1991;
Surek et al. 1994). One of the organisms of phylogenetic
signi®cance in this respect is the zygnematalean alga
Spirogyra (Fowke and Pickett-Heaps 1969). In this
®lamentous alga, a phragmoplast-like structure is involved in cytokinesis (McIntosh et al. 1995).
Spirogyra crassa, a particularly large and translucent
species, has proven useful to investigate actin-based
organelle transportation during interphase (Grolig 1990)
and the arrangement and functional interrelation of
microtubules (MTs) and micro®laments (MFs) during
cytokinesis (Sawitzky and Grolig 1995). The premitotic
nucleus is tethered in the center of the cell by an
extensive perinuclear sca€old (PNS) that resembles the
higher plant phragmosome (Grolig 1992). Nuclear
centering in the cylindrical Spirogyra cell implies
positioning in both the longitudinal direction (during
postmitotic nuclear migration) and in the transverse
direction. This report focuses on the latter aspect,
addressing in particular the contribution of MTs and
MFs to this cytomechanical task. Structural and
functional features of the PNS in vivo and the e€ects
of cytoskeletal inhibitors thereon were analysed by
F. Grolig: Cytoskeletal control of nuclear centering in Spirogyra
video-enhanced microscopy. The arrangement of MFs
and MTs in the sca€old was visualized by ¯uorescent
tagging either in situ or after gentle rupture of previously
®xed cells.
Materials and methods
Plant material. Spirogyra crassa was cultured in MXS (NeuschelerWirth 1970) as described by Grolig (1990). Non-dividing cells with
a length/width ratio >2 were obtained after three to four weeks
without change of culture medium. Cells with a ratio <1.5 were
plasmolyzed by 0.4±0.6 mol á l)1 mannitol to obtain rounded
55
protoplasts slightly smaller than the transverse cell diameter. By
shaking (50 rpm, >3 h) of such cells, protoplasts are rotated and
the protoplasts can be observed through that area of the plasma
membrane which usually faces the cross wall.
Microscopy. Live cells or ®xed, isolated PNSs were observed by
video-enhanced di€erential interference contrast (DIC) microscopy
(microscope Diaplan, objectives NPL Fluotar 40 ´, NA 0.65 and
100x, NA 1.32; Leica, Bensheim, Germany) as described by Grolig
(1990). Filter blocks used for epi¯uorescence microscopy were:
Ploemopak N2 for rhodamine, Ploemopak L3 for FITC (¯uorescein-isothiocyanate) and Ploemopak D combined with KIF 590
(Schott, Mainz, Germany) for 4¢-6-diamidino-2-phenylindole
(DAPI) ¯uorescence.
Cytoskeletal inhibitors. Cytochalasin D (CD) was applied as
described previously (Grolig 1990). Oryzalin (Morejohn et al.
1987), prepared as a 1.5 ´ 10)2 mol á l)1 stock in acetone, was
applied at concentrations up to 2.5 ´ 10)5 mol á l)1. Phalloidin
(Cooper 1987) was applied at 5 ´ 10)6 mol á l)1. Controls were
treated with equivalent concentrations of the respective stock
solvent.
Fig. 1A,B. Transverse and longitudinal positioning of the lenticular
nucleus (with central nucleolus) in Spirogyra crassa. The PNS is
associated with helical chloroplast bands in the cell periphery.
A Scheme of a longitudinal midplane section, showing postcytokinetic
daughter nuclei (right side) positioned eccentrically in the longitudinal
direction, and (left side) a both transversely and longitudinally wellcentered premitotic nucleus. The distances (a, b, c and d) as measured
for calculation of the nuclear center position are indicated. r, cell
radius; L, cell length. B Scheme of a half-cell transverse section,
showing an eccentrically positioned nucleus (with central nucleolus)
with the distances measured as in A and the trigonometric relations
used to calculate the nuclear center position. The x-coordinate of the
transverse nuclear center position was calculated as a + (c ) a)/2 +
(2r ) d)/2; distance b was used instead of a + (c ) a)/2 if the rim of the
nucleus could not be discerned clearly (in particular after convergence
of chloroplast bands towards the nucleus upon treatment with
oryzalin). The y-coordinate was calculated as r á sin a, with
a = arccos d/2r
Fig. 2. A Longitudinal mid-plane view of the PNS of interphase
Spirogyra crassa. The lenticular nucleus (n) is suspended in the center
of the cell, surrounded by the vacuole (v). Rigid, branching
cytoplasmic strands (arrows) radiate from the nuclear rim to the
chloroplast bands in the peripheral cytoplasm (pc). Total cell length is
350 lm. B The ¯uorescent, lens-shaped nucleus as stained by DAPI.
