Download The Dictyostelium cell cycle and its relationship to differentiation

FEMS Microbiology Letters 124 (1994) 123-130
© 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00
Published by Elsevier
123
FEMSLE 06280
MiniReview
The Dictyostelium cell cycle and its relationship
to differentiation
Gerald Weeks
a
and Cornelis J. Weijer b,,
a Department of Microbiology, Uniuersity of British Columbia, Vancouver, BC, V6T 1Z3 Canada, and b Zoological Institute, University
of Munich, Luisenstrasse 14, 80333 Munich, Germany
(Received 11 July 1994; revision received 28 September 1994; accepted 3 October 1994)
Abstract: The Dictyostelium vegetative cell cycle is charcaterized by a short mitotic period followed immediately by a short S-phase
(less than 30 min) and a long and variable G2 phase. The cell cycle continues during differentiation despite a decrease in cell mass:
DNA replication and mitosis occur early in development and also at the tipped aggregate stage. Cells that are in mitosis, S-phase or
early G2, when starved differentiate into prestalk cells and cells that are in the middle of G2 differentiate into prespore cells. We
postulate that there is a restriction point late in the G2 phase, about 1-2 h before mitosis, where the cells can be arrested either by
starvation and the initiation of development, by growing into stationary phase, or by prolonged incubation at low temperature.
During development, this block persists to the tipped aggregate stage, where it is specifically released in prespore cells, and these
cells then go through one more round of cell division. Genes encoding components of the cell cycle machinery have recently been
isolated and attemps to specifically block the cell cycle by reverse genetics to study the effects on differentiation have been
initiated.
Key words: Dictyostelium discoideum; Cell cycle; Differentiation; Cdc2 kinase; Cdk; Cyclin B
Introduction
The cellular slime mould Dictyostelium discoideum lies at the border between the unicellular and multicellular modes of existence. It grows
as an unicellular organism, feeding on bacteria
and multiplying by binary fission, but upon starvation it undergoes a multicellular developmental
cycle to form a fruiting body consisting of two
* Corresponding author. Tel.: (+ 49-89) 5902 469; Fax: (+ 4989) 5902 450.
SSDI0378-1097(94)00433-1
distinct cell types, stalk cells and spores. The
initial response to starvation, if the cell density is
sufficiently high, is for the cells to collect in
multicellular aggregates by chemotaxis in response to periodically generated pulses of cAMP.
Each aggregate then elongates to form a migrating pseudoplasmodium or slug, which eventually
rounds up and culminates in the terminal fruiting
body. Initial differentiation into randomly dispersed cells prestalk and prespore cells is clearly
distinguishable in the aggregate. However, the
cells sort out ehemotactically during tip formation
and by the slug stage the prestalk cells occupy the
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anterior 20% with the prespore cells at the back
of the slug [1]. Spores germinate under favourable
conditions and single amoebae emerge, whereas
stalk cells die during the differentiation process.
It was claimed originally that there is a complete
separation of growth and development in Dictyostelium, in that cells do not divide during development [2], but more recent experiments have
shown that the cell cycle continues during development. In addition, there is an important link
between cell cycle position at the time of starvation and cell type differentiation.
Cell cycle during growth
Dictyostelium cells are haploid and contain
seven chromosomes as determined by cytological
studies [3]. They can be grown on bacteria (e.g.
Klebsiella aerogenes or Escherichia coli B / r ) , either in suspension or on agar plates, with a generation time of 3 - 4 h. Axenic mutants have been
isolated that can be grown in either a complex
nutrient medium, where they double every 8 h, or
in a defined medium where they double every
10-24 h, depending on the strain ([4]; Weijer
unpublished observations). The cell cycle of growing axenic cells has been analysed by classical
techniques [5,6] and by measurement of the DNA
content of cells and nuclei by flow fluorometric
techniques [7,8]. Both approaches have shown
that the cell cycle consists of a short mitotic
period (10-20 min) immediately followed by a
short S-phase ( < 30 min). There is no detectable
G1 phase and cells are in G2 phase for the rest of
the cycle.
