* Medical Research Council Laboratory for Molecular Cell Biology, Department of Physiology, and § Department of Molecular
Medicine, University College London, London WC1E 6BT, United Kingdom; and
Department of Microbiology and
Immunology and Department of Medical Genetics, University of British Columbia, Vancouver V6T 1Z3, Canada
RasG is the most abundant Ras protein in
growing Dictyostelium cells and the closest relative of
mammalian Ras proteins. We have generated null mutants in which expression of RasG is completely abolished. Unexpectedly, RasG cells are able to grow at
nearly wild-type rates. However, they exhibit defective
cell movement and a wide range of defects in the control of the actin cytoskeleton, including a loss of cell polarity, absence of normal lamellipodia, formation of unusual small, punctate polymerized actin structures, and
a large number of abnormally long filopodia. Despite
their lack of polarity and abnormal cytoskeleton, mutant cells perform normal chemotaxis. However, rasG
cells are unable to perform normal cytokinesis, becoming multinucleate when grown in suspension culture.
Taken together, these data suggest a principal role for
RasG in coordination of cell movement and control of
the cytoskeleton.
RAS proteins are a family of small GTPases that control a range of fundamental processes in all known
eukaryotic cells. Ras was first discovered in an activated form as the product of a viral oncogene (Shih et al.,
1979 Ras proteins are active when GTP is bound and become
inactive by hydrolyzing this GTP to GDP. Two families of
proteins regulate Ras activity by controlling the bound nucleotide. Guanine nucleotide exchange factors (GEFs)1
such as CDC25 and Sos activate Ras by allowing GDP to
dissociate and be replaced by GTP. GTPase-activating proteins (GAPs), on the other hand, inactivate Ras by binding
to the active form and stimulating the hydrolysis of GTP
to GDP (Boguski and McCormick, 1993 The work described in this paper suggests a connection
between Ras proteins and cytokinesis. Correct cell division
involves two distinct processes: nuclear division (karyokinesis) and then partitioning of the cytoplasm and organelles
(cytokinesis; Rappaport, 1986 The first cytokinesis mutants to be isolated in Dictyostelium, and consequently the most extensively studied mutants, are defective in cytoskeletal proteins, for example,
myosin II heavy chain (DeLozanne and Spudich, 1987)
and coronin (de Hostos et al., 1993 Dictyostelium possesses an unusual, extended family of
ras genes (Daniel et al., 1995 Aside from their different patterns of expression, little is
known about the functions of Dictyostelium Ras proteins.
Overexpression of an activated form of RasD causes aberrant signaling and developmental arrest in aggregates (Reymond et al., 1986 Unless otherwise indicated, all chemicals were obtained from Sigma Chemical Company (St. Louis, MO) and all enzymes from New England Biolabs (Beverly, MA).
Cell Strains, Growth, and Transformation
Dictyostelium discoideum AX2 cells were maintained at 22°C in HL-5
medium (Sussman, 1987 Molecular Biology
The rasG knockout vector was constructed by inserting a version of the
pBsr Western Blotting
rasG Microscopy
Cells from axenic culture were seeded onto acid-washed coverslips and
fixed with 1% glutaraldehyde, 0.1% Triton X-100 in KK2 buffer (20 mM
potassium phosphate, pH 6.2, 2 mM MgCl2) for 10 min. Autofluorescence
was quenched using 5 mg/ml NaBH4 for 10 min. Actin filaments were
stained with 100 nM Rhodamine- or Texas red-conjugated phalloidin
(Sigma Chemical Co. and Molecular Probes, Inc., Eugene, OR, respectively) and nuclei with 500 nM Hoechst 33258 or 33342. Cells were observed using an epifluorescence microscope (Axiophot; Zeiss, Inc., Thornwood, NY) or a scanning confocal microscope (MRC1024; Bio Rad,
Hercules, CA). Images were recorded with a Hamamatsu CCD camera or
on Kodak Ektachrome 400 film, and digitized with an Agfa Arcus II scanner in conjunction with Adobe Photoshop 3.0 software.
Cells were synchronized to observe cytokinesis using aphidicolin (Pedrali-Noy et al., 1987). The medium of axenically growing cells was supplemented with 10 µg/ml aphidicolin (final concentration) for 16 h. The
aphidicolin was removed with three washes of fresh axenic medium and
the cells allowed to continue through the cell cycle. At the first signs of mitotic activity (usually about 5 h after release) cells were fixed as described
above.
