Mechanics and Control of Cytokinesis
The structural mechanics of cell division in
Trypanosoma brucei
Sue Vaughan and Keith Gull1
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, U.K.
Abstract
Undoubtedly, there are fundamental processes driving the structural mechanics of cell division in eukaryotic
organisms that have been conserved throughout evolution and are being revealed by studies on organisms
such as yeast and mammalian cells. Precision of structural mechanics of cytokinesis is however probably no
better illustrated than in the protozoa. A dramatic example of this is the protozoan parasite Trypanosoma
brucei, a unicellular flagellated parasite that causes a devastating disease (African sleeping sickness) across
Sub-Saharan Africa in both man and animals. As trypanosomes migrate between and within a mammalian
host and the tsetse vector, there are periods of cell proliferation and cell differentiation involving at least five
morphologically distinct cell types. Much of the existing cytoskeleton remains intact during these processes,
necessitating a very precise temporal and spatial duplication and segregation of the many single-copy
organelles. This structural precision is aiding progress in understanding these processes as we apply the
excellent reverse genetics and post-genomic technologies available in this system. Here we outline our
current understanding of some of the structural aspects of cell division in this fascinating organism.
Structures
The African trypanosome has a long slender shape with
a single flagellum laterally attached to the cell body in a
left-handed helix from close to the posterior end along
to the anterior [1] (Figure 1A). This is a major defining
characteristic, yet the specific cell body shape is defined by a
highly stable, highly cross-linked subpellicular microtubule
cytoskeleton that underlies the plasma membrane [2]
(Figure 1B). The microtubules of this array are equally
spaced and have an identical polarity with their plus ends
at the posterior end of the cell [3]. The only exception to
this polarity is a specific microtubule quartet (see later).
The length and number of microtubules therefore varies in
order to accommodate the change in cell diameter from the
posterior to anterior end. Hence, shorter microtubules are
inserted between longer microtubules [1].
Organelles such as the mitochondrion and Golgi are
present as single copies in specific positions resulting in a
highly reproducible, organized and polarized cellular structure (Figure 2A). The kinetoplast (containing the mitochondrial DNA) is physically connected to the proximal end of the
two basal bodies [4,5]. A mature basal body subtends the
single flagellum with the pro-basal body connected alongside
it. The pro-basal body will mature and form a new flagellum
in the next cell cycle [1]. In fact, basal bodies are both
morphologically and functionally analogous to centrioles in
mammalian cells and exhibit the same conserved maturation
and inheritance pattern (for a review see [6]). The single
Key words: basal body, cell division, cytokinesis, cytotaxis, flagellum, Trypanosoma brucei.
Abbreviations used: FAZ, flagellum attachment zone; PFR, paraflagellar rod.
1
To whom correspondence should be addressed (email [email protected])
Biochem. Soc. Trans. (2008) 36, 421–424; doi:10.1042/BST0360421
flagellum exits the cell body via the flagellar pocket, the only
reported site of endocytosis and exocytosis (for a review
see [7]). The flagellum is composed of the canonical 9 + 2
microtubule axoneme plus a specialized structure called the
PFR (paraflagellar rod) that runs along the length of the
flagellum once it exits the flagellar pocket and is essential
for motility [8–11]. The flagellum is connected along the
length of the cell body by the FAZ (flagellum attachment
zone). The FAZ is composed of a filament structure that
connects the cell body with the PFR in the flagellum plus
a set of four specialized microtubules that originate close
to the basal bodies and so have the opposite polarity to
the subpellicular microtubules [1,3,12]. The FAZ therefore
results in a ‘seam’ in the subpellicular corset with ideal
properties for defining axis and polarity for the mechanics
of cytokinesis. Since the flagellum is tethered to the cell body
via the FAZ, one can envisage a highly connected set of
internal structures (kinetoplast, mitochondrion, basal bodies,
flagellar pocket) that are physically tethered to each other
and the external flagellum/cell body via the FAZ region.
