Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Avenue, Lawrence, KS 66045-7534, USA
* Author for correspondence (e-mail: erikl{at}ku.edu)
Accepted 21 November 2002
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SUMMARY |
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Key words: Rac, UNC-115, Actin-binding protein, Axon pathfinding
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INTRODUCTION |
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Like other Ras superfamily GTPases, Racs cycle between a GTP-bound active
state and a GDP-bound inactive state
(Bourne et al., 1991).
Molecules that regulate Rho GTPase activity include GTP exchange factors
(GEFs), which stimulate exchange of GDP for GTP, promoting the active state
(Kaibuchi et al., 1999
). A
number of dbl-homology (DH) GEFs have been implicated in controlling Rac
activity, including UNC-73 Trio (Bellanger
et al., 1998
; Steven et al.,
1998
), which acts with each of the three Racs in C.
elegans and Drosophila axon pathfinding in vivo
(Awasaki et al., 2000
;
Bateman et al., 2000
;
Liebl et al., 2000
;
Lundquist et al., 2001
;
Newsome et al., 2000
).
In the developing nervous system, growth cones, which are dynamic
sensorimotor structures at the distal tip of extending axons, sense and
respond to extracellular guidance cues by modulating their actin
cytoskeletons, resulting in changes in direction of outgrowth
(Tessier-Lavigne and Goodman,
1996). Growth cones are composed of lamellipodia and filopodia,
which are dynamic, actin-based plasma membrane extensions that underlie
outgrowth and guidance (Letourneau,
1996
). Rac GTPases control cell shape at least in part by
regulating the structure and dynamics of the actin cytoskeleton
(Hall, 1998
) and Rac activity
can induce lamellipodia in cultured cells
(Ridley et al., 1992
). Recent
studies have identified a number of downstream Rac effector molecules that
adapt Rac activity to the actin cytoskeleton
(Bishop and Hall, 2000
), some
of which involve activation of the actin-nucleating Arp2/3 complex. For
example, Rac interacts with the adapter protein IRSp53 which in turn links the
Arp2/3 potentiator WAVE2 (Takenawa and
Miki, 2001
), and Rac1 along with the adapter protein Nck acitvate
the Arp2/3 potentiator Scar/WAVE (Eden et
al., 2002
). Furthermore, the actin-interacting protein cortactin,
also an Arp2/3 activator (Weaver et al.,
2001
), has been implicated as downstream Rac effector
(Weed et al., 1998
). Distinct
mechanisms involve Rac interaction with p21-activated kinase, which in turn
can influence the cytoskeletal effector merlin
(Kissil et al., 2002
;
Xiao et al., 2002
), and
potential Rac regulation of the actin-binding protein gelsolin
(Azuma et al., 1998
). Thus, Rac
activity can influence the actin cytoskeleton by a variety of pathways,
possibly reflecting the multiple and overlapping functions of Racs in
development. Undoubtedly, many downstream cytoskeletal effectors of Racs
remain to be identified. Rac activity in different morphogenetic events might
be controlled by different combinations of upstream Rac regulators (GEFs) and
downstream cytoskeletal effectors. Mechanisms by which multiple Rac
regulators, Racs and downstream effectors control different morphogenetic
events in vivo remain unclear.
In this work we provide evidence that UNC-115 is a downstream Rac effector
in C. elegans axon pathfinding. UNC-115 is similar to the human
actin-binding protein abLIM/limatin (Roof
et al., 1997) and is involved in axon pathfinding
(Lundquist et al., 1998
). We
show that UNC-115 acts with the Racs and UNC-73/Trio GEF in axon pathfinding:
UNC-115 acts in parallel to CED-10 and MIG-2, possibly in the RAC-2/3 pathway,
and UNC-115 is required for the effects of constitutively active RAC-2 on axon
morphogenesis. Furthermore, we show that UNC-115 is an actin-binding protein,
suggesting that UNC-115 is a new downstream cytoskeletal effector of Rac
signaling that acts specifically in the RAC-2 branch of the triply-redundant
Rac pathway in axon pathfinding.
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MATERIALS AND METHODS |
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LGI: unc-73(e936, rh40), kyIs5[ceh-23::gfp, lin-15(+)]
LGIV: ced-10(n1993), dpy-13(e184), lqIs3[osm-6::gfp, lin-15(+)]
LGX: unc-115(mn481, ky275), mig-2(mu28, rh17, gm38 mu133), lin-15(n765), lqIs2[osm-6::gfp, lin-15(+)], lqIs10[ceh-10::gfp, lin-15(+)], kyIs4[ceh-23::gfp, lin-15(+)]
Germline transformation of nematodes was performed by standard techniques
(Mello and Fire, 1995) using
lin-15(n765) mutants and lin-15(+) DNA
(Clark et al., 1994
) as a
marker for co-transformation. Extrachromosomal arrays were integrated into the
genome using trimethylpsoralen/UV mutagenesis
(Anderson, 1995
) and standard
techniques (Mello and Fire,
1995
). RNAi was performed by dsRNA injection (1 µg/µl) into
the body cavity or gonad as described
(Fire et al., 1998
).
Analysis of CAN and PDE axon pathfinding and CAN cell migration
CAN axon morphology and cell position was scored using the integrated
ceh-23::gfp transgenes kyIs4 X and kyIs5 I, or the
integrated ceh-10::gfp transgene lqIs10 X, as described
(Lundquist et al., 2001). A
posterior CAN axon was scored as mutant if, because of premature termination
or misguidance, the axon failed to extend further than half the distance
between the vulva and the phasmid neurons in the tail (approximately to the
region of the postdeirid ganglion). CAN cell migration was scored as mutant if
the CAN cell body was greater than two CAN cell body-widths from the
vulva.
