1 Gene Discovery Research Center, National Institute of Advanced Industrial
Science and Technology (AIST), Tsukuba, Ibaraki 305-8562, Japan
2 Department of Cell Genetics, Exelixis Inc., South San Francisco, CA
94083-0511, USA
* Author for correspondence (e-mail: t-uyeda{at}aist.go.jp )
Accepted 21 February 2002
![]() |
Summary |
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Key words: Cellular slime mold, Myosin II, AmiA, Coronin, GFP-histone H1
![]() |
Introduction |
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Interestingly, when maintained on a solid surface,
mhcA- cells were capable of efficiently dividing, despite
the absence of functional myosin II. Division of mhcA-
cells on solid surfaces was originally attributed to a cell-cycle-independent
process termed `traction-meditated cytofission', in which different parts of a
large multinucleate cell move in different directions, producing smaller cell
fragments with reduced numbers of nuclei
(Spudich, 1989). However, more
recent detailed microscopic analyses by Neujahr et al.
(Neujahr et al., 1997b
;
Neujahr et al., 1998
) revealed
that, although attached to solid surfaces, mhcA- cells are
able to divide in a cell-cycle-coupled fashion using a process termed
`attachment-assisted mitotic cleavage'. This process is extremely efficient,
with more than 90% of cells dividing successfully following nuclear division,
and the morphological changes during the division process, including formation
of an equatorial cleavage furrow, are similar to those seen in wild-type cells
grown on substrates. Moreover, this process is fairly rapid, taking
approximately 3-4 minutes at 22°C, which is only two-fold slower than that
of wild-type cells under similar conditions. It is thus evident that
cell-cycle-coupled cytokinesis can proceed in Dictyostelium grown on
solid substrates in the absence of myosin II. It is further speculated that
this myosin-II-independent cytokinesis emerged earlier in eukaryotic evolution
than the myosin-II-dependent method, although the molecular mechanism by which
the equatorial cleavage furrow is formed in this case remains unknown
(Gerisch and Weber, 2000
;
Uyeda et al., 2000
).
Unfortunately, the terms that have been used to describe the various methods
of cell division in Dictyostelium are rather confusing. In this
paper, we use the terms cytokinesis A, B and C. Cytokinesis A refers to the
myosin-II-dependent and adhesion-independent division method, which wild-type
cells in suspension use for proliferation. Cytokinesis B is the
myosin-II-independent and adhesion-dependent division method, by which
mhcA- cells on substrates divide. Both cytokinesis A and B
are coupled to the progression of the cell cycle. In contrast, cytokinesis C
is an adhesion-dependent division method that is not coupled with the cell
cycle. As will be described later, certain cell lines of
Dictyostelium appear to depend on this method of division for
proliferation, and for that reason, we call it a form of cytokinesis, even
though it is not coupled to the cell cycle.
In parallel with the apparent functional differences between cytokinesis A
and B, these two modes of cytokinesis require different protein factors. As
discussed above, cytokinesis A depends on the motor activity of myosin II,
whereas cytokinesis B does not. It is notable that cell division is partially
disrupted in cells lacking amiA
(Nagasaki et al., 1998) or
corA (de Hostos et al.,
1993
), the gene encoding coronin. These mutants are moderately
multinucleate when grown on solid surfaces, although they divide with
efficiencies comparable to those of wild-type cells in suspension, suggesting
these two genes play important roles in cytokinesis B in
Dictyostelium. Coronin contains WD repeats with sequence similarities
to the ß-subunit of trimeric G proteins
(de Hostos et al., 1991
).
Analysis of a GFP fusion protein (Fukui et
al., 1999
; Maniak et al.,
1995
; Rauchenberger et al.,
1997
) and a knockout mutant (de
Hostos et al., 1993
) showed that coronin participates in the
remodeling of the cortical actin cytoskeleton, which is required for efficient
phagocytosis and macropinocytosis. The amiA gene
(Nagasaki et al., 1998
), also
known as piaA (Chen et al.,
1997
), was originally cloned as a gene required for chemotaxis by
insertional mutagenesis of Dictyostelium, but homologous genes were
later identified in the genome databases of yeast and humans
(Nagasaki et al., 1998
) (A.N.
and T.Q.P.U., unpublished). Genetic analyses have suggested that AmiA is
involved in communication between cyclic AMP receptor and adenylyl kinase
(Nagasaki et al., 1998
;
Chen et al., 1997
). However,
little is known about how AmiA functions in vivo, since this protein has not
been purified biochemically, and its predicted amino acid sequence does not
have significant homology with known motifs or functional domains except for a
leucine zipper domain.
The findings summarized above raise the intriguing possibility that Dictyostelium has two mechanistically distinct methods of cell-cycle-coupled cytokinesis. To test this hypothesis, we constructed double mutant cell lines in which both cytokinesis A and B were anticipated to be defective. Analysis of these cells fully supported the hypothesis, providing genetic evidence for the presence of two parallel pathways via which cell-cycle-coupled division is achieved in Dictyostelium. Furthermore, microscopic observation of the division process of the mutant cell lines suggested that the two cell-cycle-coupled methods of cytokinesis are mechanistically rather different. The definitions of cytokinesis A, B and C described above are functional and do not necessarily imply mechanistic differences among the three methods. On the basis of these results, however, we will propose a re-definition of these terms from mechanistic viewpoints. The implications of these results are discussed in terms of cytokinesis in higher animal cells.