The single Golgi is also specifically positioned and, although
there is no direct evidence for a physical connection, it
certainly follows the same temporal and spatial duplication
and segregation patterning with the other interconnected
cytoskeletal structures [13,14].
Structural mechanics of cell division
The trypanosome cell division cycle is the same as other
eukaryotic cells, except that there two distinct S-phases
which must be co-ordinated; one for the single mass of
mitochondrial DNA contained within the kinetoplast and
one for nuclear DNA [1,15]. Most of the single-copy
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Figure 1 The procyclic form of T. brucei
(A) A scanning electron micrograph shows the polarity of the subpellicular microtubules, indicated with green + and −, and the polarity
of the microtubules of the flagellum is indicated by red + and
−. (B) Transmission electron micrograph cross-section illustrating the
attached flagellum and subpellicular microtubules underlying the plasma
membrane of the cell body.
Figure 2 Diagrammatic illustration of the cell cycle of the
procyclic form of T. brucei
(A) A G1 cell with a singe attached flagellum. Many of the single
copy organelles are marked. (B) The probasal body matures and a new
flagellum extends, which is attached to the old flagellum by the flagella
connector. Two new probasal bodies form alongside the mature basal
bodies. (C) The flagella connector stops at ∼0.6 of the length of the old
flagellum. The two flagella with attached basal bodies and kinetoplast
segregate, followed by mitosis. (D) Cytokinesis initiates from the anterior
end of the cell and follows a path to the posterior end between the two
separated flagella and associated organelles.
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Authors Journal compilation organelles are duplicated via an apparent templated mechanism, whereby a new organelle is built next to an old
one. The process begins with S-phase of the mitochondrial
DNA followed by basal body maturation and duplication
(Figure 2B) [1]. A filament system connecting the duplicated
DNA and a specific membrane region of the mitochondrion
is duplicated and connected to the duplicated basal bodies [5].
The new flagellum invades the flagellar pocket and the tip is
physically connected to the old flagellum by a transmembrane
mobile junction called the flagella connector (Figure 2B)
[16,17]. The discovery of the flagella connector has been
fundamental to our understanding of how these procyclic
trypanosomes divide. We now understand that growth of
the new flagellum is crucial in cell division. Perturbing
flagellum growth or attachment to the cell body results in two
unequal daughter cells, improper segregation of organelles,
and is ultimately lethal [18–22]. The flagella connector
provides a cytotaxis mechanism whereby an existing cellular
structure transmits spatial organization to the new structure.
In Trypanosoma brucei, transmission of cell polarity, axis,
alignment and helicity occurs as the new flagellum extends
along the old flagellum, guided by the flagella connector
[16,17,23,24]. We do not yet know the mechanism that
drives this mobile junction, but assembly and growth of the
new flagellar axoneme via the IFT (intraflagellar transport)
mechanism appears not to be necessary [21].
More recently, we have speculated on a further role for
the flagella connector in these procyclic cells. The flagella
connector stops at ∼0.6 of the length of the old flagellum
and we refer to this as the ‘stop point’. The timing of the
flagella connector stop point coincides with basal body and
kinetoplast segregation. The position of the flagella connector
at this stop point may actually aid in the segregation of the two
flagella with their interconnected organelles by effectively
acting as an anchor point as the two structures segregate
[3,4,21] (Figure 2C). The flagella connector has only been
characterized in the procyclic life cycle stage, but a similar
structure has been identified in differentiation divisions ([25],
and see later).
Of course, during cell division, the cell body increases
in length and diameter while the new flagellum continues
to extend. Extension of the subpellicular microtubules at
the posterior end of the cell appears to be important in
driving increases in cell body length. Remarkably, increase
in cell diameter is achieved by insertion of new microtubules
between old microtubules. This effectively results in a semiconservative inheritance of the subpellicular microtubules
to each daughter cell [26,27]. We do not yet understand
this spatially at the individual microtubule level. Nor do
we understand how the duplication and segregation of
the organelles and the subpellicular microtubules are coordinated in the formation of the two daughter cells.