PDE morphology was examined in animals harboring the integrated osm-6::gfp transgenes lqIs3 IV and lqIs2 X that were expressed in all ciliated sensory neurons, including PDE. A PDE axon was scored as having a pathfinding defect if, because of premature termination or axon wandering, the axon failed to reach the VNC. A PDE axon was scored as exhibiting ectopic axons if one or more ectopic axons were seen emanating from the normal axon or from the PDE cell body. A PDE cell was scored as having abnormal cellular morphology if sheet-like membrane extensions and/or finger-like membrane extensions were observed anywhere on the cell, including on the axons, dendrites and cell bodies. Each genotype was scored on at least three separate occasions, and >100 animals were scored. Twice the standard error of the proportion (percentage) is displayed in the tables. Genetic interactions were considered synthetic (the genes have redundant, overlapping functions) when the proportion of defects in the double mutant was greater than the additive effects of each mutant alone.
Molecular biology
Standard molecular biological techniques were used
(Ausubel et al., 1987;
Sambrook, 1989). Oligonucleotide primer sequences used for PCR are available
upon request. The sequences of all coding regions of clones derived using PCR
in this work were determined to ensure that no errors were introduced. The
osm-6::gfp transgene was produced by fusing the osm-6
promoter (bases 2680-2373 of cosmid F58H1) produced by PCR from C.
elegans genomic DNA upstream of gfp. osm-6::gfp transgenic lines
were constructed by microinjection at a concentration of 30 ng/µl.
Construction and analysis of constitutively-active rac(G12V)
transgenes
Each rac-coding region, including initiator ATG, introns and stop
codon, was amplified by PCR from genomic DNA: ced-10 (bases
36,193-34,079 of cosmid C09G12); mig-2 (bases 20,146-21,722 of cosmid
C35C5); and rac-2 (bases 105-1957 of cosmid K03D3). Each coding
region was placed downstream of the osm-6 promoter and the
unc-115 promoter. The G12V mutation (G16V in mig-2) was
introduced into the ced-10-, mig-2- and rac-2-coding regions
using the Quikchange Site-Directed Mutagenesis System (Stratagene, La Jolla,
CA). Rac transgenes were injected in germline transformation
experiments at 5 ng/µl, and the osm-6::gfp plasmid was co-injected
at 30 ng/µl. Multiple transformed lines were obtained for each construct
(>3). Each line displayed similar behavior, and representative lines from
each transgene are shown in Table
3.
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To score the effects of unc-115 mutation on PDE axons harboring rac transgenes, arrays were crossed into the unc-115(ky275) mutant background. At least two independent arrays consisting of each transgene were scored in the unc-115 background with similar results. One representative line from each is shown in Table 3. Furthermore, the transgenes were re-isolated by outcrossing from unc-115 and rescored. PDE defects were restored to a degree similar to what was observed before building the unc-115; rac-2(G12V) double (data not shown).
Construction and analysis of gfp-tagged rac-2
The gfp-coding region was placed upstream of and in frame with the
rac-2-coding region in the unc-115 promoter::rac-2(+) and
unc-115 promoter::rac-2(G12V) transgenes. Frameshifts between
gfp- and rac-2-coding regions were produced by fusing
gfp upstream of and out of frame with the rac-2-coding
regions. unc-115::gfp::rac-2 transgenes were microinjected at
concentrations of 5 ng/µl to generate extrachromosomal arrays.
Molecular modeling and actin co-sedimentation
The program MODELLER was used to model the structure of the UNC-115 VHD
from the chicken villin VHD (HP67; 1qqv.pdb). Charge distribution was
determined using SYBYL program.
For actin sedimentation assays, a fragment of the C-terminal region of the
unc-115 cDNA, including the VHD-coding region (from position 1350 in
F09B9.2b open reading frame sequence to the stop codon), was fused in frame to
the 6-histidine moeity and DHFR in the pQE-41 vector (Qiagen, Valencia, CA).
Point mutations in the VHD were constructed using the Quikchange Site-Directed
Mutagenesis System (Stratagene, La Jolla, CA). The resulting
6HIS::DHFR::UNC-115 proteins were purified by standard Ni++
chelation chromatography (Qiagen, Valencia, CA). Actin co-sedimentation assays
were based on standard procedures (Miller
et al., 1991): 5 µM 6HIS::DHFR::UNC-115 protein with and
without 20 µM rabbit skeletal muscle G-actin was mixed in 5 mM Tris pH 7.5,
0.2 mM ATP, 0.2 mM DTT, 0.2 mM CaCl2 and actin was polymerized by
adding 20 mM MgCl2, 5 mM ATP, 100 mM KCl and incubating at room
temperature for 1 hour. Actin filaments were sedimented by centrifugation
(130,000 g for 40 minutes). The pellet was analyzed by
SDS-PAGE and western blotting with anti-RGS-6HIS antibody (Qiagen, Valencia,
CA).
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RESULTS |
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We analyzed PDE axon morphology in rac single and double
loss-of-function mutants. rac-2/3 activity was perturbed using RNAi
(Fire et al., 1998). The
rac-2/3 locus is composed of two nearly identical rac genes,
and rac-2/3(RNAi) is predicted to reduce the function of both genes
(see Lundquist et al., 2001
).
ced-10, mig-2 and rac-2/3(RNAi) single mutants displayed few
defects in PDE development (Table
1). Each pairwise double mutant combination of ced-10(n1993),
mig-2(mu28) and rac-2/3(RNAi) displayed synthetic PDE axon
defects (Table 1), including
axon guidance defects (the axons failed to reach the VNC and often wandered
along the lateral body wall) (Fig.