C Frontal surface of the nucleus (n) with central nucleolus as viewed in
a plasmolysed, rotated protoplast. Mitochondria (arrows) move all
over the nuclear surface and in both directions through each radiating
strand. A, C Video-enhanced DIC microscopy. ´ 300 (A,B), ´ 1000
(C); bars = 20 lm
56
Morphometrical analysis of nuclear positioning precision. To determine the precision of positioning, the distances a, c (or b), and d
(see Fig. 1A) were measured on the video screen (magni®cation
1.130 ´) after adjustment of that longitudinal optical section of the
cell which gave the largest diameter (i.e. center) of the nucleus.
Compression of cells was avoided by use of coverslips as spacers.
Nuclear center positions were calculated (Fig. 1B) and plotted into
the half-transverse section of a cell. The nuclear diameter was
calculated as (c ) a).
Permeabilization and fractionation of ®xed cells. Spirogyra ®laments, ®xed in 1% (v/v) glutaraldehyde, 1.5% (w/v) formaldehyde
in 3 ´ 10)3 mol á l)1 EGTA, 1.5 ´ 10)2 mol á l)1 K2HPO4, pH 7.0
(20 min; modi®ed after Galway and Hardham 1991), were gently
washed in distilled water and cracked open in liquid nitrogen by
means of a strong blunt needle to give access to antibodies (Braun
and Sievers 1994; Sawitzky and Grolig 1995).
Fixed PNSs were isolated to inspect the F-actin arrangement
more closely. Algal ®laments were pre®xed in 10)4 mol á l)1
m-maleimidbenzoyl N-hydroxysuccinimide ester (MBS; Sonobe
and Shibaoka 1989) in ®xation bu€er (10 mM EGTA, 5 mM
MgSO4, 100 mM P-KOH, pH 6.9; Traas et al. 1987), which was
supplemented after 25 min by 1.5% (w/v) formaldehyde and 0.05%
(v/v) glutaraldehyde during incubation (30 min) in a nitrogen celldisruption bomb (Parr Instruments Co., Moline, Ill., USA) at 70
bar (Grolig and Wagner 1987). The released homogenate was
centrifuged di€erentially (3 min at 450 g, followed by 20 min at
3500 g) in a swing-out rotor to enrich ®xed nuclei with associated
sca€olds.
Fluorescent staining of F-actin and DNA. F-actin was visualized by
rhodamine-labelled phalloidin (RLP) in ®xed cells as described
previously (Grolig 1990). After isolation, ®xed PNSs and fragments
of them were stained with 1.6 ´ 10)7 mol á l)1 RLP in ®xation
bu€er and inspected after 10 min. Nuclear DNA was stained after
®xation [1.5% (w/v) formaldehyde, 0.2% (v/v) glutaraldehyde in
®xation bu€er; 15 min] with DAPI (1.4 ´ 10)5 mol á l)1, 45 min;
Fig. 3A±D. A series of optical sections, reaching from the longitudinal midplane of the centrally positioned nucleus (A) to more-distal
regions of its associated PNS (B±D). Video-enhanced DIC microscopy
reveals a fasciate appearance of the sca€old stalks, with elements of
the fascicle splitting into the branches (B vs. C, arrowheads).
