Cytokinesis in trypanosomatids

Cytokinesis in trypanosomatids
Tansy C Hammarton, Séverine Monnerat and Jeremy C Mottram
The process of cytokinesis, where the cytoplasm of one cell is
divided to produce two daughter cells, is intricate in
trypanosomatids because of the requirement to replicate and
segregate a number of single copy organelles, including the
nucleus, kinetoplast, Golgi apparatus, and flagellum.
Identifying regulators of the three stages of cytokinesis,
initiation, furrow ingression, and abscission is complicated by
the fact that cell division in trypanosomatids is easily perturbed
and aberrant cells are readily produced during functional
characterization of gene products. In this review, we discuss
direct and indirect effects on cytokinesis, using Trypanosoma
brucei as a model.
Addresses
Wellcome Centre for Molecular Parasitology and Division of Infection &
Immunity, Institute of Biomedical and Life Sciences, University of
Glasgow, 120 University Place, Glasgow G12 8TA, United Kingdom
Corresponding author: Mottram, Jeremy C ([email protected])
Current Opinion in Microbiology 2007, 10:520–527
This review comes from a themed issue on
Eukaryotes
Edited by Marc Ouellette
1369-5274/$ – see front matter
# 2007 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.mib.2007.10.005
Abbreviations
BB
basal body
BSF bloodstream form
K
kinetoplast
N
nucleus
PCF procyclic form
Introduction
Trypanosomatids are parasitic protozoa found within the
order Kinetoplastida. Trypanosoma brucei, which causes
African sleeping sickness, Trypanosoma cruzi, the causative
agent of Chagas disease in South America and Leishmania
major, one of the several species of Leishmania that cause
leishmaniasis, are well-studied model organisms with
sequenced genomes [1–3]. These trypanosomatids have
complex life cycles that involve differentiation between
highly polarized, morphologically distinct, stages. The
parasites have a single flagellum, which exits the cell at
a unique invagination of the plasma membrane, the flagellar pocket. In Leishmania the flagellum remains free,
whilst in trypanosomes it is attached to the body of the cell
(Figure 1). The elongated spindle shapes of the parasites
Current Opinion in Microbiology 2007, 10:520–527
are maintained by a corset of subpellicular microtubules
(MTs), which encloses the internal organelles, including
the single mitochondrion, nucleus, and Golgi apparatus.
Within the mitochondrion resides the kinetoplast, an unusual structure that contains the organelle’s DNA.
The replication and segregation of these single organelles
to produce two identical daughter cells must be precisely
controlled during the cell cycle. The typical eukaryotic cell
cycle consists of four main phases — G1, S, G2, and M. In
animal cells, cytokinesis commences before mitotic
chromosome segregation is completed, and hence mitosis
and cytokinesis overlap. Although the cell division cycle in
trypanosomatids broadly follows the general eukaryotic
model, it possesses some unique features and complexities.
Cell division in T. brucei
The order and timing of cell cycle events in T. brucei have
been extensively studied (for recent reviews see [4,5]).
Most of the work has been undertaken in procyclic form
(PCF) cells, but subtle differences may exist in different
life cycle stages. The order of events is similar in the
bloodstream form (BSF), but the length of the cell cycle
is shorter (about 6 hours compared with the 8.5 hours of the
PCF) and the kinetics are different. The cell cycle starts
with the elongation and maturation of the probasal body,
which permits the nucleation steps that initiate the formation of a new flagellum. Replication of the Golgi apparatus then follows, via a mechanism that appears to combine
both templated biogenesis and de novo assembly [6]. Kinetoplast DNA replication (SK) initiates immediately before
the onset of the nuclear S phase (SN), takes less time to
complete and thus, kinetoplast division occurs before the
start of mitosis. During G2 phase of the nuclear cycle, basal
bodies separate in a MT-mediated process, an event that is
essential for the segregation of the kinetoplasts and Golgi.