1C,D) and premature axon termination and defasciculation in the
ventral nerve cord (Fig. 1E,F).
In addition to these defects in axon pathfinding, rac double mutants
displayed ectopic axon formation (ectopic axons formed as branches from the
main axon or emanated from the cell body)
(Fig. 1G). Thus, rac
genes control multiple aspects of PDE axon development, including axon
pathfinding (guidance, outgrowth and fasciculation), as well as suppression of
ectopic axon formation. Similar results were obtained using the
mig-2(gm38mu133) allele (Table
1). Each rac double mutant displayed the entire spectrum
of defects, suggesting that all three rac genes act in each process
as opposed to individual rac gene involvement in a single aspect of
PDE axon development.
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ced-10(M+); mig-2 double mutants also displayed defects in PDE dendrite development (data not shown). The PDE dendrite was missing or misshapen (misguided, lacking a discernible cilium or exhibited ectopic branches) in 17% of ced-10(M+); mig-2 PDE neurons. rac-2/3(RNAi) double mutants did not display dendrite defects.
UNC-73 is a Trio-like molecule with two DH-GEF domains, one of which, GEF1,
is a Rac-GEF (Steven et al.,
1998). unc-73 acts with the three Racs in CAN and D-class
axon development (Lundquist et al.,
2001
), and here we find that unc-73 also affects PDE axon
development. The incomplete loss-of-function mutants unc-73(rh40) and
unc-73(e936) displayed PDE axon defects similar to rac
double mutants, including axon guidance defects, axon defasciculation and
premature termination in the VNC, and ectopic axon formation
(Table 1). unc-73(rh40) and unc-73(e936) PDE axon pathfinding defects
and ectopic axon formation were enhanced significantly by ced-10,
mig-2 and rac-2/3(RNAi)
(Table 1), suggesting that
unc-73 and the three rac genes act together in PDE axon
development. However, the fact that rac mutations enhance
unc-73(rh40), a mutation that specifically attenuates the GEF1
Rac-GEF activity of UNC-73, raises the possibility that the Racs might act in
a pathway parallel to UNC-73. To determine if RAC-2 acts in the UNC-73
pathway, we tested the ability of constitutively-active rac-2
(rac-2 harboring the G12V mutation; see below) to suppress PDE axon
defects caused by unc-73(rh40). Indeed, transgenic expression of
constitutively-active rac-2 in the PDE neuron partially suppressed
the PDE ventral axon guidance defects caused by unc-73(rh40)
(Table 1). Furthermore,
unc-73(rh40) PDE axons often wandered laterally before reaching the
VNC, and this wandering was also partially suppressed by activated
rac-2 (data not shown). These data suggest that rac-2 acts
downstream of unc-73 in the same pathway. Similar suppression of
unc-73 defects in the D-class motor axons has been observed with
constitutively active ced-10 and mig-2
(Wu et al., 2002
). These
results combined with the biochemical data that UNC-73 acts as a GEF on CED-10
and MIG-2 (Wu et al., 2002
)
strongly suggest that ced-10, mig-2, rac-2/3 and unc-73 act
in the same pathway in axon pathfinding. As described by Lundquist et al.
(Lundquist et al., 2001
),
enhancement of unc-73(rh40) axon pathfinding defects by rac
mutation could be due to Rac regulation by other molecules in addition to
UNC-73 or Rac regulation by a domain of UNC-73 apart from the GEF1 Rac-GEF
domain.
Although these data indicate that rac-2 acts in the
unc-73 pathway, they do not exclude the possibility that UNC-73 has
additional, Rac-independent roles in PDE axon development.
unc-73(e936) is predicted to affect multiple UNC-73 activities,
including that of GEF2 Rho-GEF (Steven et
al., 1998). Disruption of a rac-independent activity of
UNC-73 by unc-73(e936) might contribute to rac enhancement
of unc-73(e936).
Dendrite development was also perturbed by unc-73(rh40 and e936) and these defects were enhanced by ced-10 and mig-2 (data not shown).
UNC-115 acts with the Racs and UNC-73 in PDE and CAN axon
pathfinding
unc-115 mutations cause defects in axon pathfinding and
unc-115 encodes a putative actin-binding protein
(Lundquist et al., 1998). Racs
are thought to mediate cellular morphogenesis in part by regulating the
structure and dynamics of the actin cytoskeleton
(Hall, 1998
). To determine if
unc-115 acts with rac genes in axon pathfinding, we analyzed
the CAN and PDE axons of double loss-of-function mutants of unc-115
with ced-10, mig-2 and rac-2/3(RNAi). Neither
unc-115(ky275), a putative unc-115 null allele, nor
unc-115(mn481), an incomplete loss-of-function allele, caused defects
in CAN axon pathfinding (Table
2) [for a description of CAN axon pathfinding and defects see
Lundquist et al. (Lundquist et al.,
2001
)]. However, unc-115 animals displayed weak PDE axon
guidance defects: 12% of unc-115(ky275) PDE axons (n=174)
and 6% of unc-115(mn481) PDE axons (n=189) wandered
laterally (>45°C from straight ventrally) on their trajectory to the
VNC. All unc-115 PDE axons eventually reached the VNC. Furthermore,
unc-115 mutants alone displayed ectopic PDE axons (20% of PDEs in
unc-115(ky275)) (Table
2). Thus, unc-115 mutations alone had weak effects on PDE
axon development, most notably ectopic axon formation.