Time-lapse video microscopy of cells was used to observe cytokinesis.
Cells growing in petri dishes were synchronized as above, and a time-lapse
video recorder (Betacam; Sony, Inc., Tokyo, Japan) was used to record
phase contrast images collected by a CCD camera (Sony, Inc.) from an inverted microscope (Zeiss, Inc.).
To observe traction-mediated cytofission, rasG For the effects of azide, axenically growing cells were seeded onto coverslips and perfused with KK2 buffer for 5 min using a perfusion chamber
as described in Devreotes et al. (1987) To measure speed and persistence, cells were followed in a perfusion
chamber, and images of several cells were recorded every minute. Images
were grabbed as above, and the position of the centroids of several cells
calculated using NIH Image 1.60 software and expressed as an x,y coordinate. The coordinates were used to calculate the velocity and persistence
of each cell as described below.
Firstly, the length of the path between successive centroids was taken
as the instantaneous velocity of each cell. Secondly, the cosine of the angle
of the path between centroids was used as a measure of the persistence of
movement of a cell. A cell moving in a constant direction will produce a
path with angle 0° and therefore a cosine of one, while a cell changing direction at 90° will produce a cosine value of zero.
At least eight cells were followed for at least 20 min, and the velocities
and persistences were pooled and used to give mean values.
Chemotaxis Assay
Bacterially growing cells were washed with KK2 buffer to remove bacteria. Cells were seeded onto coverslips and submerged in KK2 buffer. A
micropipette filled with 10 µM folic acid in KK2 was placed just above the
coverslip and pressurized to expel the folic acid. The assay was performed
with the same pipette for both rasG Disruption of the Dictyostelium rasG Gene
Cells containing a disrupted rasG gene were generated by
homologous recombination by the strategy shown in Fig.
1 A. A construct was made containing 1.9 kb of rasG genomic DNA (Robbins et al., 1992
Growth of rasG Unexpectedly, rasG appears to be dispensable for growth
under most conditions. As shown in Fig. 2 A, rasG
When cells are grown in shaken suspension, however, a
different result is seen (Fig. 2 B). rasG Retransfection of rasG Aberrant Morphology and Adhesion in rasG Mutants
Observed by phase contrast microscopy, rasG
Table I.
Speed and Persistence Measurements for Wild-type
and rasG). It has since been found to be a central regulator of
mammalian cell growth and division, differentiation, shape,
and motility (for reviews see Sorcher et al., 1993
; Valencia
and Sander, 1995
). Distinct roles for Ras have been found
in several organisms. In particular, Ras proteins mediate
signaling through receptors such as the Sevenless protein in Drosophila (Fortini et al., 1992
), and a Ras homologue
(the let-60 gene product) is required for the specification
of the vulval cells of Caenorhabditis elegans (Han and
Sternberg, 1990
). The RAS1 and RAS2 genes of Saccharomyces control cell growth by regulating the activity of adenylyl cyclase (Toda et al., 1985
) and may also influence the
cytoskeleton through the CAP protein (Wang et al., 1993
).
Relatives of Ras proteins in the Rac and Rho subfamilies
act downstream of Ras; again, they appear to control both
growth and motility (Ridley et al., 1992
). Rac and Rho
seem to control separate aspects of cell architecture, with
Rac and its relative Cdc42 controlling actin-rich protrusions (ruffles and filopodia, respectively) and Rho regulating the formation of actomyosin bundles known as stress
fibers (Ridley et al., 1992
; Nobes and Hall, 1995
).
). Both may be
controlled by different stimuli. In mammalian cells, the
binding of receptor tyrosine kinases to their ligands can
cause recruitment of both GEFs and GAPs to the membrane through a family of adaptor proteins such as Grb2
and Shc (Lowenstein et al., 1992
; Pelicci et al., 1992
); the
interaction between Drosophila Sevenless and Boss proteins is transmitted to Ras through the Grb2 homologue
Drk (Olivier et al., 1993
). In yeast, the signals that regulate
Ras activity through CDC25 and IRA1&2 are not yet understood.