The replicated nucleus then undergoes mitosis, which
occurs without chromosome condensation or nuclear
envelope breakdown. In the PCF, but not the BSF, one
nucleus is repositioned between the two divided kinetoplasts. Finally, cytokinesis occurs via the ingression of a
cleavage furrow along the longitudinal axis of the cell,
initiating at the anterior end, and passing between the two
flagella to separate the daughter cells. Ingressing furrows
are observed less frequently in BSF parasite populations
compared to PCF populations, indicating that furrowing is
rapid in the BSF.
Cell cycle events are driven by the sequential activation
and inactivation of cyclin-dependent kinases and are
monitored by specific signaling checkpoints. DNA
synthesis is monitored by DNA replication/damage
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Cytokinesis in trypanosomatids Hammarton, Monnerat and Mottram 521
checkpoints, while mitosis and cytokinesis are controlled
by checkpoints that monitor spindle assembly, kinetochore attachment to the spindle, and chromosome segregation. In trypanosomes, some key checkpoints present
in yeast and mammalian cells appear to be absent. Incubation of PCF cells with the MT inhibitor rhizoxin
blocked mitosis, but not cytokinesis, generating 1N*1K
cells with 4C nuclear DNA content and 0N1K cells
(zoids) [7]. A similar phenotype was observed following
RNAi knockdown of a mitotic B-type cyclin CYC6 (also
designated CycB2) indicating that PCF trypanosomes
lack the mitosis to cytokinesis checkpoint [8,9]. In the
BSF, however, CYC6 depletion inhibited both mitosis
and cytokinesis but not kinetoplast duplication, resulting
in cells with multiple kinetoplasts [8]. Although this could
indicate the existence of an operational mitosis to cytokinesis checkpoint in BSF T. brucei, it cannot be ruled out
that the absence of mitosis physically impedes cytokinesis in this life cycle stage.
Figure 1
Other trypanosomatids
Trypanosomatids differ with respect to their cell shapes,
the position of their kinetoplast and flagellum, and the
order in which they replicate their organelles. Crithidia
and Leishmania, for instance, divide their nucleus before
their kinetoplast [10]. In T. cruzi epimastigotes, the
kinetoplast divides immediately before the nucleus,
though its replication probably begins after the start of
nuclear S phase, ending before the nucleus enters G2
[11]. Daughter flagellum elongation and flagellar pocket
division occur much later in the T. cruzi cell cycle compared to T. brucei [11]. The new flagellum starts to
extrude from the old flagellar pocket only after the
completion of S phase, and the two flagella continue to
share the same pocket until late in the cell cycle. Flagellar
pocket division commences before mitosis, with an invagination of the pocket membrane nearest to the kinetoplasts that proceeds toward the cell surface, eventually
separating the pockets during cytokinesis. The new flagellum also continues to grow to the end of the cell cycle,
finally reaching full length during cytokinesis.
Cytokinesis
Trypanosomatids carry out cytokinesis by unusual mechanisms that may differ not only between different species
but also between different life cycle stages of the same
parasite. All replicating forms of T. brucei divide, not via
the constriction of an actomyosin ring as observed in
mammalian cells, but via the ingression of a cleavage
furrow that follows the helical axis of the cell and initiates
at the anterior end (see example in Figure 1). T. cruzi
epimastigotes appear to divide in a similar manner [12],
but cytokinesis in T. cruzi and Leishmania amastigote cells
superficially resembles the purse string mechanism of
mammalian cells (although there is no evidence at present to support the formation of a contractile actomyosin
ring). Leishmania promastigote cells are different again, as
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Scanning electron micrographs of Trypanosoma brucei and Leishmania
major cells in G1 and undergoing cytokinesis (L Tetley and G Patuzzi,
University of Glasgow). Scale bar: 10 mm.
many cells appear to round up before dividing. Longitudinal furrow ingression can be observed (Figure 1), but
detailed studies of Leishmania cytokinesis have not yet
been published.