Mitosis can be blocked by the use of the mitotic inhibitors nocodazole and CIPC [9], but
D N A replication proceeds slowly under these
conditions and the cells become polyploid [3,7,9].
A high percentage (up to 50%) of cells grown
axenically in suspension are multinucleate,
whereas cells grown in Petri dishes are mostly
mononucleate [5]. This indicates that cells need
substratum contact for cytokinesis and that nuclear division can be uncoupled from cell division.
S-phase is short: only 8 - 1 0 % of an exponen-
tially growing population are labelled following a
10-min pulse with [3H]thymidine [5]. Furthermore, in flow fluorometric analyses, fewer than
10% of either exponentially growing or developing cells have a D N A content that would be
expected of S-phase cells [7,8]. The DNA measurements have also shown that half of the total
cellular DNA is mitochondrial [7,8], and this
replicates asynchronously during the cell cycle [7].
A detailed analysis of S-phase has been hampered in the past by the relatively slow incorporation of thymidine into DNA, necessitating high
concentrations in the medium and long exposure
times of autoradiograms. A major improvement
has been the development of 5-bromodeoxyuridine (BudR) incorporation and its detection by
specific antisera [10,11]. Using this technique, it
has been confirmed that less than 10% of the
population are in S-phase.
The G2 phase is the longest part of the cell
cycle but may vary between 4 and 8 h as determined by labelled mitosis experiments using populations of randomly growing exponential axenic
cells [5,7]. Furthermore, the total cell cycle time
varies considerably with growth conditions. The
doubling time of bacterially grown cells is only 3
h, whereas it is 8 h for axenic cells growing in rich
nutrient media. Therefore, G2 has to be, on
average, very much shorter in bacterially grown
cells (2-2.5 h) than in axenic cells ( > 7 h).
Cell cycle during development
During development of axenically grown cells,
the cell number more than doubles [6]. The increase is partly due to the division of multinucleate cells to mononucleate cells that occurs during
the first hours of development. However, there is
almost a complete doubling in cell number due to
real mitotic division [6]. Katz and Bourguignon
[12] have shown, using a temperature-sensitive
cell cycle mutant, that cells starved late in G2
double in cell number before aggregation and
cells starved in middle G2 show no increase in
cell number before aggregation. When exponentially growing cells are starved to inititate devel-
125
opment, the number of cells entering mitosis and
S-phase drops rapidly to low levels during early
aggregation. Less than 20% of the cells go through
mitosis before aggregation, but following aggregation most of the remaining cells go through mitosis [6]. The second round of mitosis does not
occur in a mutant blocked at the mound stage of
development, suggesting that this mitotic event is
linked to a specific developmental stage [6]. In a
different set of experiments using BudR labelling,
the continuation of the cell cycle during development has been confirmed. The number of cells
entering S-phase decreases during aggregation,
but then rises at the mound stage, showing that
the cells that go through mitosis also go through
S-phase (Fig. 1) [11]. Double label experiments
monitoring BudR incorporation and expression
of a prespore specific antigen have shown that all
cells that replicate DNA during the mound stage
express the prespore antigen [11]. In contrast, the
cells that go through S-phase during the early
stages of development become prestalk in the
slug. These experiments show that cell cycle progression is temporarily blocked during early aggregation but is specifically reinitiated in prespore cells at the mound stage. Prestalk cells do
not re-enter the cell cycle. There is no evidence
as yet that mitosis is essential for spore cell
formation. In fact, under some conditions spore
differentiation can take place in the absence of
detectable cell division: in the presence of mitotic
inhibitors [13] or under conditions of low density
[141.
The cell cycle of bacterially grown cells is not
so well characterized. It has, however, been shown
that bacterially grown cells are mostly mononucleate [3,5] and that their cell number increases
by 50% during development to the fruiting body.