). Karyokinesis is mainly accomplished by the microtubules that form the spindle,
whereas cytokinesis is apparently based around actin and
myosin II (classical, double-headed myosin). Soon after
karyokinesis, a concentration of polymerized actin (F-actin)
is visible at the equator of the cell, and a cleavage furrow
containing myosin II forms which pinches the daughter
cell in two (Fishkind and Wang, 1995
). Mutants in several
species affected in myosin II function lose the ability to
perform cytokinesis properly, despite apparently normal
karyokinesis (De Lozanne and Spudich, 1987
; Karess et
al., 1991
). The simple eukaryote Dictyostelium discoideum
has recently proved to be an excellent subject for the study
of cytokinesis, in particular because the cells possess an alternative method of partitioning cell contents when normal partition cannot take place (De Lozanne and Spudich,
1987
). This process, which has been named "traction-mediated cytofission" (Fukui et al., 1990
), allows the survival of
mutants with strong cytokinesis phenotypes, which would
be inviable in other systems. Dictyostelium also offers relatively simple gene disruption by homologous recombination, and is a much-studied target for analysis of cytoskeletal proteins.
). However, several recent reports have identified two GAP proteins of unknown
substrate specificity (Faix and Dittrich, 1996
; Lee et al.,
1997
) and the small GTP-binding protein RacE (Larochelle,
1996) as putative regulators of cytokinesis. Neither GAP
proteins nor RacE are likely to be directly involved in the physical separation of the daughter cells; instead they must
lie on the signaling pathways controlling the process.
). Two of the products (RasG
and RasD; Reymond et al., 1984
; Robbins et al., 1989
) are
closely related to mammalian Ras proteins (68% and 65%
overall identity to human H-ras, respectively), whereas the
RasB, RasC, and RasS gene products (Daniel et al., 1993
,
1994
) are more divergent (though still clearly members of
the Ras subfamily). Dictyostelium cells grow unicellularly but aggregate and form a multicellular fruiting body upon
starvation. The rasD and rasS genes are only expressed
during multicellular development (Reymond et al., 1984
;
Daniel et al., 1994
). RasD, furthermore, is expressed at
higher levels in the stalk cell precursors than the prespore
cells. The gene products are therefore presumed to have a
function specifically connected with aggregation or cell-type differentiation. RasG, RasB, and RasC are expressed during growth (Robbins et al., 1989
; Daniel et al., 1993
).
RasG mRNA expression ceases as soon as multicellular
development begins, and RasG protein is lost from the
cells during development, suggesting a specific requirement during growth (Khosla et al., 1990
, 1996
).
). Similar activating mutants of RasG cause
a block in aggregation (Khosla et al., 1996
) and cytoskeletal changes (Rebstein et al., 1997
). Neither of these lines
has allowed elucidation of the function of the Ras proteins in normal Dictyostelium cells. In this work we report the
disruption of the gene that encodes RasG; disruptants
show a range of phenotypes based around the control of
the actin cytoskeleton, in particular during cytokinesis. This
suggests that one major role of RasG is to control cell architecture, rather than growth or differentiation.
Materials and Methods
). For bacterially grown cells, SM plates were inoculated with 105-106 Dictyostelium cells plus 200 µl of a suspension of
Klebsiella aerogenes in L-broth. Transformation was performed by a modification of Howard et al. (1988)
; briefly, cells in log phase growth were mixed with 25 µg of linearized DNA and electroporated at 1.0 or 1.1 mV,
3 µF with a 5
resistance in series. After 10 min incubation on ice, cells
were warmed for 15 min in the presence of 2 µl healing solution (100 mM
MgCl2, 100 mM CaCl2), and then HL-5 was added. Blasticidin (ICN, Irvine,
CA) or G418 (GIBCO BRL, Gaithersburg, MD) at 10 µg/ml final concentration were added 24 h after electroporation. Transformants appearing after 4-5 d were cloned on lawns of Klebsiella growing on SM agar plates.
Bam marker (Adachi et al., 1994
) with BamHI linkers into the single BglII site of pRASG (Robbins et al., 1992
). Transformants in which
the marker was oriented in the same direction as the rasG gene were selected to ensure the presence of a terminator between the actin promoter
and the rasG coding sequence. From the resulting vector a 3.2-kb fragment was digested with EcoRI and HindIII restriction enzymes and used
to transform Dictyostelium strain AX2 as described above. To analyze
clones, AX2 or rasG
genomic DNA prepared by the method of Sun et al.