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Materials and Methods |
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Construction of GFP-histone
Dictyostelium histone H1 cDNA was amplified by RT-PCR using a pair
of oligonucleotides, 5'-GGATCCAATGGGTCCAAAAGCAC-CAAC-3' and
5'-GAGCTCCTATTTTTTGGCAGCGACTT-3'. These oligonucleotides add
BamHI and SacI recognition sites at either end of the PCR
product, enabling it to be subcloned into GFP/pBig downstream of the GFP
coding sequence. Subsequent expression of GFP-H1 was driven by the promoter of
actin 15.
Generation of knockout mutants
Each cell line was generated by homologous recombination in wild-type,
mhcA- (Ruppel et al.,
1994) or corA-
(Fukui et al., 1999
) cells.
Disruption of amiA in wild-type or mhcA- cells
was achieved using pKO amiA(Bsr) (Fig.
2A), which resulted in the blasticidin resistance gene being
inserted into the coding region of amiA.
AmiA-/corA- and
amiA-/mhcA- double knockout cells were
generated using the targeting vector pKO amiA(neo) in
corA- or mhcA- cells. This gene
disruption construct consisted of an amiA gene with its promoter and
part of the coding sequence being replaced by a cassette conferring neomycin
resistance. The insertion position of the neomycin resistance gene was the
same as that of the original REMI mutant
(Nagasaki et al., 1998
). To
create an mhcA-/corA- double knockout
strain, the Neo selectable marker cassette was inserted between the two
EcoRV restriction sites within the motor domain of mhcA; the
resultant plasmid was then used to knockout mhcA in
corA- cells.
|
Fluorescence microscopy
Cells were transfected with GFP-H1/pBig by electroporation, after which the
transfectants were incubated on a plastic Petri dish with a thin glass bottom
(IWAKI, Japan). The modified HL-5 culture medium, which minimized background
fluorescence, contained 3.85 g/l of glucose, 1.78 g/l of Proteose Peptone
(Difco), 0.45 g/l of yeast extract (Difco), 0.485 g/l of KH2PO4 and 1.2 g/l of
Na2HPO4·12H2O and was sterilized by
filtration (pore size, 0.25 µm). Cells expressing GFP-H1 were observed
under a fluorescence microscope (IX50; Olympus, Japan) equipped with a UPlan
Apo 40X oil immersion objective lens (Olympus). Time-lapse images were
acquired with a CCD camera (C5985; Hamamatsu Photonics, Japan) for 10 hours
with intervals of 30-120 seconds between frames using a time-lapse recording
system (ARGAS-20, Hamamatsu Photonics). For montage sequences, video images
were digitized using NIH image software version 1.61.
![]() |
Results |
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|
Hereafter we use the terms `cytokinesis A' and `cytokinesis B' to describe the two cell-cycle-coupled methods of cytokinesis. Cytokinesis A refers to the myosin-II-dependent and adhesion-independent division method, which wild-type cells in suspension rely on for proliferation. Cytokinesis B is the myosin-II-independent and adhesion-dependent division method, by which mhcA- cells on substrates divide. Wild-type cells on substrates probably utilize both cytokinesis A and B, although the relative contributions made by each is not known and possibly variable, as will be discussed later. We refer to the cell-cycle-independent division as `cytokinesis C' (Fig. 1C). Because the division process of cytokinesis A or B of mononucleate cells is typically completed within 3-4 minutes under our experimental conditions, we operationally judge division that occurred more than 30 minutes after the nuclear division to be cell cycle independent, and hence, cytokinesis C. Cytokinesis C, like cytokinesis B, depends on adhesion to solid substrates and does not require myosin II.
Disruption of mhcA, amiA and corA in
Dictyostelium
Inactivation of the mhcA or amiA gene in wild-type,
mhcA- or corA- cells was achieved by
homologous recombination (Table
1). The gene-disruption constructs consisted of the coding region
and/or promoter of mhcA or amiA, part of which was replaced
by a gene conferring resistance to the antibiotic blasticidin S or G418
(Fig. 2A). Selective disruption
of targeted genes was confirmed by genomic PCR
(Fig. 2B); the primers used are
indicated by arrows in Fig. 2A.
The generation of corA- cells was described previously
(Fukui et al., 1999).
|
Cytokinesis defects in single and double mutants affecting
cytokinesis A or B
When amiA- and corA- cells were
cultured on solid surfaces, they tended to become larger and flatter than
wild-type or mhcA- cells
(Fig. 3C-E,G). Most notably,
staining of the nuclei with 4,6-diamidino-2-phenylindole (DAPI) revealed that
some of these mutants had become moderately multinucleate. When assayed three
days after dilution to new plate cultures, 63% and 68% of
amiA- and corA- cells had more than
two nuclei, respectively (Fig.