Regardless of the exact physical mechanism of cytokinesis,
the process can be subdivided into three stages. Firstly,
signaling events are required to ensure cytokinesis initiates
at the appropriate time (e.g. following duplication and
segregation of organelles), which may involve the inactivation of one or more checkpoints. Secondly, a cleavage
furrow ingresses to bisect the cell. This must involve
remodeling of the MT cytoskeleton and cell membranes
as the cleavage furrow progresses. Motor proteins and
katanins are likely to be crucial for this process; endocytic
vesicles are required in mammalian cells to bring additional
membrane and proteins to the furrow, but it is not clear if
vesicular transport is required during furrowing of trypanosomatids. Some form of physical force is also likely to be
required, though the exact nature of this force (e.g. whether
the ingressing furrow is ‘pushed’ from behind or ‘pulled’
from in front) and exactly what generates it is unknown.
Basal body (BB)/flagella separation is a vital prerequisite
for cytokinesis to occur in T. brucei, and it is conceivable
that this may either trigger the process or result in a
rearrangement of cell morphology that is particularly conducive to furrowing. However, the different furrowing
mechanisms utilized by different parasites and life cycle
stages will probably involve different forces and be
Current Opinion in Microbiology 2007, 10:520–527
522 Eukaryotes
regulated distinctly. Thirdly, abscission brings about the
final separation of the two daughter cells. This event
probably also requires cytoskeleton and membrane remodeling, and it is possible that the proteins responsible
accumulate at the join between the daughter cells, analogous to proteins accumulating at the midbody of mammalian cells. Physical force may also be required to bring about
abscission, as in T. brucei it has been suggested that
rotational forces arising from flagellar beat contribute to
abscission [13].
Cytokinesis and the mutant phenotype
A number of experimental approaches have been taken to
investigate cell division processes in trypanosomatids,
including inducible RNAi (T. brucei), inducible overexpression, and treatment with a variety of chemical inhibitors (see Tables 1 and 2). Regardless of the approach taken,
it is the phenotype analysis of mutant cell lines that is most
important. The time at which the analysis is carried out is
critical. Often, investigating the phenotype as a growth
defect becomes visible is too late, as the deficiency that
ultimately causes the growth defect occurs at an earlier
time point. Indirect effects also accrue over time, and
distinguishing between direct and indirect effects is vital
(particularly for the study of cytokinesis). For example,
cytokinesis in T. brucei is inhibited by defects in BB [14],
flagellum [15], or Golgi duplication [16] that occur earlier in
the cell cycle, as well as by an inhibition of mitosis (BSF
only) [8]. It is also inhibited by reduced availability of
surface GPI-anchored proteins and VSG [17,18]. Hence,
although downregulating a variety of proteins can be said to
inhibit cytokinesis in T. brucei, very few actually play a
direct role in this process, either by signaling the initiation
of cytokinesis, regulating furrow ingression or controlling
abscission. The appearance of ‘monster’ cells (with
multiple nuclei, kinetoplasts, and flagella) does not imply
that a particular protein is involved in cytokinesis, but
merely indicates that functional analyses should be carried
out at an earlier time point in order to determine the precise
role of a particular protein.