(1990)
was digested with HindIII and EcoRI, blotted onto nylon membrane (Qiagen Inc., Chatsworth, CA), and probed with the [
-32P]dATP
labeled BglII-HindIII fragment from the rasG-coding sequence (Robbins
et al., 1989
) as described in Sambrook et al. (1989)
.
+ AX2 cell lysates were separated by SDS-PAGE and blotted onto
0.45 µm PVDF membrane (Amersham Life Science, Pittsburgh, PA) by
standard procedures (Sambrook et al., 1989
). The membrane was probed
with rat
-Ras mAb Y13-259 (Oncogene Science, Mineola, NY) at 10 µg/ml
final concentration, or with a polyclonal antibody to RasG as described in
Khosla et al. (1994)
. Secondary HRP-conjugated anti-rat antibody or anti-
rabbit antibody (Pierce, Rockford, IL) was used at 1:2,500 dilution. The signal was detected using an enhanced chemiluminescence kit (Amersham
Corp., Arlington Heights, IL).
cells were grown for
5 d in shaking flasks and transferred to tissue culture plates. Phase-contrast photomicrographs were taken using an inverted microscope (Zeiss,
Inc.) and Kodak T-Max 100 film. Alternatively, electronic images were obtained using a Panasonic video camera and a frame grabber connected to
a Macintosh computer running NIH Image 1.60 software.
. The buffer was replaced with
0.01% sodium azide in KK2 and an inverted microscope (Zeiss, Inc.) with
phase contrast optics used to observe adhering cells.
and wild-type cells.
Results
), with a bsr marker (Sutoh, 1993
) inserted between the promoter and coding sequence of the gene, in the same orientation so the strong
act8 terminator blocked any read-through from the rasG or bsr promoters. The construct was transfected into AX2
cells, and transformants were cloned after 7 d of blasticidin
selection. 2 independent clones, out of 74 examined, were
found to contain a disruption in rasG (Fig. 1 B), with the
rest apparently containing nonhomologous integration of
the vector. Both independent lines were found to behave
similarly, so one (IR15) was used for all the work described here. When Western blots are probed with the broad-spectrum Ras antibody Y13-259 (Furth et al., 1982
),
disrupted cells show approximately half the wild-type level
of Ras proteins (Fig. 1 C, left). Y13-259 detects the diverged RasB and RasC proteins, albeit less well than RasG
or RasD. The Ras protein seen in IR15 therefore presumably derives from the rasB and rasC genes (which are expressed at lower levels than rasG in wild-type cells) or rasD (which is barely expressed in growing wild-type cells).
No RasG protein is found in disruptants when a specific
RasG antibody is used (Fig. 1 C, right).
Fig. 1.
Disruption of the
rasG gene. (a) Schematic
representation of the cloning
strategy employed to disrupt
the rasG gene. A 1.7-kb fragment encoding the cDNA for
the blasticidin resistance
gene (bsr) driven by the constitutive actin15 promoter
was inserted by homologous
recombination into the rasG
promoter between the promoter and the ATG start
codon. A probe from the
rasG coding sequence
(shaded bar) was used to detect correct disruptants by Southern blotting of genomic
DNA. The expected bands in
the parental strain and disruptants are indicated by
dotted lines. (b) Southern blot of rasG and wild-type
parental genomic DNA. Nuclear DNA from strains IR15
(rasG
) and AX2 (wt) was
digested with EcoRI and
HindIII, separated on an
0.8% agarose gel, blotted
onto nylon, and probed with
the rasG coding sequence
(see above). The 1.9-kb parental band and 3.2-kb rasG
disrupted band are marked. (C) Western blot of rasG
and AX2 wild-type cells. Whole cell lysates were separated by PAGE using a
15% acrylamide gel, blotted onto PVDF, and probed with the general Ras antibody Y13-259 (left) and a RasG specific antibody (right).
Y13-259 recognizes several different Dictyostelium Ras proteins with varying efficiency.
[View Larger Versions of these Images (12 + 41 + 62K GIF file)]
Cells
cells
in liquid medium grow only slightly more slowly than wild-type (14.5 vs 13.4 h doubling time). rasG
cells also grow
somewhat more slowly than wild-type on bacteria, forming
colonies that are a little smaller than AX2 (data not shown).
Fig. 2.