3A). Less than 4% of wild-type cells maintained on substrates were
multinucleate. In contrast, when cultured in suspension for three days, 44% of
the wild-type cells became multinucleate, indicating the limited efficiency of
cytokinesis A. Suspension culture of amiA- and
corA- mutant cells yielded multinucleate cells. The
fractions of multinucleate cells (52% and 41%, respectively) were somewhat
smaller than but similar to those on substrates
(Fig. 3B). These values are
also comparable to that of wild-type cells grown in suspension. In summary,
wild-type cells on substrates divide with nearly 100% efficiency, whereas
amiA- and corA- cells either in
suspension or on substrates and wild-type cells in suspension divide at
similarly compromised efficiencies (3060%). In other words,
amiA- and corA- cells are more
severely disrupted in cytokinesis than wild-type cells on substrates, but they
divide with efficiencies similar to that of wild-type cells in suspension.
|
That amiA- and corA- cells exhibit more severely disrupted cytokinesis than wild-type cells only when cultured on substrates. This is in sharp contrast to mhcA- cells, which exhibit severely defective cytokinesis only in suspension. These results suggest that AmiA and coronin might play important roles in cytokinesis B and that we now have mutations that selectively affect cytokinesis A (mhcA-) and cytokinesis B (amiA- and corA-). On the basis of that premise, we set out to dissect genetically the mechanism of cytokinesis in Dictyostelium. When cultured on solid substrates, cytokinesis in cells lacking both AmiA and myosin II (amiA-/mhcA-) was much more disrupted than that in cells lacking only AmiA (Fig. 3F,J) or double mutants lacking coronin and myosin II (corA-/mhcA-) (Fig. 3H,J). The efficient suppression of cytokinesis in amiA-/mhcA- and corA-/mhcA- cells appeared to reflect a synergistic effect of the loss of myosin II and AmiA or coronin rather than being merely an additive effect of two gene disruptions; indeed amiA-/corA- double mutants were no more severely affected than cells carrying a single mutation (Fig. 3I,J).
Morphological changes during the mitotic phase in mutants and
wild-type cells
Detailed analyses of the morphological changes that occur during
cytokinesis revealed subtle but reproducible differences among the wild-type
and mutant cell lines. For example, amiA- and
corA- cells grown on substrates always rounded up and
became detached from the substrate when they carried out cytokinesis
successfully as judged by their refractile appearance in phase
contrast micrographs (Fig.
4C,D) - and in most cases they remained so throughout the cleavage
process, until the two daughter cells were completely separated. Furthermore,
dividing amiA- and corA- cells often
drifted over the substrate, suggesting that substrate adhesion is greatly
decreased during this division process. This manner of cell division is
similar to that of wild-type cells cultured in suspension, in agarose
(Fig. 1A), or on hydrophobic
surfaces (Zang et al., 1997).
Under these conditions, cytokinesis A powered by myosin-II-dependent active
furrowing of the contractile rings is the sole means of division, which is
consistent with our premise that AmiA and coronin are required for cytokinesis
B.
|
MhcA- cells also first retracted pseudopods and temporarily rounded up slightly when entering mitosis, but in contrast to amiA- or corA- cells, they remained attached to the substrate during the entire division process. Moreover, during anaphase or telophase, mhcA- cells would resume their flat appearance, adhering to the substrate over their entire ventral surfaces (Fig. 1B, Fig. 4B). Mitotic wild-type cells grown on substrates were initially adherent, much like mitotic mhcA- cells, but like amiA- or corA- cells, they rounded up by the time cleavage furrows formed and remained detached until division was complete. Thus the morphological changes seen during cytokinesis in wild-type cells grown on substrates are intermediate between those of amiA- and corA- cells and those of mhcA- cells, which suggests that, on substrates, wild-type cells may employ both cytokinesis A and B to guarantee efficient and faithful bisection.
When amiA-/mhcA- cells were
cultured on solid surfaces for three days, most became multinucleated, with
some becoming extremely large and highly multinucleate
(Fig. 3J). Moreover, among the
mononucleate amiA-/mhcA- cells, most
did not divide during a time-lapse observation period of 8 hours (not shown).
Given that their doubling time is normally about 8 hours
(Zada-Hames and Ashworth,
1978), these observations suggest that the majority of
amiA-/mhcA- cells do not divide in a
cell-cycle-coupled manner. Time-lapse observation of large, multinucleate
amiA-/mhcA- cells showed that a
portion of these cells often moved in different directions by assembling
multiple leading edges, which sometimes tore the large cell into several
fragments (Fig. 4E). This
method of cytokinesis resembles the cytokinesis C observed when large,
multinucleate mhcA- cells produced by culture in
suspension were placed on a solid surface
(Fig. 1C). That type of
division is apparently uncoupled from the cell cycle, as most of the large
cells initiated this process immediately following adhesion to the substrate
(data not shown). We suggest that the morphologically similar division of
large amiA-/mhcA- cells is also
uncoupled from the cell cycle.
Visualization of cell cycle progression using GFP-histone
To further investigate the relationship between cell cycle progression and
division events, we visualized progression of the cell cycle as a function of
the expression of histone H1 fused with the green fluorescent protein (GFP-H1;
Fig. 5A). Using time-lapse
observation of GFP-H1 fluorescence, we determined that during interphase
GFP-H1 was distributed uniformly within nuclei
(Fig. 5B). When cells entered
the mitotic phase, however, the pattern of GFP-H1 fluorescence varied in a
characteristic fashion that enabled it to be used to track the cell within the
cell cycle. First the contour of the nucleus became obscure and the
chromosomes condensed (Fig.