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We found that unc-115 synergized with ced-10 and
mig-2 but not rac-2/3 in CAN and PDE axon pathfinding.
unc-115; ced-10 and unc-115 mig-2 double mutants were viable
and fertile, but displayed synthetic defects, including a withered tail (Wit),
similar to but weaker than rac double mutants and unc-73
animals. The Wit phenotype is associated with perturbed CAN neuron development
(Forrester et al., 1998) and
indeed, ced-10; unc-115 and mig-2 unc-115 animals displayed
failure of the posterior CAN axon to extend into the tail due to axon
wandering or premature termination (Table
2). Furthermore, unc-115; ced-10 and unc-115
mig-2 animals displayed synthetic PDE defects, including ventral axon
guidance errors (Table 2) and
axon defasciculation in the VNC (data not shown). These data indicate that
unc-115 has overlapping function with ced-10 and
mig-2 in CAN and PDE axon pathfinding.
By contrast, the CAN and PDE axons of unc-115; rac-2/3(RNAi)
animals resembled those unc-115 alone
(Table 2), suggesting that
unc-115 and rac-2/3 affect the same pathway in CAN and PDE
axon pathfinding, possibly a pathway in parallel to mig-2 and
ced-10. However, we cannot rule out the possibility that
unc-115 also acts in the ced-10 pathway, because
ced-10(n1993) is not a null allele
(Soto et al., 2002) (E. A. L.,
unpublished results).
In addition, we found that unc-115 acts with unc-73 in axon pathfinding. unc-115(ky275) and unc-115(mn481) mutations significantly enhanced the CAN and PDE axon pathfinding defects caused by unc-73(e936) and unc-73(rh40) mutations (Table 2), including CAN posterior guidance errors and PDE ventral guidance errors. PDE defects of unc-115; unc-73 double mutants were not further enhanced by rac-2/3(RNAi) (Table 2), confirming that unc-115 and rac-2/3 act in the same pathway. Taken together, these results indicate that unc-115 acts with the rac genes and unc-73 to mediate CAN and PDE axon pathfinding.
While unc-115 and unc-73 mutants and rac double mutants displayed ectopic axon formation, double mutants of unc-115 with ced-10, mig-2 and unc-73 did not display enhanced ectopic axon formation (Table 2). Possibly, unc-115 acts specifically with rac-2/3 in axon pathfinding and with all three rac pathways in the suppression of ectopic axon formation.
The three racs and unc-73 affect other actin-based
morphogenetic processes, including migration of the CAN cell body
(Lundquist et al., 2001).
unc-115 alone or in double mutant combinations with the racs
and unc-73 had no effect on CAN cell migration
(Table 2). Indeed,
unc-115 doubles with ced-10, mig-2 and unc-73
showed no enhanced CAN cell migration defect despite enhanced CAN axon
pathfinding defects. ced-10, mig-2 and rac-2/3 also control
the migrations of the distal tip cells of the hermaphrodite gonad, and
ced-10 and rac-2/3 are involved in phagocytosis of cells
undergoing programmed cell death
(Lundquist et al., 2001
;
Reddien and Horvitz, 2000
).
unc-115 had no effect on distal tip cell migration or phagocytosis
alone or in any double mutant combination with the racs and
unc-73 (P. Reddien, personal communication). Thus, unc-115
is required for CAN and PDE axon pathfinding but is apparently not involved in
CAN and distal tip cell migration and cell corpse phagocytosis.
Constitutively active Racs dominantly perturb PDE morphogenesis
In order to understand the molecular relationships UNC-115 and the three
Racs, we studied the effects of constitutively active Racs on axon
pathfinding. The glycine to valine mutation at position 12, which is canonical
for constitutive activation of Ras superfamily GTPases, attenuates GTPase
activity, thus favoring the GTP-bound, active state
(Bourne et al., 1991).
mig-2(rh17), an allele with the equivalent G16V activating
mutation, disrupts HSN and CAN axon pathfinding, and ced-10(G12V)
dominantly perturbs CAN axon pathfinding and cell migration
(Lundquist et al., 2001
;
Zipkin et al., 1997
). To test
if constitutively active rac-2 can also dominantly perturb axon
pathfinding, we constructed a transgene containing the rac-2-coding
region harboring the G12V mutation under the control of the
osm-6 promoter. The osm-6 promoter is expressed exclusively
in ciliated neurons, including PDE, and shows no other discernible expression
(Collet et al., 1998
). Similar
constructs were made using the ced-10(G12V)- and
mig-2(G16V)-coding regions [collectively referred to as the
rac(G12V) transgenes], as well as the wild-type coding region of each
rac. Animals harboring the rac-2(G12V) transgene displayed
dominant defects in PDE axon development that resembled the defects of
rac double loss-of-function mutants, including axon guidance errors
(Table 3), axon defasciculation
and ectopic axon formation (Fig.
2A,B; Table 3).
ced-10(G12V), mig-2(G12V) and mig-2(rh17) showed similar
defects (Table 3). The
pathfinding and fasciculation errors caused by the rac(G12V)
transgenes were generally weaker than the equivalent defects caused by
rac loss-of-function, whereas ectopic axon formation was generally
more severe in rac(G12V) animals than in the rac
loss-of-function mutants (compare Table
2 with Table 3, and
Fig. 1E with
Fig. 2A,B). The PDE dendrite
also displayed ectopic branching in each of the three rac(G12V)
animals (Fig. 2B). The
wild-type versions of each rac transgene caused PDE axon defects
similar to but weaker than the rac(G12V) transgenes, most often weak
ectopic axon formation (Table
3). rac(G12V) expression driven by a different promoter,
the neuron-specific unc-115 promoter, caused similar defects (data
not shown).