This increase in cell number is dependent upon
the formation of a mound stage [15]. All these
Fig. 1. Pattern of B u d R incorporation in the prespore zone of a slug after a 45-min BudR pulse label. A slug was pulse-labelled for
30 rain by placing it on agar containing 1 m M BudR. Incorporation of B u d R can be seen in some cells in the prespore zone (right
side of slug). From double label experiments detecting B u d R incorporation and prespore antigen expression it is known that all the
cells that are labelled are prespore cells [11].
126
observations are consistent with there being a
close link between cell cycle progression and differentiation.
Cell cycle position at the time of starvation initiates cell type determination
During the development of Dictyostelium, an
apparently homogeneous population of cells (derived from one clone) develops into both spore
and stalk cells and several of presumptive morphogens have been described that promote this
cell type differentiation [16]. However, an initial
inhomogeneity in the population needs to be
generated and then amplified during development. Several types of evidence indicate that this
inhomogeneity is established by differences in
cell cycle position at the time of starvation. Sorting experiments with synchronized cells that are
starved in different cell cycle phases show that
cells starved at around mitosis will sort to the
prestalk region of the slug, while cells starved in
A
other parts of the cell cycle, sort to the prespore
region of the slug [17-21]. These experiments
were performed with cells synchronized by different methods; release from stationary phase arrest
[27], preferential detachment of mitotic cells [18],
and release from low temperature arrest [19-21].
Cells that sort to the prestalk zone are prestalk
cells, while cells that sort to the prespore zone
can be either prespore or anterior-like cells. Experiments in which exponentially growing cells
were pulse-labelled with BudR and starved immediately or after a variable period of further
growth clearly showed that S-phase and early
G2-phase cells differentiate preferentially into
prestalk cells, while cells in the rest of G2 differentiate into prespore cells [11].
Furthermore, it has been shown that cells
starved close to mitosis in the presence of cyclic
A M P and a conditioned medium factor under
low density conditions differentiate into prestalk
cells, while those starved in the middle of G2
differentiate into prespore cells. This led to the
suggestion that cell type determination is cell
differentiation
TT!rO
T1T~"1"2
"1"4. . . . .
"S-p~e
< 30 min
Fig. 2. Model for the relationship between cell cycle and differentiation. (A) Model for the control of cell cycle progression during
development as proposed by Maeda and co-workers[20,25]. It shows that the cells have to reach a point in the cell cycle, called the
PS point, either during growth or during starvation, before they leave the cycle and start to differentiate. (B) Model, as proposed in
the text, of the relationship between cell cycle progression and differentiation. Cells which are past the restriction point in the cell
cycle at the moment of starvation go through mitosis before aggregation. These cells will differentiate to prestalk cells and
then to stalk cells, without going through mitosis and S-phase again. Cells which are in G2 but before the restriction point at
starvation, accumulate at the restriction point during early aggregation and differentiate into prespore cells. The cell cycle block
is released in these prespore cells at the mound stage. They undergo one final round of division and DNA synthesis and then
differentiate into spores.
127
autonomous and cell cycle linked [22]. In addition, even under certain conditions of normal
development the developmental pattern can be
altered. Weijer et al. [17] showed that synchronized cell populations starved around mitosis
formed slugs with 50% prestalk ceils, while populations of cells starved in the middle of G2 formed
slugs with 90% prespore cells. Similar results
have since been obtained by other workers [23,24].
However, although there is a reproducible correlation between the cell cycle stage at the time
of starvation and differentiation into prespore
and prestalk cells, the cell cycle is not solely
responsible for cell type determination. Dictyostelium slugs can regulate their cell type proportions such that if a slug is cut into prespore
and prestalk segments, both parts will regulate to
form normally proportioned slugs, given sufficient
time, presumably under the influence of morphogens such as the well-characterized differentiation inducing factor (DIF) [16].