5C); after which the two sets of chromosomes separated, and two
daughter nuclei were formed (Fig.
5D). These changes in patterns of GFP fluorescence (see Movie 1 at
http://jcs.biologists.org/supplemental
), and direct observation of nuclear division in particular, allowed us to
determine the onset of anaphase unambiguously within each cell.
|
Of the cells entering mitosis, more than 90% of the wild-type and mhcA- (Fig. 5D) cells on substrates successfully divided into two daughter cells (n=20 and 40, respectively). Of 17 amiA- cells that we were able to follow throughout the mitosis process, as judged by GFP-H1 localization, 10 successfully divided within 30 minutes (59% success). This number may not be accurate, however, since we were unable to follow the majority of the mitosis events of amiA- cells in video sequences, owing to detachment of the mitotic cells from the surface and the resultant drifting. Also, detachment of the mitotic cells carried nuclei out of the focal plane, which obscured the fluorescence images of GFP-H1.
In contrast, the majority of the mononucleate
amiA-/mhcA- cells failed to complete
cell division, even though nuclear division proceeded normally and each of the
two new nuclei temporarily migrated to the respective lobes of an elongated
cell (Fig. 5E). Of 30
mononucleate amiA-/mhcA- cells that
went through nuclear division, only seven successfully divided within 30
minutes (23% success) (see also Movies 2 and 3 at
http://jcs.biologists.org/supplemental
for failed cytokinesis of mononucleate and multinucleate
amiA-/mhcA- cells). When mononucleate
or binucleate amiA-/mhcA- cells
successfully divided following nuclear division, two or four mononucleate
daughter cells were generated, respectively. This indicates that cleavage
furrows were formed between every nascent nucleus. On the other hand, when
large highly multinucleate amiA-/mhcA- cells
successfully divided following nuclear division (three out of 12 cases), only
a small number of large, unilateral furrows were formed, resulting in
generation of two or three multinucleate cell fragments
(Fig. 5F) (Movie 4 at
http://jcs.biologists.org/supplemental
) (see also Neujahr et al.,
1998). Conversely, when large multinucleate
amiA-/mhcA- cells on substrates divided,
retrospective examination of the time-lapse images demonstrated that in most
cases there were no preceding nuclear division within 30 minutes (data not
shown), and these division processes were judged to be cytokinesis C
(Fig. 5F).
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Discussion |
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To achieve that goal, we used gene disruption to generate cell lines that
were unable to carry out cytokinesis A and/or B. The mhcA-
mutation selectively inhibited cytokinesis in cells grown in suspension, which
is indicative of cytokinesis A impairment, whereas the
amiA- (Nagasaki et
al., 1998) and corA-
(de Hostos et al., 1993
)
mutations more severely affected cells cultured on substrates, which is
indicative of cytokinesis B impairment. Both amiA- and
corA- were originally cloned in Dictyostelium,
but homologs were subsequently identified in the yeast and human genomes
[coronin (Heil-Chapdelaine et al.,
1998
; Iizaka et al.,
2000
; Parente et al.,
1999
; de Hostos,
1999
), amiA/piaA (Chen
et al., 1997
; Nagasaki et al.,
1998
)]. Coronin was originally purified from precipitated
actin-myosin complexes as an actin-binding protein
(de Hostos et al., 1991
) and
was later implicated in phagocytosis on the basis of corA gene
knockout experiments (de Hostos et al.,
1993
). AmiA, also known as piaA, was cloned as a
chemotaxis-related gene (Chen et al.,
1997
; Nagasaki et al.,
1998
). Although their functions in vivo are not yet fully
understood, earlier phenotypic analyses clearly indicated that the process we
have termed cytokinesis B was impaired in knockout mutants lacking either
amiA or corA and that these genes can be used to dissect the
mechanism of cytokinesis in Dictyostelium. In addition, we engineered
and expressed GFP-H1, which allowed easy visual identification of mitotic
cells among a population of cells using fluorescence microscopy
(Fig. 5). Armed with these
tools, we set out to examine cytokinesis defects caused by disruption of
mhcA, amiA and corA, alone or in combination.
Cytokinesis in double mutants lacking myosin II and either AmiA or coronin was severely disrupted in cells grown on substrates (Fig. 3). In contrast, the phenotype of cells lacking both AmiA and coronin was no worse than those of cells lacking one or the other (Fig. 3J). The severe defects observed in mhcA-/amiA- and mhcA-/corA- cells were thus synergistic rather than additive, which strongly suggests the presence of two independent pathways leading to mitotic cytokinesis in Dictyostelium. Furthermore, the fact that mhcA-/amiA- and mhcA-/corA- cells only infrequently undergo cell-cycle-coupled division under the present experimental conditions (23% in the case of mhcA-/amiA-) indicates that the inhibition of cytokinesis B by either the amiA- or corA- mutation is fairly strong, just as the mhcA- mutation strongly inhibits cytokinesis A. That amiA- or corA- cells divide as efficiently as wild-type cells in suspension indicates that these proteins play only unessential roles, if any, in cytokinesis A.