Molecules directly involved in cytokinesis
Defining molecules that directly regulate initiation of
cytokinesis is difficult. In PCF T. brucei, the best candidate to date for a cytokinesis initiator is the aurora kinase,
AUK1 [19]. Depletion of this kinase resulted in the
rapid accumulation of 1N2K cells, as well as an approximate twofold increase in 2N2K cells by 24 hours postinduction. The 1N2K cells all contained a mitotic
spindle, suggesting mitotic exit was affected. PCF trypanosomes lack a mitosis to cytokinesis checkpoint, and
usually, inhibition of mitosis in this life cycle stage does
not prevent cytokinesis, resulting in a 1N2K cell dividing
to give a 1N1K cell and a zoid (0N1K) [7,8]. However,
zoid formation was not observed following AUK1 RNAi,
suggesting cytokinesis was inhibited, and that AUK1
may play a key role in triggering exit from mitosis and
Current Opinion in Microbiology 2007, 10:520–527
entry into cytokinesis, at least in this life cycle stage
[19]. In the BSF, AUK1 RNAi resulted in a transient
increase in 1N2K cells (indicating inhibition of mitosis),
followed by the appearance of cells with a single enlarged
nucleus but multiple kinetoplasts (because of a block in
cytokinesis) [20]. However, in BSF trypanosomes, mitotic inhibition is known to prevent cytokinesis [8], so it is
not possible from these data to assign a role for AUK1 in
initiating cytokinesis in this life cycle stage. It might also
be expected that the anaphase promoting complex (APC)
would play a key role in the mitosis:cytokinesis transition. However, RNAi of two APC components, CDC27
and APC1, in PCF T. brucei, though preventing mitosis by
arresting cells at metaphase, did not prevent cytokinesis
[21]. In the BSF, depletion of these components resulted
in the accumulation of cells at anaphase (observed as
2N2K cells with a long spindle linking the nuclei),
suggesting a delay or block in cytokinesis. The observed
inhibition of mitosis means once again it is not possible to
imply a direct role for the APC in cytokinesis. Recent
work has also raised the intriguing possibility that metacaspases may play a role in signaling entry into cytokinesis, since combined knockdown of MCA2, MCA3, and
MCA5 in BSF T. brucei prevented initiation of cytokinesis [22]. In L. major, overexpression of the single metacaspase leads to a delay in growth because of deficiencies
in kinetoplast segregation, nuclear division, and cytokinesis [23].
Although little is known about the signals that trigger
entry into cytokinesis, some information has emerged
concerning checkpoints that must be overcome in order
to divide the cell. In the BSF, depletion of VSG synthesis
results in the accumulation of 2N2K cells lacking cleavage furrows, suggesting a precytokinesis arrest [18].
However, unlike downregulation of GPI8, which prevented cytokinesis in BSF T. brucei, but did not stop
the re-replication of DNA and organelles [17], ‘monster’
cells were not observed following VSG RNAi. BSF cells
may have evolved a novel checkpoint that senses VSG
synthesis, reflecting the vital role VSG plays in evading
the host immune response, to ensure cells do not divide in
the absence of adequate cell surface protection.
Depletion of phosphatidyl-inositol 4-kinase III-b
(PI4KIIIb) in PCF T. brucei also resulted in the accumulation of 2N2K cells that lacked a cleavage furrow without
the generation of ‘monster’ cells [24], suggesting that
phosphoinositides may be required for cytokinesis. However, PI4KIIIb knockdown severely affected cell
morphology, cell ultrastructure, and organelle positioning, so the effect on cytokinesis may be indirect. Ablation
of dynamin-like protein (DLP) in PCF T. brucei blocked
mitochondrial fission and arrested cells midfurrow in a
NKKN configuration [25]. Once again, this arrest was
precise, with no accompanying monster formation,
suggesting that mitochondrial fission may constitute a
checkpoint that must be overcome during cytokinesis.
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Cytokinesis in trypanosomatids Hammarton, Monnerat and Mottram 523
However, the factors that determine whether a cell rereplicates its DNA following a block in cytokinesis are not
well understood yet. Almost certainly, the exact point at
which cytokinesis is blocked and the parasite life cycle
stage will be key factors, but because of the low number
of cytokinesis proteins identified till date, we are a long
way from being able to confirm the existence of any
molecular cytokinesis checkpoint.
Three proteins have been demonstrated to be required
for furrow ingression in T. brucei: MOB1, TRACK, and
Polo-like kinase (PLK). RNAi of MOB1 or PLK in BSF
Table 1
T. brucei gene products with an RNAi cytokinesis phenotype
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Current Opinion in Microbiology 2007, 10:520–527
524 Eukaryotes
Table 1 (Continued )
The proposed functions of genes reported to display a cytokinesis phenotype following their downregulation via RNAi are listed. Phenotypes of RNAi
mutants in BSF and PCF T. brucei are given. In the ‘Comments’ column, ‘Possible regulatory function’ refers to a potential direct molecular role in one
of the three main stages of cytokinesis (initiation signaling events, furrowing and abscission). Many RNAi mutants are defective in cytokinesis through
an indirect mechanism, for example, as a result of a defect in flagellar motility, mitosis (in the case of BSF), or basal body (BB) duplication/segregation.