Numerous small vesicles (arrows) move on the nuclear surface and
on the stalks. ´ 1000; bar = 10 lm
F. Grolig: Cytoskeletal control of nuclear centering in Spirogyra
Katsuta et al. 1990) in ®xation bu€er and washed under the
coverslip (3 ´ 0.1 ml bu€er, 5 min).
Indirect immuno¯uorescence. Shattered cells were incubated with
monoclonal anti-a or anti-b tubulin (N.356 and N.357, respectively;
Amersham, Braunschweig, Germany; dilution 1/1.000), followed
by FITC-labelled secondary antibody (N.1031, Amersham; dilution
1/800) according to Galway and Hardham (1991); in some
preparations, detergent [0.1% (w/v) Triton X-100] was omitted.
For a control, either another antibody (anti-a-amylase) was used or
the primary antibody was omitted.
Results
Structural features of the PNS. An extensive PNS
(Fig. 2A) suspends the interphase nucleus of S. crassa
in the center of the large central vacuole. Staining of
S. crassa cells with DAPI clearly depicted the compressed shape of the nucleus (Fig. 2B). Rigid cytoplasmic
strands (stalks) radiate from the rim of the lenticular
nucleus, frequently showing branches of ®rst and second
order, and taper o€ towards attachment sites on the
mid-segment of chloroplast bands arranged in lefthanded helices in the peripheral cytoplasm (Fig. 2A,
C). The mean number of stalks was 13.8‹1.9 (‹SD;
n = 23), with branching giving rise to some 30 distal
attachment sites on 14±16 chloroplast bands in an
elongate cell. Most branchings occurred in the distal
third of the cell radius. The branches of each stalk were
attached either to a single or to two adjacent chloroplasts, usually close to the chloroplast midline and very
often close to a pyrenoid. No stalks were attached to the
cross-walls, which are devoid of chloroplasts. Viewed
from the side (Fig. 2A), the PNS fanned out during
longitudinal growth of the cell, which was accompanied
by a decreasing pitch of the chloroplast helices. In
contrast to the unidirectional long-range translocation in
the peripheral cytoplasm, changes in the direction of
mitochondrial movement occurred rather frequently
within the strands. Plasmolysis had little e€ect on the
F. Grolig: Cytoskeletal control of nuclear centering in Spirogyra
relative arrangement of the PNS and the associated
chloroplasts. Rotated protoplasts (obtained by shaking)
permitted the convex surface of the nucleus to be viewed
through the chloroplast-free windows of the protoplast
(Fig. 2C). On the nuclear surface, no prominent longrange tracks were observed and mitochondrial net
translocation was low. Optical conditions favorable for
high-resolution DIC microscopy showed the proximal
Fig. 4A±G. Arrangement of F-actin in situ and in the ®xed, isolated
nucleus-positioning PNS of Spirogyra crassa as visualized by RLP.
A Longitudinal optical section in situ, focussed slightly above the
nucleus (n), shows RLP ¯uorescence throughout the stalks of the
sca€old as well as in the connected peripheral cytoplasm (pc). B Focus
set slightly above the chloroplast bands (alignment of plastid long axes
indicated by arrowheads) in the peripheral cytoplasm, with subcortical
bundles of F-actin and short bundles of micro®laments (small arrows)
normal to the chloroplast surface. C,D Front, surface views of nuclei
(each with central nucleolus) with associated fragments of the sca€old
as set free upon fractionation of ®xed cells; video-enhanced DIC
microscopy. E,F Distribution of F-actin in isolated sca€olds. E The
nuclear surface is covered by a dense layer of ®ne, non-bundled
F-actin. F F-actin follows the radiating, splitting stalks in the form of
discrete micro®lament bundles that protrude from the nuclear surface
and merge into the stalks (arrows). G Arrangement of F-actin bundles
in a fragment of a split stalk with a clear gap (arrow) between the
bundles. ´ 350 (A), ´ 800 (B), ´ 550 (C), ´ 900 (D), ´ 750 (E), ´ 450
(F), ´ 1000 (G); bars = 20 lm (A±F), 10 lm (G)
57
parts of the stalks to be composed of bundles of parallel
longitudinal elements, which split upon branching of the
stalks (Fig. 3).