Mitosis occurs leaving one of the two nuclei positioned
between the two kinetoplasts (Figure 2C), so ensuring
two ‘clusters’ of cytoplasmic organelles ready for cleavage.
Cleavage furrow ingression is unidirectional along the cell
body from anterior to posterior between the old and new
Mechanics and Control of Cytokinesis
flagella. The exact selection site for furrow ingression is
not known, but we have suggested that the position and
end of the FAZ is implicated since it marks a unique seam
in the cytoskeleton coincident with growth of the new
flagellum to the anterior end of the cell where ingression
initiates [3] (Figure 2D). The mechanism of furrow ingression
is unknown and a functional characterization of actin in
T. brucei failed to find a role in cytokinesis [28]. As
in mammalian cells, the two daughter cells remain attached
to each other for some time before final cell abscission.
the trypanosome to orchestrate its cytokinetic mechanics to
fit both the proliferative and differentiation type of divisions
required of its life cycle.
Although we have good descriptions of the positional
changes and cell shape alterations during differentiation, we
do not understand how these are achieved within the confines
of the existing cytoskeleton. We also lack an understanding of how the cell extends in length and how flagellum length
is regulated in order to produce cells with shorter or longer
flagella.
Structural mechanics of differentiation
Conclusions and future perspectives
In this overview we have outlined how two essentially
identical daughter cells are formed within the confines of
the existing cytoskeleton in T. brucei. However, when these
cells receive certain cues, cell proliferation stops and cell
differentiation occurs, giving rise to a new cell type that
is both morphologically and biochemically distinct. This
cell type is often pre-adapted for invasion of a new tissue
or host and there are at least five morphologically distinct
developmental forms in the life cycle of T. brucei. They are
classified by the relative positions of the kinetoplast, nucleus
and flagellum along the posterior–anterior axis of the cell
[29]. The changes in the position of these structures are again
performed within the confines of the existing cytoskeleton.
An extreme example of these positional changes and
dramatic asymmetric cell division occurs in the differentiation
event from the procyclic to epimastigote form that takes place
in the tsetse fly vector. Procyclic cells undergo a period of
cell proliferation in the tsetse fly midgut and after 2–3 days
they start migrating towards the salivary glands, during which
differentiation to the epimastigote life cycle form occurs [30–
32]. As they arrive at the proventriculus, an almost doubling
of cell body length has occurred. Growth of a short new
flagellum takes place, and at mitotic anaphase kinetoplast
segregation occurs. After mitosis, furrow ingression initiates
at the anterior end of the short new flagellum, resulting in one
very long and one very short epimastigote cell (Figure 3). The
kinetoplast of the short cell is now anterior to the nucleus,
rather than posterior to the nucleus in procyclic cells. It is
thought that only the short epimastigote cells migrate to the
salivary glands where cell proliferation initiates once more
[25]. This highly asymmetric division illustrates the ability of
The mechanisms and control of cell proliferation and
differentiation is essential to the parasite life cycle and as
such this is a fascinating organism to study. Understanding
these processes is important in trying to find targets to
combat this disease. Sequencing of the genome of T. brucei
and other related parasites are complete [33] and we have
many biochemical and genetic tools (see [34–37]). These tools
have enabled a molecular dissection of genes involved in
cell proliferation and differentiation (for reviews see [38,39]).
However, in order to better understand the phenotypes that
have arisen from these experiments our next challenge is
to understand the three-dimensional spatial organization of
these cytoskeletal structures, how co-ordination of assembly
and segregation is performed and, no doubt, the dependency
relationships that exist between the events.
Figure 3 A scanning electron micrograph of an asymmetrical
division giving rise to one short and one long epimastigote cell
The arrow and arrowhead marks the distal end of the short and
long flagella respectively. Scale bar, 1 μm. Reprinted from [25] with
permission from Elsevier.
Work in our laboratory is funded by The Wellcome Trust and the EP
Abraham Trust. K.G. is a Wellcome Trust Principal Research Fellow.
We thank members of our group for generous discussions.
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Received 20 February 2008
doi:10.1042/BST0360421