Genes listed are color coded by category — pink: potential signaling molecules (see Refs. [8,14,19–22,24,25,26,27,28,36–39]); blue: flagellar
proteins (see Refs. [13,15,33,34,35,40–45]); yellow: centrins (see Refs. [16,46]); green: cell surface molecules (see Refs. [17,18]); white: other
(see Ref. [47]). ND: not done; N/A: not applicable. *Overexpression.
trypanosomes resulted in an accumulation of furrowing
2N2K cells six to eight hours postinduction [14,26],
indicating that furrow ingression is delayed following
depletion of either of these proteins. Different phenotypes were observed following RNAi of these regulators
in PCF T. brucei. MOB1 appears to be required for
accuracy of furrow ingression rather than being required
for furrow ingression per se [26]. PLK was reported
to inhibit initiation of cytokinesis in procyclic cells
[27], but subsequent analyses have shown that PLK
actually inhibits BB duplication earlier in the cell cycle,
which itself blocks cytokinesis [14]. Although an
additional direct role for PLK in cytokinesis in the
PCF cannot at present be ruled out, the available data
only support an indirect involvement. RNAi of TRACK
resulted in cells undergoing multiple rounds of incomplete furrow ingression during PCF cytokinesis and in
an accumulation of 2N2K cells lacking furrows in BSF
trypanosomes [28]. Hence, TRACK is essential
for furrow ingression in PCF cells, but required for
progression into cytokinesis in BSF parasites. Based on
this limited data set, it appears that cytokinesis is
regulated very differently in these two life cycle stages,
probably reflecting the different morphology, organelle
Current Opinion in Microbiology 2007, 10:520–527
positioning, and cytoskeleton composition in these
forms.
MTs and MT-associated proteins (MAPs) are likely to be
key players during cytokinesis. The composition of the
cytoskeleton in T. brucei differs between life cycle stages
[3,29], with the result that the cytoskeletons of BSF and
PCF trypanosomes differ in their stability. Modification
of MAPs, for example, by phosphorylation can alter their
affinity for MTs leading to changes in MT stability [30].
In T. cruzi, incubation with Taxol, a MT-stabilizing agent,
blocks cytokinesis during furrow ingression [12], while
incubation with vinca alkaloids, MT-destabilizing agents,
inhibits initiation of cytokinesis (Table 2) [31]. Tubulin
inhibitors also disrupt cytokinesis in Leishmania donovani
[32]. Further investigation of the role of MT stability
during cytokinesis is clearly warranted.
Proteins with a direct role in abscission have not yet been
identified. Rotational flagellar forces may be required for
this process because defects in flagellar beat caused by
depletion of radial spoke and central pair proteins are
accompanied by impaired abscission in PCF T. brucei
[13,33], but other PCF flagellar motility mutants do
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Cytokinesis in trypanosomatids Hammarton, Monnerat and Mottram 525
Table 2
Inhibitors that cause a cell cycle phenotype in trypanosomatids
Key to shading — pink: microtubule (MT) inhibitors (see Refs. [7,12,31,48]); yellow: DNA replication inhibitors (see Refs. [7,48,49]); blue: cell cycle
regulator inhibitors (see Refs. [50–53]); unshaded: other inhibitors (see Refs. [54,55]).
not display cytokinesis defects [34]. However, in the
BSF, flagellar motility defects are lethal, resulting in the
formation of highly contorted cells containing multiple
nuclei and flagella [34,35]. These cells were unable to
complete cytokinesis, but it is not clear in all cases
whether initiation, furrowing, abscission, or all stages of
cytokinesis were affected.