Growth of wild-type and rasG cells. (a) Growth on
surfaces. Wild-type (
), rasG
(
), and rasG
cells rescued by
expression of rasG (
) were seeded in Petri plates (105 cells/
plate) with 10 ml axenic medium. At intervals the cells were detached from the surface, and the number of cells in a small aliquot was counted. (b) Growth in suspension culture. Wild-type (
), rasG
(
), and rasG
cells rescued by expression of rasG
(
) were transferred from Petri plates into axenic medium at
time zero and counted at intervals thereafter.
[View Larger Versions of these Images (19 + 19K GIF file)]
cells double far
more slowly than wild-type (27 vs 15 h doubling time), and
cease growing at a considerably lower cell density (Fig. 2
B). This weak growth in suspension culture, despite nearly normal growth on surfaces, is reminiscent of the phenotype of mhcA
cells (De Lozanne and Spudich, 1987
),
which do not produce myosin II, and therefore cannot perform cytokinesis. The role of rasG in cytokinesis was examined further, as described below.
cells with a genomic copy of the
rasG gene and promoter reverses both growth phenotypes, allowing a normal doubling time whether cells are
grown on plates, in shaken suspension (Fig. 2, A and B), or
on bacteria (data not shown). The mutant phenotypes are
therefore solely due to a lack of RasG.
cells differ
from wild-type in several respects. Cells grown on bacteria
(which behave more consistently than axenically grown
cells) and then allowed to migrate on a glass coverslip (Fig.
3 A) are far more flattened than wild-type and adhere
more tightly. They are also less polar; most wild-type cells
possess a clear orientation, whereas rasG
cells are nearly
all circular (Fig. 3 A). When the motion of cells was recorded by video microscopy, rasG
cells travelled considerably slower than wild-type (64% of the mean speed; Table I). Two aspects of the motion of rasG
cells are
surprising. The defect in polarization of rasG
cells would
suggest that they are unable to maintain a specific direction, yet they move with nearly the same persistence (a
measure of the cell's ability to move in a constant direction) as wild-type (Table I). Secondly, wild-type cells alternate between polarized and rounded morphologies, with
nearly all movement taking place in the polar phase.
rasG
cells seem to move nearly as fast when rounded as
when polar, which explains how they are able to move at
as much as 64% of wild-type speeds despite the majority
being apparently unpolarized.
Fig. 3.
Morphology of wild-type and rasG cells. (a) Phase-contrast micrographs of cells adhering to glass. Wild-type cells (top) show a rounded and polarized morphology, while rasG
cells (bottom) are more flattened and nonpolar. (b and c) Scanning confocal micrographs of cells stained with rhodamine-phalloidin to visualize F-actin. (b) Moving wild-type cells (top) and rasG
cells (bottom). (c) Stationary wild-type (top) and rasG
cells (bottom). Bar: (a) 20 µm; (b and c) 10 µm.
[View Larger Versions of these Images (125 + 74 + 98K GIF file)]
Cells
The distribution of F-actin is also aberrant in axenically
grown rasG cells. Staining with fluorescent phalloidin
(Fig. 3 B) reveals two major differences between rasG
and wild-type. Firstly, the lamellipodia seen at the leading edges of polarized wild-type cells are replaced by large
numbers of elongated filopodia. While similar filopodia
are seen in a proportion of wild-type cells (~10-15%; data
not shown), they are present in nearly all rasG
cells, frequently in large numbers, often reaching considerably greater lengths than in wild-type cells (Fig. 3 B). Again,
nearly all rasG
cells show no obvious polarity; the filopodia appear to be spread randomly around the perimeter of
most cells. Wild-type cells in the rounded phase frequently
show a continuous, broad cortex of F-actin, with no actin-rich protrusions; equivalent rasG
cells exhibit a similar
cortex, but usually also possess long filopodia (Fig. 3 C).
Various highly unusual morphologies are also common in
rasG
cells, in particular crescent and hour glass shapes
(Fig. 4). In these cells the cortex, rather than being continuous, is divided into discrete F-actin-rich lobes, as if
the different ends of the cell were trying to move in opposite directions. The nuclei are usually found between the
lobes of F-actin, and the two halves of the cells pulling in
opposite directions can squeeze the nuclei into cylindrical
shapes.