Arrangement of MFs and MTs within the PNS. Branched
stalks of many PNSs appeared less straight and tense
after extended ®xation. Gentle disruption of ®xed
S. crassa cells by means of a nitrogen bomb yielded
many fragments of stalks, but also a reasonably high
proportion of structurally more or less intact PNSs
(Fig. 4C,D). The diverse fragments could be roughly
subfractionated and further concentrated by di€erential
centrifugation.
Labelling of the actin cytoskeleton in situ with RLP
showed staining of the PNS stalks (Fig. 4A). In accordance with continuous organelle movements in vivo
(Fig. 2A,C), the MFs of the stalks appeared to be in
close contact with the extensive system of peripheral
MFs (Fig. 4A,B). Surface views of ®xed, isolated nuclei
with associated PNSs (Fig. 4C,D) revealed that the
lenticular interphase nucleus was encased in a dense
cover of F-actin without structural details (Fig. 4E,F).
Bundles of MFs emerged at the rim of the lenticular
nucleus (Fig. 4F), ran within each stalk and split into the
branches (Fig. 4G).
Indirect immuno¯uorescence showed that MTs (or
MT bundles) extended from the nuclear surface
(Fig. 5A) to the attachment sites of the stalks on the
58
F. Grolig: Cytoskeletal control of nuclear centering in Spirogyra
Fig. 5A±D. Indirect immuno¯uorescence of MTs in cell fragments of
®xed, frozen and shattered Spirogyra
crassa cells. After thawing, cells were
treated without (A) or with (B±D)
detergent prior to incubation with
monoclonal anti-b-tubulin. A, B Nuclei, adhering with their PNSs to large
cell fragments. Even staining of the
nuclear surface is observed (A,B), but
only in PNSs treated with detergent
after ®xation (B) is the fasciate construction of the stalks apparent, with
bundles of MTs extending in parallel
(arrows) over large distances in the
proximal parts of the PNS. The distal
branches of the stalks end on the
inner surface of the same or of
neighboring chloroplast bands (arrowheads). C Detailed view of the rim
of a nucleus with bundles of MTs
merging into the stalks (arrows).
D Detailed view of the distal branches
(arrows) of a stalk attached to neighboring chloroplast bands (arrowheads). The pyrenoids of the
chloroplast bands show a high level of
®xation-derived auto¯uorescence.
´ 250 (A), ´ 450 (B), ´ 600; (C,D)
bars = 20 lm (A,B), 10 lm (C,D)
inner side of the chloroplast envelope (Fig. 5B). In the
thicker, proximal parts of the PNS the stalks comprised
a bundle of parallel MTs (or MT bundles) which,
however, could be resolved only if the specimen had
been detergent-treated (Fig. 5B±D). The bundles split
upon distal branching of the stalks (Fig. 5B,D). Tubulin
immuno¯uorescence was evenly spread over the convex
side of the lens-shaped nucleus.
E€ects of cytoskeletal inhibitors on the structural integrity
of the PNS. Cytochalasin D (2 ´ 10)5 mol á l)1) reversibly inhibited the transportation of small organelles in
the cytoplasm of S. crassa. These organelles accumulated in the form of mostly one to three large blebs very
close to the nucleus (Fig. 6A). The extensive peripheral
MF system (Fig. 4B) disrupted upon application of CD
into very short, punctate MF residues (as indicated by
RLP ¯uorescence; not shown). The structure of the PNS
and the nuclear shape did not change upon CD
treatment (Fig. 6A). Phalloidin (5 ´ 10)6 mol á l)1)
slowly caused an increased number of transportation
tracks in the peripheral cytoplasm (not shown), and
membrane tubules began to extend in the form of short
loops from the PNS into the surrounding vacuolar space
(Fig. 6B).
Only extended application of the plant-MT-depolymerizing drug oryzalin (2.5 ´ 10)5 mol á l)1, >2 h) progressively a€ected the PNS, in particular in elongate cells.