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These data indicate that Rac(G12V) molecules dominantly perturb PDE axon development. Several lines of evidence suggest that rac(G12V) transgenes produced gain-of-function, constitutively active Rac molecules: similar mutations in other Ras superfamily GTPases produce constitutive activation; the effects of rac(G12V) were dominant to wild type; the effects of ced-10(G12V) did not require wild-type ced-10 (data not shown); and the effects of rac(G12V) transgenic expression were more severe than rac(+) transgenic expression.
Constitutively active Racs induce ectopic plasma membrane
extensions
In addition to pathfinding defects and ectopic axon formation,
constitutively active racs caused PDE cell shape alterations not seen
in rac loss-of-function double mutants. Animals harboring any of the
three rac(G12V) transgenes displayed sheet-like extensions of plasma
membrane and multiple, thin processes emanating from the cell bodies, axons
and dendrites (Fig. 2C,D).
Ectopic axons often emanated from the sheet-like extensions. The thin
processes were generally thinner and shorter than the normal or ectopic axons
and were often observed at the edges of the sheet-like membrane extensions.
Despite abnormal cellular morphology, the PDE axons usually completed their
extensions to the VNC.
rac(G12V)-induced ectopic extensions displayed dynamic morphology even in adult animals (Fig. 2E,F). For example, the sheet-like structures were observed to form single or multiple neurite-like extensions, and the thin processes were apparently extended and retracted over time. These results demonstrate that constitutively active racs induced dynamic cellular structures, plasma membrane extensions and thin processes, not seen in rac, unc-73 or unc-115 loss-of-function mutants. However, these structures resemble lamellipodia and filopodia normally found on growth cones during axon outgrowth (Knobel et al., 2001). Possibly, Rac(G12V) molecules ectopically induce these morphogenetic structures.
UNC-115 is required for the morphogenetic effects of
constitutively-active RAC-2
The loss-of-function studies described above place unc-115 in the
rac-2/3 pathway. To test if UNC-115 acts downstream of RAC-2, we
determined if loss of unc-115 function could suppress the effects of
constitutively active rac-2(G12V). We found that the null mutation
unc-115(ky275) suppressed the dominant effects of
rac-2(G12V) (Table 3).
The strong ectopic axon formation induced by rac-2(G12V) was
suppressed by unc-115(ky275) to a level similar to
unc-115(ky275) alone, and unc-115(ky275) suppressed the
ectopic plasma membrane extensions induced by rac-2(G12V). Whereas
the axon pathfinding defects caused by rac-2(G12V) were weak (1%), we
saw no axon pathfinding defects in unc-115(ky275); rac-2(G12V) double
mutants. By contrast, unc-115(ky275) did not suppress the effects of
constitutively-active ced-10(G12V), mig-2(G16V) or
mig-2(rh17) (Table 3).
Instead, unc-115(ky275) slightly enhanced the effects of
ced-10(G12V) and mig-2(G16V). These results indicate that
UNC-115 activity mediates the effects of constitutively active RAC-2(G12V).
Although it is possible that RAC-2(G12V) perturbs a process in which Racs are
not normally involved, the loss-of-function data that place rac-2/3
and unc-115 in the same pathway combined with unc-115
suppression of rac-2(G12V) strongly suggest that UNC-115 acts
downstream of RAC-2 in PDE axon development.
GFP::RAC-2 accumulates at the plasma membrane
Rho GTPases, including Racs, are anchored to the plasma membrane by
covalent attachment of prenyl group mediated by a C-terminal CAAX box
(Zhang and Casey, 1996), and
GFP::MIG-2 and GFP::CED-10 accumulate at the plasma membrane
(Lundquist et al., 2001
;
Zipkin et al., 1997
). The
potential RAC-2 polypeptide contains a consensus C-terminal CAAX box (CTVL).
To determine if RAC-2 accumulates at the plasma membrane, a rac-2
transgene was generated that consisted of the wild-type rac-2-coding
region fused in-frame downstream of the green fluorescent protein
(gfp) (Chalfie et al.,
1994
) coding region (Fig.
3A). This transgene is predicted to encode a full-length RAC-2
molecule tagged with GFP at the N terminus. The expression of the
gfp::rac-2 transgene was placed under the control of the
unc-115 promoter, which is expressed in most if not all neurons and
neuroblasts (Lundquist et al.,
1998
). Animals harboring the unc-115::gfp::rac-2
transgene showed expression of GFP::RAC-2 that accumulated at the cell margins
of neuroblasts and neurons as well as in the nerve ring
(Fig. 3B-D). The nerve ring is
composed of the axons of many neurons
(White et al., 1986
), and
nerve ring accumulation suggests that GFP::RAC-2 localized to the plasma
membranes of axons. GFP::CED-10 and GFP::MIG-2 show similar plasma membrane
and nerve ring accumulation (Lundquist et
al., 2001
; Zipkin et al.,
1997
). Wild-type rac-2
(Fig. 3) and
rac-2(G12V)-coding regions displayed indistinguishable localization,
and gfp::rac-2(G12V) caused dominant effects on PDE development as
described above (data not shown). Expression of GFP from a transgene that
contained a frameshift between the gfp- and rac-2-coding
regions failed to accumulate at the cell margins and in the nerve ring
(Fig. 3E-G) and instead was
uniformly distributed in the cytoplasm, indicating that RAC-2 sequences
mediate accumulation of GFP::RAC-2 at the plasma membrane, presumably via
C-terminal CAAX motif.
|
UNC-115 is an actin-binding protein
The predicted UNC-115 polypeptide consists of three N-terminal LIM domains
and a C-terminal region similar to the actin-binding headpiece domain of
villin (VHD) (Fig. 4A,B)
(Lundquist et al., 1998). Not
all VHDs can bind to actin, and those VHDs with demonstrated actin-binding
ability have conserved basic residues that form a `positive patch' that might
mediate molecular contacts with actin
(Vardar et al., 2002
). We
modeled the structure of the UNC-115 VHD based upon the NMR structure of the
chicken villin VHD (called HP67) (Fig.