The very low rate of cell-cycle-coupled cytokinesis in cells defective in
both cytokinesis A and B further suggests that these two are the only two
major pathways of cell-cycle-coupled division in Dictyostelium. When
these cultures become dense, however, the success rates of cell-cycle-coupled
cytokinesis increase, and consequently, the fraction of multinucleate cells
decreases. Time-lapse observation showed that when these double mutants in
dense cultures go into mitotic phase, a neighbor cell often crawls towards the
equatorial region of the mitotic cell and apparently helps the mitotic cell to
divide by walking over the equatorial region (A.N. and T.Q.P.U., unpublished).
A similar phenomenon was recently reported for another amoeba, Entamoeba
invadens (Biron et al.,
2001). The authors suggested that the Entamoeba cells
chemotax towards equatorial region of mitotic cells. We speculate that perhaps
a similar, third, cell-cycle-coupled process of cytokinesis exists in
Dictyostelium, and we are currently investigating this point.
Because cleavage furrows are formed in the equatorial regions during
telophase in both cytokinesis A and B, the positioning and timing signals of
both mechanisms must derive from the mitotic apparatus. This suggests the
possibility that cytokinesis A and B may share the same upstream regulatory
mechanism. The final stage of cytokinesis in both cases involves scission of
the cytoplasmic strand connecting the two daughter cells, another process that
may be shared by the two cytokinesis mechanisms. Most probably, the two
mechanisms diverge from one another in the middle stages of cytokinesis,
during furrow formation in particular (Fig.
6C). The present genetic evidence does not address how extensive
is the divergence between the two pathways, however. In one extreme case, AmiA
and coronin, factors essential for cytokinesis B, might complement the loss of
myosin II by providing contractile force in the equatorial region. Robinson
and Spudich (Robinson and Spudich,
2000) and Gerisch and Weber
(Gerisch and Weber, 2000
)
proposed that the equatorial region of mitotic mhcA- cells
may contract actively without myosin II. According to these hypotheses,
cytokinesis A and B are mechanistically similar, differing only in terms of
the force-generating element that constricts the furrow. Alternatively,
cytokinesis A and B might achieve furrow formation via physically distinct
mechanisms.
|
To further investigate the difference between cytokinesis A and B, we
carried out detailed morphological analyses of each mutant cell line during
cytokinesis. Mitotic mhcA- cells were flat and adhered to
the surface of the culture dish during the furrowing and separation processes.
After separation, the two daughter cells were elongated laterally rather than
longitudinally. These observations led us to speculate that division of these
cells (cytokinesis B) is achieved by passive contraction of the equatorial
region driven by radial traction forces produced along the polar peripheries
against the surface (Uyeda et al.,
2000). A similar model has been proposed by Neujahr et al.
(Neujahr et al., 1997b
) on the
basis of their observation of morphological changes in large
mhcA- cells during cytokinesis with respect to the
behavior of mitotic apparatus. In contrast, division of
amiA- or corA- cells, which are unable
to carry out cytokinesis B, is primarily dependent on active constriction of
the contractile ring powered by myosin II
(Fig. 6A; cytokinesis A). When
amiA- or corA- cells cultured on
substrates divide successfully, they usually detached from the surface and
rounded up before forming cleavage furrows
(Fig. 4C,D); the two daughter
cells remained detached and more or less spherical until they had completely
separated. These differences in the sequence of morphological changes during
cytokinesis A and B favor the possibility that the two processes are
mechanistically rather different.
This view is further substantiated by three other pieces of information.
First, AmiA and coronin are not motor proteins and are not known to activate
other motor proteins. Second, the intracellular localization of GFP-AmiA (A.N.
and K. Sutoh, unpublished) and native coronin
(de Hostos et al., 1991) is not
consistent with their primary function in the furrow region, although a more
recent analysis of cells overexpressing GFP-coronin demonstrated its presence
in the region of the equatorial furrow as well as in the polar regions
(Fukui et al., 1999
). Third, if
AmiA and coronin support active constriction of the contractile ring,
mhcA- cells would be expected to show some equatorial
furrowing even in the absence of supporting substrates. However, analysis of
mhcA- cells on non-adherent, hydrophobic surfaces clearly
demonstrated that these cells elongate axially in telophase but show no sign
of furrowing (Zang et al.,
1997
). We therefore propose that Dictyostelium cells
possess two parallel and mechanistically distinct pathways leading to
cell-cycle-coupled division. One mechanism involves active,
myosin-II-dependent constriction of the contractile ring, while the other
depends on radial traction forces generated along the polar peripheries to
indirectly cause passive constriction of the equatorial region. This leads to
re-definition of cytokinesis A and B from a general and mechanistic viewpoint:
cytokinesis A as the cell-cycle-coupled division method that is driven by
active equatorial constriction, and cytokinesis B as the cell-cycle-coupled
division method that is driven by opposing traction forces generated along the
polar peripheries. More work is needed to further examine this proposal.
Wild-type cells cultured on solid substrates have the potential to carry
out both cytokinesis A and B, which raises a question about how they
coordinate these apparently distinct processes. Neujahr et al.