Shortening of the stalks led to convergence of the
associated chloroplast bands in the mid-segment of the
cell towards the nucleus (Fig. 7); the stalks lost their distal,
tapering branchings and got slightly thicker. Flexible,
¯accid cytoplasmic threads were drawn out between the
peripheral cytoplasm and the chloroplast segments converging towards the nucleus. Prolonged application of
oryzalin (>3 h) reduced the extent of actin-based organelle motility in the peripheral cytoplasm. In contrast to
application of oryzalin only, the chloroplast bands did not
converge towards the nucleus when CD and oryzalin were
applied simultaneously. The diameter of the lenticular
nucleus (3.21 ‹ 0.4 ´ 10)5 m; ‹ SD) was not signi®cantly a€ected by these cytoskeletal inhibitors (CD:
3.37 ‹ 0.55 ´ 10)5 m; oryzalin: 3.16 ‹ 0.44 ´ 10)5 m;
CD/oryzalin: 3.12 ‹ 0.44 ´ 10)5 m).
Precision of nuclear positioning, and e€ects of cytoskeletal
inhibitors. The mean relative transverse center position
of the nucleus in S. crassa cells with a length/width ratio
of two to three was 0.5 ‹ 0.034 (‹SD; n = 146) of the
cell diameter (156.4 ‹ 1.6 ´ 10)6 m; ‹SD). All nuclei
were found within a central area enclosed by a radius of
about 3 ´ 10)5 m, which was close to the mean nuclear
diameter (Fig. 8a). Nuclear displacements upon application of cytoskeletal inhibitors occurred only slowly,
but were signi®cant (Fig. 8).
Upon application of CD, many nuclei left the central
area of the transverse cross-section of the cell in an
apparently random fashion (Fig. 8b). Upon application
of oryzalin, precision of transverse nuclear centering
decreased only slightly (Fig. 8c). As in case of CD, all
F. Grolig: Cytoskeletal control of nuclear centering in Spirogyra
59
nuclei were still suspended after 19 h. In contrast to
treatment with either CD or oryzalin alone, a considerable number (approx. 30%) of (mostly still lenticular)
nuclei were found without a PNS in the peripheral
cytoplasm when CD and oryzalin were applied simultaneously over 19 h. Moreover, in the latter case, the
decreased precision in the positioning of still-suspended
nuclei was larger than that of cells treated with oryzalin
only (Fig. 8d).
Discussion
Fig. 6A,B. Changes in organelle distribution within the PNS of
Spirogyra crassa upon treatment with CD or phalloidin. A Application of CD (2 ´ 10)5 mol á l)1, 45 min) caused accumulation of
organelles on the PNS, resulting in massive blebs (open arrows) close
to the nucleus (n). B Treatment with phalloidin (5 ´ 10)6 mol á l)1,
3 h) caused loops of membrane tubules (arrows) to extend from the
sca€old around the nucleus (n) into the vacuolar space. Videoenhanced DIC. ´ 300 (A), ´ 350 (B); bars = 20 lm
Fig. 7A±C. Advanced state of PNS breakdown after treatment of an
elongate Spirogyra crassa cell with oryzalin (2.5 ´ 10)5 mol á l)1,
2.5 h). Focal series (video-enhanced DIC) through the mid-segment of
the cell, ranging from the cell periphery (A) to the cell center (B,C).
Shortening of the sca€old stalks leads to convergence of the attached
chloroplast bands (chl ) towards the nucleus (n), which stays roughly in
the cell center. Flaccid cytoplasmic strands (arrows) were drawn out
between the peripheral cytoplasm (A) and the distal surface of the
chloroplast band (B,C) during this process. ´ 500; bar = 20 lm
Di€erential arrangement and stability of MTs and MFs.