4B,C). The UNC-115 VHD had hallmark features of all VHDs,
including a hydrophobic `cap' and a charged `crown'
(Fig. 4C) (see
Vardar et al., 2002
).
Furthermore, the UNC-115 VHD had a prominent `positive patch' similar to other
actin-binding VHDs. We next tested the ability of the UNC-115 VHD to
co-sediment with F-actin in vitro. A bacterially expressed fragment of UNC-115
containing the VHD sedimented in the presence of but not in the absence of
actin filaments (Fig. 4D). We
generated a mutant form of the UNC-115 VHD in which basic residues that
contribute to the `positive patch' were changed to acidic residues
(Fig. 4B,C). This mutant VHD
failed to co-sediment with actin filaments
(Fig. 4D). These results
demonstrate that the UNC-115 VHD is an actin filament binding domain.
|
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DISCUSSION |
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Three C. elegans rac genes define three overlapping pathways that
regulate the development of CAN and D-class axons, and unc-73 Trio
GEF is likely to control all three rac pathways in this process
(Wu et al., 2002;
Lundquist et al., 2001
).
Furthermore, UNC-73 Trio is known to act as a GEF for multiple Racs, including
the canonical Racs and the MIG-2-like Racs
(Wu et al., 2002
;
Newsome et al., 2000
;
Steven et al., 1998
). We have
shown that the three racs act redundantly in PDE axon development.
Multiple aspects of PDE development are affected by rac
loss-of-function, including axon guidance, axon fasciculation and axon
outgrowth as well as suppression of ectopic axon formation, indicating that
rac genes control multiple aspects of axon development. We also
provide evidence that the three racs act with unc-73 in axon
pathfinding: each rac loss-of-function mutation enhanced weak
unc-73 mutations and rac-2(G12V) suppressed
unc-73(rh40). Together with results showing that UNC-73 GEF1 acts as
a GEF on CED-10 and MIG-2 (Wu et al.,
2002
), these data indicate that the racs and
unc-73 act in the same pathway in PDE axon pathfinding.
unc-115 acts with the rac genes and unc-73
Trio in CAN and PDE axon pathfinding
UNC-115 is a molecule similar to the human actin-binding protein
abLIM/limatin (Lundquist et al.,
1998; Roof et al.,
1997
) and consists of three N-terminal LIM domains, which are
thought to mediate protein-protein interactions
(Dawid et al., 1998
), and a
C-terminal villin headpiece domain, an actin-binding domain found in a variety
of proteins (Vardar et al.,
2002
). UNC-115 is required for pathfinding of many but not all
axons in C. elegans (Lundquist et
al., 1998
), and dominant-negative abLIM/limatin can perturb RGC
axon pathfinding in the developing mouse visual system
(Erkman et al., 2000
). UNC-115
might act as a cytoskeletal adapter protein that interacts with actin via the
VHD and with other molecules via the LIM domains. We show that UNC-115 acts
downstream of Rac signaling during axon pathfinding, suggesting that UNC-115
might adapt Rac activity to the growth cone actin cytoskeleton.
In C. elegans, unc-115 is expressed in most if not all neurons
throughout development, yet many neurons display normal or near-normal axon
pathfinding and development in unc-115 null mutants
(Lundquist et al., 1998). Our
results explain this paradoxical observation by demonstrating that
unc-115 acts in the rac-2/3 branch of the tripartite
rac cascade and has overlapping function with the ced-10 and
mig-2 pathways in axon pathfinding. However, ced-10(n1993)
is not a null allele and unc-115 might enhance
ced-10(n1993). Therefore, unc-115 might act in both the
rac-2/3 and ced-10 pathways. If this is the case, then there
must be other genes that act in parallel to unc-115 in the
ced-10 pathway, as the unc-115 null phenotype is viable and
fertile and does not resemble the ced-10 null. Although
unc-115 might act in the rac-2/3 pathway in axon
pathfinding, unc-115 might act with all three racs in the
suppression of ectopic axons, as unc-115 mutants alone display
ectopic axon formation that is not enhanced by mutations in the three
rac genes.
The genetic relationships between the rac genes, unc-73
and unc-115 in axon pathfinding are shown in
Fig. 5. Although our results
indicate that UNC-73 controls the three Racs in axon development, they do not
exclude the possibility that UNC-73 has additional, Rac-independent effects on
axon pathfinding. In addition to the GEF1 Rac GEF domain, UNC-73 has a second
GEF domain (GEF2) that acts on the Rac-related small GTPase Rho
(Spencer et al., 2001;
Steven et al., 1998
).