(Neujahr et al., 1997b)
reported that the majority of the wild-type NC4 cells cultured on solid
surfaces rounded up and detached from the substrate during most phases of
mitosis and cytokinesis. This sequence of shape changes is very similar to
that of mitotic amiA- and corA- cells
undergoing cytokinesis A. For a minority of cells, however, division on
substrates was indistinguishable from that of mhcA- cells,
suggesting that each mitotic NC4 cell undergoes cell division either by
cytokinesis A or B. Fig. 4A
shows a mitotic cell from the AX2 cell line, which was established as an
axenic strain from NC4 cells subjected to mutagenesis
(Watts and Ashworth, 1970
) but
which are nonetheless widely used as a `wild-type' cell line. It appears that
the morphological changes in mitotic AX2 cells grown on substrates contain
features of both cytokinesis A and B and that these cells probably use both
mechanisms. Interestingly, Neuhjahr et al. (Neuhjahr et al., 1997a) discovered
that levels of myosin II in the cleavage furrows of AX2 cells flattened by
being sandwiched between a glass surface and a sheet of agarose are higher
than in the furrows of cells cultured otherwise, suggesting that these cells
are likely to employ more of the cytokinesis A pathway under physically
demanding conditions. In contrast, the fact that practically all wild-type
cells on substrates are mononucleate whereas
40% of those grown in
suspension are multinucleate suggests that cytokinesis B contributes
significantly to efficient cytokinesis of wild-type cells on substrates. This
is most likely why amiA- and corA-
cells on substrates show moderate cytokinetic defects. However, it is not
clear why the fraction of multinucleate amiA- or
corA- cells is consistently somewhat larger on substrates
than in suspension.
Cells that cannot carry out cytokinesis A or B, that is,
amiA-/mhcA- and
corA-/mhcA- cells, are still viable on solid
surfaces. How then do they multiply? For the first few days, mononucleate
amiA-/mhcA- cells inoculated onto plastic
dishes grew in size with a very low rate of cytokinesis. The resultant large,
multinucleate cells then occasionally divided through apparently two different
routes, depending on the density of cells. In dense cultures, they often
divided in a cell-cycle-coupled manner with the help of neighboring cells in a
manner similar to Entamoeba invadens
(Biron et al., 2001). In
contrast, when the cell densities were low, division of large, multinucleate
amiA-/mhcA- cells was not coupled with cell
cycle progression (Fig. 5F). If
removed from the support of a solid surface, mhcA- cells
became extremely large and highly multinucleate. When returned to a solid
surface, many of these multinucleate mhcA- cells
immediately begin to divide by forming several leading edges. Originally
called `traction-mediated cytofission'
(Spudich, 1989
), we renamed
this process cytokinesis C by redefining it as an attachment-dependent,
cell-cycle-independent division process
(Uyeda et al., 2000
). The
division sequences of multinucleate amiA-/mhcA-
or corA-/mhcA- cells are very similar to those
of giant mhcA- cells prepared by culture in suspension
(Fig. 1C, Fig. 4E,
Fig. 5F), which suggests to us
that, on substrates, these cells employ a common mechanism of division, namely
cytokinesis C. Cytokinesis C appears mechanistically similar to cytokinesis B.
However, cytokinesis B is distinct from cytokinesis C in that it requires a
number of gene products that are dispensable for cytokinesis C (reviewed by
Uyeda et al., 2000
).
Furthermore, cytoplasmic bridges connecting separating cell fragments during
cytokinesis C are often extremely long
(Fig. 1C,
Fig. 4E), whereas those
connecting daughter cells during cytokinesis B are severed before the two
cells move away from each other, suggesting that cytokinesis B has an
additional mechanism to regulate the cortical stiffness of the cytoplasmic
bridges.
We do not believe cytokinesis B is specific to Dictyostelium; it
is probably conserved in higher animal cells as well. O'Connell et al.
(O'Connell et al., 1999)
reported that microinjection of C3 toxin, which inhibits endogenous rho in
adherent, mitotic cells (normal rat kidney cells and 3T3 fibroblasts), did not
inhibit equatorial furrow formation and even induced additional ectopic
furrows, often resulting in multiple anucleate cell fragments. The equatorial
and ectopic furrows formed in C3-injected cells were wider than normal
cleavage furrows and did not contain higher levels of actin and myosin II
filaments, properties reminiscent of cleavage furrows in mhcA-
Dictyostelium cells undergoing cytokinesis B. In contrast, furrows were
not formed when C3 was injected into interphase cells; similarly
microinjection of C3 into poorly adherent HeLa cells failed to induce ectopic
furrows and, rather, inhibited cytokinesis. Thus when rho is inactivated,
mitotic cells in culture are able to carry out adhesion-dependent,
cell-cycle-coupled cytokinesis without concentrating myosin II to the furrow
regions. More recently, O'Connell et al.