The stalks of the PNS contained fascicles of both MT
and MF bundles, which followed the splitting of the
stalks (Figs. 4G; 5B,D), much like the longitudinal
elements visible by DIC microscopy within the stalks
(Fig. 3). None of the three structures crossed-over upon
divergence of the stalk branches. The fasciate appearance of the stalks (Fig. 3) probably derives from parallel
alignment of refractile membrane tubules within the
stalks. These tubules exhibit actin-based motility
(Fig. 6B) and emerge from the PNS into the peripheral
cytoplasm (Grolig 1990). The observation of only shortrange actin-based movements of mitochondria on the
nuclear surface (Fig. 1C) is in accordance with a low
degree of MF bundling as indicated by non-structured
perinuclear RLP labelling (Fig. 4E,F). This arrangement
of F-actin is similar to that found in Mougeotia (Grolig
et al. 1990) but di€ers from the MF bundles around
higher plant nuclei (e.g. Traas et al. 1987).
While CD readily stopped actin-based translocations
along the sca€old stalks, oryzalin (and nocodazole; not
shown) slowly a€ected the structure of the PNS. The
intriguing stability of the PNS is characteristic of
interphase cells; its susceptibility to MT depolymerization increases dramatically when the cell enters prophase
(Sawitzky and Grolig 1995). The low eciency of the
60
F. Grolig: Cytoskeletal control of nuclear centering in Spirogyra
dynamics of cortical interphase MTs as observed in
higher plant cells (Hush et al. 1994), no cortical MTs
were found after treatment with oryzalin (not shown). In
S. crassa, the process of MT bundling probably turns the
dynamic, postmitotic PNS into a highly stabilized
interphase structure (McNally 1996) of enhanced ¯exural rigidity.
The radial arrangement and characteristic splitting
suggest that the arrangement of cytoskeletal elements
within the PNS is common to all stalks. Bidirectional
actin-based movements of mitochondria indicate
MF(bundle)s of opposite polarity in each stalk. The
disappearance of the distal branches and increased
thickness of the distal ends of the stalks upon treatment
with oryzalin indicate that the stalks shorten from their
distal ends. Since they are probably derived from a
persistent telophase spindle (Sawitzky and Grolig 1995)
and in analogy to the MT-nucleating capacity of higher
plant cell nuclei (Lambert 1993), the MTs of the stalks
are probably oriented with their plus (fast growing) ends
towards the cell periphery. These distal ends may be
stabilized by their attachment to the chloroplast envelope. The PNS-ensheathing tonoplast and the integrated
membrane tubules seem to contribute to the bundling of
MT(bundle)s within each stalk, as indicated by detergent
sensitivity of this complex after aldehyde ®xation (cf.
Fig. 5A vs. 5B).
Fig. 8a±d. Precision of transverse nuclear centering in Spirogyra crassa.
Distributions of nuclear center positions were determined in control
cells and cells treated with cytoskeletal inhibitors over 19 h in darkness;
nuclear center positions (s) are plotted into the half cross section of a
cell. a Control cells (n = 146); b cells treated with 2 ´ 10)5 mol á l)1 CD
(n = 145); c cells treated with 2.5 ´ 10)5 mol á l)1 oryzalin (n = 142),
d cells treated will both inhibitors simultaneously (n = 205). The inset
in (d) comprises those nuclei (71), which were no longer suspended and
found (still with a lenticular shape) in the peripheral cytoplasm. The
exact positions of these nuclei were not determined
MT drugs indicates low dynamics (high stability) of the
PNS's interphase MT bundles, constituting the structural stability of the PNS. In accordance with the high
Transverse nuclear centering depends on MF-mediated
balance/integration of forces conveyed along stabilized
MT bundles. The PNS in S. crassa appears to re¯ect a
balance of forces. Indirect anchorage of the nucleus via
the peripheral chloroplast bands allowed direct monitoring of forces acting within the PNS: cytoskeletal
perturbation resulted either in nuclear or in plastid
dislocation (cf. Fig. 9). Chloroplasts were drawn towards the nucleus upon application of oryzalin only in
the presence of intact MFs. This shows that the
necessary tension was conveyed by MFs within the
stalks. Since antiparallel MFs occurred in each strand,
the actomyosin system within the stalks probably
contributes to generation of tension.