Possibly, rho-1, the single C. elegans gene that encodes
Rho, acts downstream of unc-73 in parallel to the racs to
control PDE axon development.
|
We found that unc-115 was not required for other morphogenetic events involving the racs and unc-73, including migration of the CAN cell bodies, migration of the distal tip cell of the hermaphrodite gonad and phagocytosis of cell corpses undergoing programmed cell death. unc-115 might be a rac effector that acts specifically in axon pathfinding. Possibly, other genes with roles similar to unc-115 act with the racs and unc-73 in other morphogenetic events.
Constitutive Rac signaling can induce plasma membrane extensions in
neurons
PDE neurons of animals harboring ced-10(G12V), mig-2(G16V) and
rac-2(G12V) constitutively-active transgenes displayed ectopic
structures not seen in rac loss-of-function mutants, including
extensive networks of ectopic axons. rac(G12V) animals also displayed
ectopic plasma membrane extensions consisting of large, sheet-like structures,
as well as thin, finger-like projections emanating from cell bodies, axons and
dendrites. These structures were dynamic over time, even in adult animals.
Although we have no direct evidence of the nature of these structures, their
form and dynamics resemble actin cytoskeleton-based lamellipodia and filopodia
found in growth cones of developing animals
(Knobel et al., 1999). Rac
activity is known to induce ectopic lamellipodia in cultured cells
(Ridley et al., 1992
),
consistent with our observation of lamellipodia-like structures induced by Rac
activity in vivo. However, we also observed filopodia-like extensions induced
by Rac activity in PDE neurons, suggesting that Rac activity might induce the
formation of both lamellipodia-like and filopodia-like structures in vivo.
Possibly, Rac activity is required to produce these structures in growth cones
and is normally precisely controlled by upstream regulators such as UNC-73
Trio.
Loss of rac function caused both axon pathfinding defects and ectopic axon formation. Constitutive rac activity also caused both axon pathfinding defects and ectopic axon formation, suggesting that Rac activity is required for both axon initiation and growth, as well as for inhibition of superfluous axons and branches. Alternatively, constitutive activation of one rac might trigger a negative regulatory system that attenuates all rac signaling. If racs are required for both axon initiation and axon suppression, the mechanism of ectopic axon formation caused by rac loss-of-function and rac(G12V) constitutive activation might be distinct: rac loss-of-function ectopic axons might be due to a failure to prune spurious axon initiation, whereas Rac(G12V) activity might induce ectopic axons via uncontrolled axon initiation.
UNC-115 activity is required for the effects of constitutively-active
RAC-2(G12V)
Rac(G12V) molecules might persistently signal to their downstream effectors
leading to the ectopic formation of cellular structures that are normally
precisely controlled during axon pathfinding. We found that UNC-115 is
required for all of the morphogenetic effects of RAC-2(G12V), indicating that
UNC-115 acts downstream of RAC-2 to mediate these events and that UNC-115 is
normally involved in the formation of Rac-induced membrane extensions.
unc-115 did not suppress mig-2(G12V) or
ced-10(G12V), consistent with the idea that UNC-115 acts with RAC-2
specifically. However, unc-115 might act in the ced-10
pathway in parallel to another gene with overlapping function. Possibly,
unc-115 does not suppress ced-10(G12V) because
ced-10(G12V) can exert its influence through a downstream effector
gene that is redundant with unc-115. Inherent in both models is the
existence of other molecules, possibly other actin-binding proteins, that act
downstream of MIG-2 and CED-10 in parallel to UNC-115. unc-115 is the
only member of the unc-115/abLIM family present in the C.
elegans genome (The C.
elegans Genome Sequencing Consortium, 1998), so
unc-115 redundancy will be at the functional level and not a result
of homologous genes (as observed with rac redundancy).
UNC-115 is a new downstream cytoskeletal effector of Rac
signaling
Our results show that UNC-115 is required for the formation of plasma
membrane extensions that resemble lamellipodia and filopodia, actin-based
structures that normally regulate cell shape. We show that the modeled UNC-115
VHD structure contains a `positive patch' found in other actin-binding VHDs
(Vader et al., 2002) and that the UNC-115 VHD binds to actin filaments in
vitro, indicating that UNC-115 is an actin-binding protein. UNC-115 might
control axon pathfinding by directly interacting with the actin cytoskeleton
of growth cones.
The loss-of-function and epistasis suppression experiments described here implicate UNC-115 as a downstream effector of Rac signaling in axon pathfinding. In response to a signal (possibly an extracellular guidance signal), UNC-73 Trio might act on all three Racs, which then influence the actin cytoskeleton to achieve morphogenetic change underlying growth cone outgrowth and steering. UNC-115 might respond to RAC-2 and possibly CED-10 by binding to and modulating the actin cytoskeleton of the growth cone. Other actin binding proteins might act redundantly with UNC-115 to mediate cytoskeletal change in response to CED-10 and MIG-2 signals. Furthermore, UNC-115 appears to act downstream of Rac signaling specifically in axon pathfinding, indicating that Racs might use different downstream effectors to mediate different morphogenetic events.
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ACKNOWLEDGMENTS |
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anderson, P. (1995). Mutagenesis. InCaenorhabditis elegans: Modern Biological Analysis of an Organism (Methods in Cell Biology ), Vol.48 (ed. H. F. Epstein and D. C. Shakes), pp.31 -58. San Diego, CA: Academic Press.[Medline]
Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J. and Struhl, K. (ed.) (1987). Current Protocols in Molecular Biology. John Wiley and Sons.