(O'Connell et al., 2001
)
discovered that local application of cytochalasin D to the equatorial region
of dividing normal rat kidney cells accelerated the furrowing process instead
of inhibiting it, although its application to the polar region inhibited the
furrowing. This observation again supports the idea that contractile
activities in the equatorial region are not essential for the equatorial
furrowing and cytokinesis of these adherent cells in culture. In addition,
Zurek et al. (Zurek et al.,
1990
) observed that injection of anti-myosin antibodies into
epitheloid kidney cells, which diminished levels of myosin II in the
equatorial region, only delayed cytokinesis, and all of the injected cells
eventually divided successfully. This finding was interpreted by those
investigators to mean that there was sufficient residual myosin II in the
equatorial region to drive the cleavage slowly, but we propose an alternative
interpretation: that adherent epitheloid cells are able to divide in the
absence of myosin II, albeit more slowly than in its presence, just as
mhcA- Dictyostelium cells take two-fold longer to divide
than the wild-type cells.
This is not to say that animal cells do not in general require
myosin-II-dependent constriction of the contractile ring for successful
cytokinesis. For example, microinjection of antimyosin antibodies into
starfish eggs inhibited their division
(Mabuchi and Okuno, 1977).
This would be analogous to the failure of mhcA-
Dictyostelium cells to divide in suspension, a condition under which
cytokinesis A is the only mechanism of cell division. Another situation in
which myosin II was shown to be essential for cytokinesis in animal cells is
embryogenesis of Drosophila melanogastor
(Young et al., 1993
) and
Caenorhabditis elegans (Guo and
Kemphues, 1996
). Cells in developing animal tissues are surrounded
by other cells and the extracellular matrix and in that sense are adherent to
substrates, a condition which allows cytokinesis B in Dictyostelium.
One reason why these cells may require functional myosin II for cytokinesis
despite the presence of a substrate is that they are physically confined
within small spaces so that the daughter cells cannot tear themselves apart by
moving away from each other. This scenario is reminiscent of the observation
that mhcA- Dictyostelium cells are unable to carry out
cytokinesis B when sandwiched between a glass surface and a sheet of agarose
(Neujahr et al., 1997a
;
Yumura and Uyeda, 1997
).
Alternatively, cells in developing tissues may be inherently less motile than
cells cultured in vitro and therefore cannot generate sufficient traction
forces. Future investigation aimed at clarifying the roles of myosin II during
cytokinesis in higher animal cells should address these and other
questions.
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Acknowledgments |
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References |
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Biron, D., Libros, P., Sagi, D., Mirelman, D. and Moses, E. (2001). Asexual reproduction: `midwives' assist dividing amoebae. Nature 410,430 .[Medline]
Chen, M., Long, Y. and Devreotes, P. (1997). A
novel cytosolic regulator, Pianissimo, is required for chemoattractant
receptor and G protein-mediated activation of the 12 transmembrane domain
adenylyl cyclase in Dictyostelium. Genes Dev.
11,3218
-3231.
de Hostos, E. L. (1999). The coronin family of actin-associated proteins. Trends Cell Biol. 9, 345-350.[Medline]
de Hostos, E., Bradtke, B., Lottspeich, F., Guggenheim, R. and Gerisch, G. (1991). Coronin, an actin binding protein of Dictyostelium discoideum localized to cell surface projections, has sequence similarities to G protein beta subunits. EMBO J. 10,4097 -4104.[Abstract]
de Hostos, E. L., Rehfuess, C., Bradtke, B., Waddell, D. R., Albrecht, R., Murphy, J. and Gerisch, G. (1993). Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility. J. Cell Biol. 120,163 -173.[Abstract]
De Lozanne, A. and Spudich, J. A. (1987). Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. Science 236,1086 -1091.[Medline]
Egelhoff, T. T., Manstein, D. J. and Spudich, J. A. (1990). Complementation of myosin null mutants in Dictyostelium discoideum by direct functional selection. Dev. Biol. 137,359 -367.[Medline]
Fukui, Y., Engler, S., Inoue, S. and de Hostos, E. L. (1999). Architectural dynamics and gene replacement of coronin suggest its role in cytokinesis. Cell Motil. Cytoskeleton 42,204 -217.[Medline]
Gerisch, G. and Weber, I. (2000). Cytokinesis without myosin II. Curr. Opin. Cell Biol. 12,126 -132.[Medline]
Glotzer, M. (1997). The mechanism and control of cytokinesis. Curr. Opin. Cell Biol. 9, 815-823.[Medline]
Guo, S. and Kemphues, K. (1996). A non-muscle myosin required for embryonic polarity in Caenorhabditis elegans.Nature 382,455 -458.[Medline]
Heil-Chapdelaine, R., Tran, N. and Cooper, J. (1998). The role of Saccharomyces cerevisiae coronin in the actin and microtubule cytoskeletons. Curr. Biol. 8,1281 -1284.[Medline]
Iizaka, M., Han, H., Akashi, H., Furukawa, Y., Nakajima, Y., Sugano, S., Ogawa, M. and Nakamura, Y. (2000). Isolation and chromosomal assignment of a novel human gene, CORO1C, homologous to coronin-like actin-binding proteins. Cytogenet. Cell Genet. 88,221 -224.[Medline]
Knecht, D. A. and Loomis, W. F. (1987). Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum. Science 236,1081 -1086.[Medline]
Mabuchi, I. and Okuno, M. (1977). The effect of
myosin antibody on the division of starfish blastomeres. J. Cell
Biol. 74,251
-263.