Models for the relationship between the cell cycle
and differentiation
Maeda and co-workers have proposed that
Dictyostelium cells need to go to a particular
point (PS point) in the cell cycle, about 2 h before
mitosis, before they can enter development. This
is shown in Fig. 2A. This hypothesis is based on
experiments with cells synchronized by low temperature arrest. They show that cells starved just
prior to this PS point aggregate more rapidly than
cells starved just after the PS point [19]. In addition, cells that are 7 h past the PS point and cells
that are 1 h after the PS point, and then starved
for 6 h, show the same sorting behavior to the
prespore zone [20,21]. It is proposed that the cells
that exit the cell cycle first differentiate into
prespore cells and the ceils that exit the cell cycle
last differentiate into prestalk cells [25].
It must be emphasized, however, that the
Maeda model ignores the fact that cells do not
exit the cell cycle upon starvation and that most,
if not all, cells complete an additional round of
the cell cycle during development. In addition,
immediately post mitotic cells, synchronized by
mitotic wash off, developed more rapidly than
other cells and showed a higher incidence of
centre formation [26,27]. Consistent with this resuit, mitotic cells, synchronized by release from
stationary phase, show high levels of cAMP receptor and cAMP phosphodiesterase expression
during early development [24]. However, in experiments with a ts cell cycle mutant it was found
that the time of aggregation was independent of
the phase of the cell cycle where the cells were
starved [12]. Thus, the reported correlation between the rate of development and cell type
differentiation [19,25] may be an artifact of the
method used to achieve cell synchronization. We
propose, therefore, an alternative model to explain the relationship between the cell cycle and
differentiation. We agree with Maeda that there
is a control point 1-2 h before mitosis, where
cells are arrested in stationary phase and at low
temperature (Fig. 2B). At the onset of development, the cells that have passed this point will
continue mitosis and D N A replication before aggregation and will become prestalk. These cells
do not go through a second round of mitosis and
S-phase during development. Those cells that
have not yet reached this cell cycle control point
at the time of starvation halt at this point during
early development. These cells will become prespore cells. The cell cycle block is released at the
mound stage of development by which time prespore cell differentiation has been initiated. This
model suggests that there is a cell cycle regulatory event, the release of which is under developmental control. Aggregation minus strains that do
not proceed as far as this regulatory event show
no sign of cell division later in development [6].
This suggests that there is a feedback from cell
type-specific differentiation to cell cycle control
and it will be very important to investigate the
molecular biology of this control point.
This model implies that there is a difference in
the molecular make up of cells in the cell cycle at
around mitosis, relative to those in the remainder
of the cycle, which predisposes cells to become
prestalk rather than prespore. It is interesting in
this respect, that the Mud9 antigen is preferentially expressed during growth in cells that are
close to the mitotic stage of the cell cycle and is
128
restricted to prestalk cells at the slug stage of
development [10]. Furthermore it has been suggested that cell cycle progression is dependent on
pH changes and these correlate with subsequent
cell differentiation [28].
Molecular characterisation of the Dictyostelium
cell cycle
Recently, attempts have been made to understand the cell cycle in more molecular terms. The
Cdc2 and cyclin B proteins have been selected for
study, because the major control point for the
Dictyostelium cell cycle seems to be close to the
G 2 / M transition and these proteins are known to
regulate this control site in other eukaryotic cells.
A Dictyostelium PCR fragment encoding a
peptide with a high level of identity to the Cdc2s
of other species was used as a probe to isolate a
full length cDNA [29]. This c D N A encoded a
protein that exhibited approximately 60% identity
to the Cdc2s of other species and the gene was
able to complement a yeast cdc28 mutant. Cdc2
protein levels, as detected by antibodies either
against the conserved P S T A I R E motif or against
the variable C-terminal region of the protein,
remained constant during the cell cycle and during development ([30]; Luo and Michaelis, unpublished observations), a result not unexpected in
view of studies previously performed with other
organisms
A second gene, encoding a protein that is over
60% identical to other proteins of the cyclin-dependent kinase (Cdk) family, has also been identified. However, the encoded protein contains a
P C T A I R E sequence rather than the characteristic P S T A I R E motif of the cell cycle regulating
Cdks and does not contain either the equally
characteristic G D S E I D domain or a threonine at
position 161. In addition, this gene is unable to
complement the yeast cdc28 mutant. The available evidence is consistent with the Cdc2 protein
being the only cell cycle regulating Cdk protein in
Dictyostelium, but the possibility of there being
additional proteins, as in higher organisms, cannot be totally ruled out.