A second unusual feature of rasG cells is the abundance of small, punctate F-actin structures within the cell
body. These are conspicuously present in the majority of
cells (Figs. 3, B and C, and 4; particularly clear actin structures are also visible in Fig. 6 A). Similar structures are
seen in a small proportion of wild-type cells, but in lower
numbers (1-2 per cell; data not shown). Three-dimensional reconstruction of confocal Z-series indicates that
the structures are roughly spherical and are localized to
the cortex of the base and the upper surface of the cells
(data not shown; a QuickTime video showing an example
of a three-dimensional reconstruction is available on the
World Wide Web at http://www.ucl.ac.uk/~dmcbrob/movies.html). The precise nature of these structures is as
yet unknown. Mutant and wild-type cells contain similar
quantities of F-actin per cell (data not shown); the differences appear to reflect a failure of organization, rather
than an inability to polymerize actin.
Normal Chemotaxis in rasG Cells
Chemotaxis up chemical gradients involves both cell polarity and changes in the actin cytoskeleton. Since both the
cell polarity and cytoskeleton of the rasG cells are aberrant (Fig. 3), and in the light of the observation that the
disruption of the Aimless RasGEF demonstrates a requirement for a ras pathway for Dictyostelium chemotaxis
(Insall et al., 1996
), we measured chemotaxis of rasG
cells towards folic acid, which is used by growing cells to
detect bacteria. As documented in Fig. 5, rasG
cells are
almost equal to wild-type in their ability to migrate towards a micropipette filled with folic acid. Considering
their impaired cytoskeletal structure and slow movement,
they perform chemotaxis surprisingly successfully.
Cytokinesis and Cell Fission
The poor growth of rasG cells in shaken suspension suggests a defect in cytokinesis. When cells are transferred
from growth on petri plates, where they maintain a wild-type number of nuclei (data not shown), to shaken culture,
they rapidly become multinucleate (Fig. 6 A). Within 3 d
of shaken growth, the majority of cells has several nuclei,
and many have >10. Multinucleate cells the size of the one
shown in Fig. 6 A are common, and considerably larger examples are sometimes observed. After 6 or 7 d of growth in shaken suspension, cells become so large that they lyse,
presumably from the shear forces generated by shaking.
When multinucleate cells are allowed to adhere to a surface such as a glass coverslip, they rapidly tear themselves
into mononucleate fragments. The single cell shown in the
process of splitting in Fig. 6 B had divided into at least 11 daughters within 30 min of plating on a surface. This process, which has been named traction-mediated cytofission
(Fukui et al., 1990
), appears not to greatly harm the cells,
and is the mechanism which allows Dictyostelium cytokinesis mutants to divide at nearly normal rates on a surface
(a QuickTime video showing a rasG
cell splitting by traction-mediated cytofission after growth in suspension culture can be viewed at the World Wide Web site http://www.ucl.ac.uk/~dmcbrob/movies.html).
Nearly all the cytokinesis mutants that have been previously described in Dictyostelium have defects in the structural proteins of the cytoskeleton. The most comprehensively studied mutants lack myosin II function (De
Lozanne and Spudich, 1987; Knecht and Loomis, 1987
;
Chen et al., 1995
). These cells are unable to complete cytokinesis because of an inability to generate the contractile ring which pinches the daughter cells apart. The phenotype of rasG disruptants is not caused by a lack of myosin
II activity. Treatment of cells with sodium azide causes a
rapid depletion of cellular ATP levels, which causes myosin II to bind irreversibly to actin (Patterson and Spudich,
1995
). When wild-type cells in a perfusion chamber are
treated with 0.01% sodium azide, they round up and are
washed off the substratum. mhcA
cells, which lack the
heavy chain of myosin II, remain flattened and stuck
down. Azide treatment caused nearly normal rounding and loss of rasG
cells (Fig. 7). The small number of mutant cells that did not wash away had rounded up in the
same way as wild-type cells but were still adhering to the
glass coverslip. This seems to be caused by the aberrant
adhesion of rasG
cells rather than any lack of myosin II
function.
We wished to discern whether rasG cells were unable
to perform cytokinesis because of a mechanical defect preventing the physical separation of the daughter cells, like
that in mhcA
cells, or because of a problem with the initiation of cytokinesis due to the loss of a signal communicating the completion of nuclear division to the cytoplasm. To
answer these questions, synchronized cells in mitosis were
required, but we found existing methods of synchronizing
Dictyostelium cells unsatisfactory. By releasing cells whose
cell cycle progression was blocked by aphidicolin (an inhibitor of DNA polymerase; Pedrali-Noy et al., 1980
), we observed good synchrony. 50% of cells passed through mitosis ~4 h after release. When synchronized wild-type cells
are observed by time-lapse video microscopy, cytokinesis
is observed as a rounding of the cell followed by a rapid
pinching movement, taking ~5 min to complete (Fig. 8 A,
top rows). The initial stages of cytokinesis in rasG
cells
appear indistinguishable from those in wild-type cells (Fig.