Future perspectives
Trypanosomatid cell division is easily perturbed at many
stages of the cell cycle. Some perturbations block cytokinesis, while others are apparently disregarded by the
cell and cytokinesis proceeds unimpeded, resulting in the
generation of aberrant progeny such as zoids. This has led
to speculation concerning the existence or not of classical
cell cycle checkpoints in T. brucei. Certainly, evidence
suggests that a mitosis to cytokinesis checkpoint is absent
in PCF [7,8], though it may be present in BSF parasites,
and there is evidence that VSG synthesis may be monitored in a cytokinesis initiation checkpoint in the BSF
[18]. It is also possible that there is a midfurrowing
checkpoint, given that in several RNAi mutants (TRACK,
MOB1, and PLK), cells displaying partially ingressed
cleavage furrows accumulate, suggesting that these gene
functions are only required for the latter stages of furrowing [14,26,28]. However, it could also be argued that
residual protein following RNAi is sufficient to allow
furrowing to begin, but it becomes rate-limiting midway
through cell cleavage. T. brucei cells also take some time to
complete abscission, and it is tempting to speculate that a
checkpoint may exist here to ensure that final separation
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only occurs once the accuracy of the preceding cell cycle
has been verified. However, there is some way to go
before it will be possible to define the mechanisms and
molecular participants of any checkpoint in T. brucei and
even further until an understanding of checkpoints in
other trypanosomatids is realized.
As highlighted in this review, cell cycle data must be
carefully interpreted in order to distinguish between
direct and indirect effects on cytokinesis. In our opinion,
in order to classify a protein as playing a direct role in
cytokinesis, following its downregulation or overexpression, cells must progress normally through the cell cycle
up until nuclear division is complete. Hence, for T. brucei,
the first cell type to accumulate in a cytokinesis mutant
would, in most cases, be of a 2N2K configuration, with or
without a furrow. If the accuracy of furrow positioning is
affected, then a 2N2K cell might divide to give
2N1K + 0N1K cells. Here, dividing trypanosomes must
be ‘caught in the act’ to confirm this. Since most cytokinesis defects will ultimately result in the accumulation of
abnormal cell types, a cell lineage analysis (involving the
examination of nuclei, kinetoplasts, flagella, basal bodies,
and Golgi, as necessary) needs to be carried out at
multiple time points, for example, at least one cell cycle
before and after the first defects are observed, in order to
accurately map the progenitors of ‘monster’ cells.
Although this analysis will prevent proteins being falsely
assigned a role in cytokinesis, some direct regulators with
additional essential functions earlier in the cell cycle will
be missed, as earlier defects will mask later cytokinesis
Current Opinion in Microbiology 2007, 10:520–527
526 Eukaryotes
defects. Unfortunately, until populations of actively
dividing cells can be isolated in a given cell cycle stage,
this problem will remain difficult to address.
11. Elias MC, da Cunha JP, de Faria FP, Mortara RA, Freymuller E,
Schenkman S: Morphological events during the Trypanosoma
cruzi cell cycle. Protist 2007, 158:147-157.
This paper reports the initial characterization of the T. cruzi cell cycle with
respect to the order of organelle division. Shown to differ from T. brucei.
Despite the advances in our understanding of cell division
in T. brucei, there remain many unanswered questions. For
example, we know nothing about the proteins that determine the position of cleavage furrow initiation at the
anterior end of the flagellum/FAZ, or that cleave the
cytoskeleton and remodel membranes during furrowing
and abscission, or indeed where and when the extra membrane required for generating two daughter cells is sourced.
These questions will undoubtedly be the focus of future
research into the intriguing cell biology of the trypanosomatids, and given the obvious differences between trypanosomatid and mammalian cytokinesis, identification of
the molecular effectors of parasite cytokinesis will probably yield much-needed novel drug targets.
12. Baum SG, Wittner M, Nadler JP, Horwitz SB, Dennis JE, Schiff PB,
Tanowitz HB: Taxol, a microtubule stabilizing agent, blocks the
replication of Trypanosoma cruzi. Proc Natl Acad Sci U S A
1981, 78:4571-4575.
Acknowledgements
16. He CY, Pypaert M, Warren G: Golgi duplication in Trypanosoma
brucei requires Centrin2. Science 2005, 310:1196-1198.
This work was funded by the Wellcome Trust and the MRC. TCH holds an
MRC Career Development Fellowship (ref G120/1001).
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