The tension conveyed by MFs appears to be balanced
(for the most part) by the MT bundles of the stalks,
which seem to act like the struts of a tent. Comparison of
the e€ect of oryzalin alone with that of simultaneously
applied CD suggests that the MTs of the stalks depolymerized more easily when tension exerted longitudinal
compression. Stabilization of the MTs by bundling and
by attachment at the plus-end may counteract compression-mediated MT depolymerization (Hill 1981) in the
untreated cell. A balance of tension and compression has
been suggested for the MF and MT cytoskeletons
involved in neurite outgrowth (Dennerll et al. 1988).
Since in oryzalin-treated S. crassa cells the nucleus stayed
roughly in the center of the cell during distal shortening
of the stalks, MT depolymerization must have occurred
in a coordinate way that roughly maintained the balance
of forces responsible for central positioning.
Another force contributing (a probably minor part)
to counteraction of the tension conveyed along the stalks
F. Grolig: Cytoskeletal control of nuclear centering in Spirogyra
Fig. 9A±D. Schematic summary of the di€erential e€ects of the
cytoskeletal inhibitors cytochalasin D (CD, B) and oryzalin (oryz, C),
applied alone or in combination (CD/oryz, D), on the structural
integrity of the PNS and on the precision of nuclear centering in
Spirogyra crassa. A control. B Application of CD leaves the structure
of the PNS intact but causes randomization of transverse nuclear
positioning. C In the presence of oryzalin, the nucleus stays in the
transverse center of the cell (though less precisely than in untreated
cells). Chloroplast bands are drawn most eciently towards the
nucleus in the mid-segment of the cell, apparently because here
the vectorial component of tension oriented normal to the plane of the
chloroplast band is maximal. D When CD is applied in addition to
oryzalin, the chloroplasts are no longer drawn towards the nucleus
and their arrangement in the peripheral cytoplasm is not disturbed
presumably resides in tethering the chloroplast bands to
the cell cortex. The strands, drawn out between chloroplast
and peripheral cytoplasm upon treatment of cells with
oryzalin (Fig. 7), probably correlate to MF structures
extending from the cell cortex to the upper chloroplast
surface (Fig. 4B; cf. Figs. 8E,F in Grolig 1990). Unfortunately, it has not yet been possible to preserve the
labile extended strands during ®xation. Observation of
Spirogyra cells during longitudinal centrifugation revealed enhanced fastening of the chloroplast bands in
the mid segment of the cell (Kuroda and Kamiya 1991).
Increased fastening may balance the tension which
draws the mid segments of the chloroplast bands
towards the central nucleus. Decreased organelle motility in the peripheral cytoplasm after application of
oryzalin for more than 3 h suggests a stabilizing in¯uence of cortical MTs on peripheral MFs in S. crassa.
This e€ect is in accordance with the potentiation by
oryzalin of the e€ect of cytochalasin on cytoplasmic
streaming in Nitella (Collings et al. 1996). Such indirect
destabilisation of peripheral MFs may have weakened
chloroplast fastening in the periphery.
Randomization of the transverse nuclear position
after disruption of MFs demonstrates that (i) F-actin is
indispensible for attaining a central position of the
nucleus and that (ii) non-actin-based forces exist which
can dislocate the nucleus out of the cell center. Control
of central nuclear positioning apparently depends on the
integration of all these forces within the PNS. Loss of
(MF-mediated) tension conveyed along the PNS stalks
may lead to uncontrolled, and with respect to central
nuclear positioning, unbalanced tubulin polymerization.
Polymerization of tubulin could generate pushing forces
(Hill 1981), which would be focused on the lenticular
nucleus. Irregular PNS de¯ections, observed more fre-
61
Fig. 10A±C. Hypothetical scheme of the structure and function of the
PNS in Spirogyra crassa (quarter transverse section), illustrating in the
order of increasing complexity (A±C) the arrangement of MTs (thick
lines) and MFs (thin lines) and their functional integration within the
PNS. The polarity of MTs (+: plus end) and MFs (
: barbed
end) is indicated. While the nuclear diameter is of appropriate size, for
the sake of clarity other components are not drawn to scale.