8 A, lower rows). However, the daughter cells appear unable to separate completely and remain attached by a thin
bridge of cytoplasm, which is eventually resolved by traction between the daughter cells (Fig. 8 A).
Wild-type cells that are fixed during cytokinesis and
stained for F-actin and chromosomes show a characteristic
binucleate hour-glass shape with the chromosomes condensed throughout the process (Fig. 8 B, top rows). Again,
with fixed cells the initial stages of cytokinesis in rasG
cells appear identical to those in wild-type cells (Fig. 8 B, lower rows), but the bridge of cytoplasm that results from
the failure of the rasG
daughter cells to separate is shown
clearly. The chromosomes of the two daughters decondense (Fig. 8 B, lower right image) indicating that the cells
can remain attached for some time. In contrast, wild-type
cells show condensed nuclei even as the daughters separate completely (Fig. 8 B, upper right image). Staining with an anti-tubulin antibody indicates that the microtubules
also return to an interphase morphology before complete
separation in the rasG
cells but remain in a mitotic morphology throughout cytokinesis in wild-type cells (data not
shown).
The failure of rasG cells late in cytokinesis suggests
that they have a mechanical defect. RasG does not appear
to be required for the spatial or temporal specification of
the cleavage furrow. As demonstrated by Fig. 7, myosin II
activity is present in rasG
cells, but RasG may be required for the correct regulation of myosin II or other cytoskeletal proteins during cytokinesis. Alternatively, RasG
may be required for the correct regulation of actin polymerization during cytokinesis with the result that an aberrant F-actin framework is established in the cleavage furrow that prevents the necessary contractile forces being
generated by myosin II and other cytoskeletal proteins.
The most obvious phenotype of rasG mutant cells is, unexpectedly, a defect in cytokinesis. Cells also exhibit several other cytoskeletal defects, including alterations in cell
polarity, morphology, motility, and F-actin structures. The
slightly reduced growth rate of mutants is most probably
due to the defects in motility and cytokinesis, although we
cannot rule out other minor difficulties. Clearly rasG is not
essential for growth. These results could be interpreted in
two ways. Firstly, RasG may be concerned solely with the
regulation of motility, while differentiation and growth
control are mediated through other Ras proteins, or not by
a Ras at all. Alternatively, RasG could be important for the control of a wide range of cellular processes, just like
its mammalian homologues, but the phenotype of rasG
disruption can be moderated by partial functional redundancy. If the latter is true, RasG might normally be central
to regulation of growth but be substituted by RasB, RasC,
or ectopically expressed RasD in rasG
cells. RasD is highly
homologous to RasG: 100% identical in the effector and
effector-proximal domains and 82% identical over the entire length (Daniel et al., 1995
), and therefore might be expected to substitute for some of the functions of RasG if
expressed during growth. The low recovery of homologous
recombinants compared to random integrants might suggest a central role for RasG, but it is notoriously hard to
predict the efficiency of homologous integration. On the
other hand, the phenotypes observed after overexpression
of mutant forms of RasG are mainly concerned with cell
morphology. Cells containing an activated RasG appear
flattened, while those transformed with inhibitory mutants
are polarized (Rebstein et al., 1997
). Whichever explanation is true, cytokinesis and actin organization appear to
be the only processes that cannot survive rasG disruption.
Ras and Actin
The role for Ras proteins in motility has been observed in
a range of species, though until recently it has been overshadowed by the importance of growth control. Ras activation leads to enhanced motility of endothelial cells (Fox
et al., 1994) and apparently mediates the chemotactic response of fibroblasts to PDGF (Kundra et al., 1995
). When
the genes encoding RasGAP and/or NF1 are disrupted in
mice, one of the causes of embryonic lethality is malformation of blood vessels due to hypermotile endothelial cells
(Henkemeyer et al., 1995
). Likewise, Saccharomyces CDC42,
which controls actin polymerization during bud formation,
is regulated through Ras (Mosch et al., 1996
).