Maniak, M., Rauchenberger, R., Albrecht, R., Murphy, J. and Gerisch, G. (1995). Coronin involved in phagocytosis: dynamics of particle-induced relocalization visualized by a green fluorescent protein Tag. Cell 83,915 -924.[Medline]
Manstein, D. J., Titus, M. A., De Lozanne, A. and Spudich, J. A. (1989). Gene replacement in Dictyostelium: generation of myosin null mutants. EMBO J. 8, 923-932.[Abstract]
Nagasaki, A., Sutoh, K., Adachi, H. and Sutoh, K. (1998). A novel Dictyostelium discoideum gene required for cAMP-dependent cell aggregation. Biochem. Biophys. Res. Comm. 244,505 -513.[Medline]
Neujahr, R., Heizer, C., Albrecht, R., Ecke, M., Schwartz, J.
M., Weber, I. and Gerisch, G. (1997a). Three-dimensional
patterns and redistribution of myosin II and actin in mitotic
Dictyostelium cells. J. Cell Biol.
139,1793
-1804.
Neujahr, R., Heizer, C. and Gerisch, G.
(1997b). Myosin II-independent processes in mitotic cells of
Dictyostelium discoideum: redistribution of the nuclei,
re-arrangement of the actin system and formation of the cleavage furrow.
J. Cell Sci. 110,123
-137.
Neujahr, R., Albrecht, R., Kohler, J., Matzner, M., Schwartz, J.
M., Westphal, M. and Gerisch, G. (1998). Microtubule-mediated
centrosome motility and the positioning of cleavage furrows in multinucleate
myosin II-null cells. J. Cell Sci.
111,1227
-1240.
O'Connell, C., Wheatley, S., Ahmed, S. and Wang, Y.
(1999). The small GTP-binding protein rho regulates cortical
activities in cultured cells during division. J. Cell
Biol. 144,305
-313.
O'Connell, C. B., Warner, A. K. and Wang, Y. (2001). Distinct roles of the equatorial and polar cortices in the cleavage of adherent cells. Curr. Biol. 11,702 -707.[Medline]
Parente, J. J., Chen, X., Zhou, C., Petropoulos, A. and Chew,
C. (1999). Isolation, cloning, and characterization of a new
mammalian coronin family member, coroninse, which is regulated within the
protein kinase C signaling pathway. J. Biol. Chem.
274,3017
-3025.
Rauchenberger, R., Hacker, U., Murphy, J., Niewohner, J. and M., M. (1997). Coronin and vacuolin identify consecutive stages of a late, actincoated endocytic compartment in Dictyostelium.Curr. Biol. 7,215 -218.[Medline]
Robinson, D. and Spudich, J. (2000). Towards a molecular understanding of cytokinesis. Trends Cell Biol. 10,228 -237.[Medline]
Ruppel, K. M., Uyeda, T. Q. and Spudich, J. A.
(1994). Role of highly conserved lysine 130 of myosin motor
domain. In vivo and in vitro characterization of site specifically mutated
myosin. J. Biol. Chem.
269,18773
-18780.
Spudich, J. A. (1989). In pursuit of myosin function. Cell Regul. 1,1 -11.[Medline]
Sussman, M. (1987). Cultivation and synchronous morphogenesis of Dictyostelium under controlled experimental conditions. In Dictyostelium discoideum: Molecular approaches to Cell Biology, vol. 28 (ed. J. A. Spudich), pp. 9-29. Orlando: Academic Press.
Uyeda, T. Q., Kitayama, C. and Yumura, S. (2000). Myosin II-independent cytokinesis in Dictyostelium: its mechanism and implications. Cell Struct. Func. 25,1 -10.[Medline]
Watts, D. and Ashworth, J. (1970). Growth of myxameobae of the cellular slime mould Dictyostelium discoideum in axenic culture. Biochem. J. 119,171 -174.[Medline]
Wolf, W. A., Chew, T. L. and Chisholm, R. L. (1999). Regulation of cytokinesis. Cell Mol. Life Sci. 55,108 -120.[Medline]
Young, P., Richman, A., Ketchum, A. and Kiehart, D. (1993). Morphogenesis in Drosophila requires nonmuscle myosin heavy chain function. Genes Dev. 7, 29-41.[Abstract]
Yumura, S. and Uyeda, T. Q. (1997). Transport
of myosin II to the equatorial region without its own motor activity in
mitotic Dictyostelium cells. Mol. Biol. Cell
8,2089
-2099.
Zada-Hames, I. M. and Ashworth, J. M. (1978). The cell cycle during the vegetative stage of Dictyostelium discoideum and its response to temperature change. J. Cell Sci. 32,1 -20.[Abstract]
Zang, J. H., Cavet, G., Sabry, J. H., Wagner, P., Moores, S. L.
and Spudich, J. A. (1997). On the role of myosin-II in
cytokinesis: division of Dictyostelium cells under adhesive and
nonadhesive conditions. Mol. Biol. Cell
8,2617
-2629.
Zurek, B., Sanger, J. M., Sanger, J. W. and Jockusch, B. M. (1990). Differential effects of myosin-antibody complexes on contractile rings and circumferential belts in epitheloid cells. J. Cell Sci. 97,297 -306.[Abstract]