A Bundle(s) of MTs with branches of ®rst and second order reach
from the central nucleus to the peripheral chloroplasts (dotted ovals).
B Bundles of MFs tether the chloroplasts to the cell periphery and
connect the peripheral cytoplasm with the nucleus. The directionality
(arrows) of particle (ellipsoids) movements implies an antiparallel
arrangement of MFs running within the PNS stalks. C Prevailing
cytomechanical function of the MFs (generation of tension, biheaded
arrow) and of the MTs (compression-bearing strut, bar) within each
stalk. The pattern covering the nucleus (surface view) indicates nonbundled MFs and MTs
quently after disruption of MFs (Grolig 1990) may
derive from forces generated by di€erential elongation/
shrinkage of MT bundles in the stalks. The fact that
nuclear translocation and randomization of the transverse position occurred rather slowly seems to indicate
that the MT-generated forces initially were fairly well
balanced when CD disrupted the F-actin. The di€erent
positioning status of nuclei after prolonged application
of both inhibitors (Fig. 8d) may re¯ect stages of
progressive PNS breakdown (possibly related to progress in the cell cycle).
The various components and functional aspects of
the PNS, as discussed above, are summarized in order
of increasing complexity in the hypothetical scheme of
Fig. 10. In accordance with their di€erential ¯exural
rigidity and tensile strength (Bereiter-Hahn 1987; Weiss
et al. 1987), the combined mechanical properties of MTs
and MFs appear to demonstrate the cytoskeletal property of tensional integrity (Ingber 1993).
Comparison of the PNS of Spirogyra with the higher plant
phragmosome. The phragmosome, attached directly to
the cell cortex, contains both MTs and MFs in its radial
62
strands (Lloyd 1991), much like the PNS of S. crassa.
Premitotic migration of the nucleus to the prospective
division site appears to be mediated by dynamic MTs;
disruption of MFs only seems to retard translocation (or
centering?). However, when the nucleus has attained its
central position, both MT- and MF-depolymerizing
drugs (applied over 2±6 h) cause only a small decrease in
the percentage of cells with a central nucleus (Katsuta et
al. 1990). Taking into account the comparatively short
treatment and low spatial resolution in determining the
nuclear position, the cytoskeletal situation during
phragmosomal nuclear anchoring (not migration) may
be similar to that described here for S. crassa, where
stabilized MTs allowed deviations of the nucleus from
the central position to occur only slowly. Tension in
phragmosomal strands has been indicated by laser
microsurgery (Goodbody et al. 1991), but without direct
experimental discrimination between the contribution of
MTs and MFs.
The PNS of Spirogyra and the higher plant phragmosome appear to be functional equivalents with respect to
premitotic transverse nuclear centering. In contrast to
the relatively short-lived (several hours) phragmosome
in the higher plant cell, the PNS of Spirogyra persists
during interphase, possibly because of the higher cell
division frequency. Further comparative studies, addressing the cell-cycle-dependent di€erential stability of
the MTs in the strands and the regulatory mechanism(s)
behind the capability of the PNS to approach a balance
of forces, may shed light on the phylogenetic origin of
the phragmosome.
I thank Dr. Geo€rey O. Wasteneys (Research School of Biological
Sciences, Canberra, Australia), Prof. Paul Galland (Botanisches
Institut, Marburg, Germany) and Dr. Albert J. Duschl (TheodorBoveri-Institut, WuÈrzburg, Germany) for critical reading of the
manuscript, Prof. Diedrik Menzel (Botanisches Institut, Bonn,
Germany) for helpful comments during early parts of this work and
Andrea Weisert for technical assistance in analysis of nuclear
positioning precision. The generous, kind gift of rhodamine
phalloidin by Prof. Theodor Wieland (Max-Planck-Institut fuÈr
Medizinische Forschung, Heidelberg, Germany) and the donation
of oryzalin by Eli Lily (Bad Homburg, Germany) are gratefully
acknowledged. This work was ®nancially supported by the
Deutsche Forschungsgemeinschaft.
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