In Dictyostelium rasG cells, all of the phenotypes we
observe are connected with the control of the actin cytoskeleton. The lamellipodia at the leading edge of wild-type
cells, and the filopodia found in rasG
cells, are both transitory structures made of freshly polymerized actin (Hall
et al., 1988
), and the cleavage furrow is also actomyosin rich
(Fukui and Inoue, 1991
). The absolute quantity of F-actin in rasG
cells is normal, though the part played by the unusual punctate actin structures is unknown.
A range of experiments has shown that small GTPases
such as Rac and Cdc42 proteins are controlled by Ras
pathways (Nobes and Hall, 1995). Dictyostelium contains a
large number of rac homologues (Bush et al., 1993
), so the
different effects of RasG on the cytoskeleton and motility
could be transmitted through several Rac proteins with
separate roles. The specific role of the racE gene in cytokinesis (Larochelle et al., 1996
), described below, is a possible example. Another known effector of Ras proteins is
p110 PI 3-kinase (Rodriguez-Viciana et al., 1994
), which
may constitute the direct connection between Ras and Rac
proteins. Three different p110 homologues have recently been cloned in Dictyostelium (Zhou et al., 1995
), and it is
significant that mutants also show defects in the control of
the cytoskeleton. We look forward to observing the effects
of rasG disruption on PI 3-kinase and Rac activity.
Ras and Cytokinesis
Ras proteins in other organisms have been associated with
both control of the cytoskeleton and cell proliferation but
not usually with cytokinesis. However, since in other systems Ras is essential for cell growth, and therefore nuclear
division, its role in the division of the cytoplasm thereafter
has been difficult to study. Despite this, there are several
reports connecting Ras activity with cytokinesis. Schizosaccharomyces byr4, a suppressor of ras1 mutations (Song
et al., 1996), appears to encode a regulated inhibitor of cytokinesis. Furthermore, activating Ras mutants increase
the rate of cytokinesis in mammalian cells (Ng et al., 1992
).
There is also an increasing amount of evidence that the small GTPases Rac, Rho, and Cdc42 are centrally involved in cytokinesis (Dutartre et al., 1996
), and Ras appears to act as an upstream regulator of their activities. It
is therefore likely that the central role of RasG in Dictyostelium cytokinesis is mirrored in other organisms.
There are several likely candidates for proteins downstream of RasG. Interestingly, the disruption of one GAP
gene (Lee et al., 1997) and overexpression of the other
(Faix and Dittrich, 1996
) both result in deficiencies in cytokinesis, as does overexpression of activating RasG mutants (Rebstein et al., 1997
). This may reflect a need for
limited, local activation of RasG during cytokinesis. RasG
activation at an inappropriate place or time could be just as harmful to efficient cytokinesis as the complete lack of
RasG in disrupted cells. The protein encoded by the racE
gene is required for cytokinesis (Larochelle et al., 1996
);
unlike rasG
cells, however, racE
mutants appear normal in all other respects, including the formation of lamellipodia, adhesion, and rate of motility. Again, since Rac
proteins in other systems can be activated by Ras, a direct connection between RasG and RacE seems possible. Similarly, cells containing concurrent deletions in two PI 3-kinase
genes (pik1 and pik2) lose the ability to grow in shaken
culture (Zhou et al., 1995
), despite nearly normal growth
on surfaces, which suggests that they have similar defects
in cytokinesis to rasG
cells. Alternatively, the defect in
cytokinesis in rasG
cells may be indirect, caused by the
general disruption of the actin cytoskeleton rather than
the loss of a specific signaling pathway.
Like the ras genes themselves, the biochemical pathways between Ras and the cytoskeleton are apparently conserved between Dictyostelium and mammalian cells. This again suggests that cytokinesis in mammalian cells may turn out to be affected by Ras as it is in Dictyostelium discoideum. The fascinating prospect is that Ras proteins through their involvement in the signaling pathways that commit cells to division and by regulating the cytoskeleton during cytokinesis are major players at the start and at the end of the cell cycle.
Received for publication 28 February 1997 and in revised form 5 May 1997.
1. Abbreviations used in this paper: GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factors.We thank Dr. David Drechsel for helpful discussions and Drs. Jeff Williams and Adrian Harwood for constructive comments on the text.
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