A full length cyclin B cDNA has also been
isolated, again by PCR technology [31]. The cyclin box motif of the encoded product is more
related to the B-type cyclins of other species than
to the A-type cyclins. The cyclin B gene (clbl) is
present as a single copy gene and low stringency
blots suggest no other related genes. The expression of clbl shows marked oscillations during the
cell cycle with maximum levels of m R N A being
observed just prior to cell division and maximum
levels of Cyclin B protein occurring about 2 h
later. It is interesting that the cdc2 mRNA levels
show similar, but less pronounced, oscillations
during the cell cycle, whereas Cdc2 protein levels
remain unchanged (Luo and Michaelis, unpublished observations).
The expression of both the clbl and cdc2
genes is developmentally regulated ([29]; Luo and
Michaelis, unpublished observations), clbl mRNA
levels decrease following the onset of differentiation and then rise dramatically at the tipped
aggregate stage of development. Although this
increase coincides approximately with the DNA
replication and mitotic periods, Cyclin B protein
does not change dramatically during development. Thus the exit of the cells that are destined
to become prestalk cells from mitosis during early
development does not appear to require the
degradation of cyclin B protein, cdc2 mRNA
levels also increase during aggregation, the increase slightly preceding that for clbl mRNA, but
the level of Cdc2 protein remains constant.
Interfering with cell cycle progression by reverse
genetics
Experiments designed to specifically block the
cell cycle by the introduction of dominant negative mutants of cdc2 and clbl have recently been
initiated. A dominant negative clbI gene was
created by deleting the portion of the gene encoding the degradation box and the resulting
truncated gene was placed under the control of
the folate repressable discoidin promoter [32].
Transformants containing a high copy number of
this construct were selected in the presence of
G418 and folate. In the presence of folate, the
transformants grew to normal cell density but in
129
its absence, growth ceased approximately two
generations after its removal [31]. The majority of
the growth arrested cells had condensed chromosomes, indicating that the arrest was during mitosis. Large amounts of truncated cyclin B protein
were detectable by the time of growth arrest.
These results are consistent with a requirement
for cyclin B degradation for cells to exit mitosis.
The development of the mitotically arrested
transformants on Millipore filters is delayed considerably and the fruiting bodies are small with
clear spore heads. When developed on glass or
plastic surfaces, development is slightly accelerated and the fruiting bodies are of normal size.
The spore heads are still clear, however, and
spore formation is only 5% of the wild-type values (Luo, unpublished observations). While more
studies are clearly necessary, these preliminary
results suggest that spore formation is specifically
blocked in these mutants. At present, two possible explanations cannot be distinguished. First,
cells may be incapable of undergoing the normal
round of division that occurs in prespore ceils at
the tipped aggregate stage of development, because they still contain significant amounts of the
truncated cyclin B and remain arrested in mitosis.
Alternatively, the transformants may have such a
strong tendency to follow the stalk cell pathway,
since they are in mitosis at the time of starvation,
that it is not possible for normal pattern regulation to occur during development and the final
fruiting bodies are predominantly stalky. Regardless of the actual explanation, the transformant's
phenotype is consistent with the cell cycle having
an important role in Dictyostelium differentiation.
Concluding remarks
It is clear that there is a relationship between
cell cycle position at the time of starvation and
cell determination. It seems to involve a control
point shortly before mitosis, but the molecular
mechanisms responsible for this control are as yet
unknown. In addition, cell cycle progression continues during differentiation, despite a decrease
in cell mass, and it is important to determine
whether this progression is essential for terminal
differentiation to occur. With the the identification of critical cell cycle components and with the
aid of reverse genetics this should be possible.
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