Awasaki, T., Saito, M., Sone, M., Suzuki, E., Sakai, R., Ito, K. and Hama, C. (2000). The Drosophila trio plays an essential role in patterning of axons by regulating their directional extension. Neuron 26,119 -131.[Medline]
Azuma, T., Witke, W., Stossel, T. P., Hartwig, J. H. and
Kwiatkowski, D. J. (1998). Gelsolin is a downstream effector
of rac for fibroblast motility. EMBO J.
17,1362
-1370.
Bateman, J., Shu, H. and van Vactor, D. (2000). The guanine nucleotide exchange factor trio mediates axonal development in the Drosophila embryo. Neuron 26, 93-106.[Medline]
Bellanger, J. M., Lazaro, J. B., Diriong, S., Fernandez, A., Lamb, N. and Debant, A. (1998). The two guanine nucleotide exchange factor domains of Trio link the Rac1 and the RhoA pathways in vivo. Oncogene 16,147 -152.[CrossRef][Medline]
Bishop, A. L. and Hall, A. (2000). Rho GTPases and their effector proteins. Biochem J. 348,241 -255.[CrossRef][Medline]
Bourne, H. R., Sanders, D. A. and McCormick, F. (1991). The GTPase superfamily: conserved structure and molecular mechanism. Nature 349,117 -127.[CrossRef][Medline]
Brenner, S. (1974). The Genetics of
Caenorhabditis elegans. Genetics
77, 71-94.
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. and Prasher, D. C. (1994). Green fluorescent protein as a marker for gene expression. Science 263,802 -805.[Medline]
Clark, S. G., Lu, X. and Horvitz, H. R. (1994).
The Caenorhabditis elegans locus lin-15, a negative
regulator of a tyrosine kinase signaling pathway, encodes two different
proteins. Genetics 137,987
-997.
Collet, J., Spike, C. A., Lundquist, E. A., Shaw, J. E. and
Herman, R. K. (1998). Analysis of osm-6, a gene that
affects sensory cilium structure and sensory neuron function in
Caenorhabditis elegans. Genetics
148,187
-200.
Dawid, I. B., Breen, J. J. and Toyama, R. (1998). LIM domains: multiple roles as adapters and functional modifiers in protein interactions. Trends Genet. 14,156 -162.[CrossRef][Medline]
Dickson, B. J. (2001). Rho GTPases in growth cone guidance. Curr. Opin. Neurobiol. 11,103 -110.[CrossRef][Medline]
Didsbury, J., Weber, R. F., Bokoch, G. M., Evans, T. and
Snyderman, R. (1989). rac, a novel ras-related family of
proteins that are botulinum toxin substrates. J. Biol.
Chem. 264,16378
-16382.
Eden, S., Rohatgi, R., Podtelejnikov, A. V., Mann, M. and Kirschner, M. W. (2002). Mechanism of Regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Nature 418,790 -793[CrossRef][Medline]
Erkman, L., Yates, P. A., McLaughlin, T., McEvilly, R. J., Whisenhunt, T., O'Connell, S. M., Krones, A. I., Kirby, M. A., Rapaport, D. H., Bermingham, J. R. et al. (2000). A POU domain transcription factor-dependent program regulates axon pathfinding in the vertebrate visual system. Neuron 28,779 -792.[Medline]
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature 391,806 -811.[CrossRef][Medline]
Forrester, W. C., Perens, E., Zallen, J. A. and Garriga, G.
(1998). Identification of Caenorhabditis elegans genes
required for neuronal differentiation and migration.
Genetics 148,151
-165.
Haataja, L., Groffen, J. and Heisterkamp, N.
(1997). Characterization of RAC3, a novel member of the Rho
family. J. Biol. Chem.
272,20384
-20388.
Hakeda-Suzuki, S., Ng, J., Tzu, J., Dietzl, G., Sun, Y., Harms, M., Nardine, T., Luo, L. and Dickson, B. J. (2002). Rac function and regulation during Drosophila development. Nature 416,438 -442.[CrossRef][Medline]
Hall, A. (1998). Rho GTPases and the actin
cytoskeleton. Science
279,509
-514.
Kaibuchi, K., Kuroda, S. and Amano, M. (1999). Regulation of the cytoskeleton and cell adhesion by the Rho family GTPases in mammalian cells. Annu. Rev. Biochem. 68,459 -486.[CrossRef][Medline]
Kishore, R. S. and Sundaram, M. V. (2002). ced-10 Rac and mig-2 function redundantly and act with unc-73 trio to control the orientation of vulval cell divisions and migrations in Caenorhabditis elegans. Dev. Biol. 241,339 -348.[CrossRef][Medline]
Kissil, J. L., Johnson, K. C., Eckman, M. S. and Jacks, T.
(2002). Merlin phosphorylation by p21-activated kinase 2 and
effects of phosphorylation on merlin localization. J. Biol.
Chem. 277,10394
-10399.
Knobel, K. M., Jorgensen, E. M. and Bastiani, M. J.
(1999). Growth cones stall and collapse during axon outgrowth in
Caenorhabditis elegans. Development
128,4079
-4092.
Letourneau, P. C. (1996). The cytoskeleton in nerve growth cone motility and axonal pathfinding. Perspect. Dev. Neurobiol. 4,111 -123.[Medline]
Liebl, E. C., Forsthoefel, D. J., Franco, L. S., Sample, S. H., Hess, J. E., Cowger, J. A., Chandler, M. P., Shupert, A. M. and Seeger, M. A. (2000). Dosage-sensitive, reciprocal genetic interactions between the Ab1 tyrosine kinase and the putative GEF trio reveal trio's role in axon pathfinding. Neuron 26,107 -118.[Medline]
Lundquist, E. A., Herman, R. K., Shaw, J. E. and Bargmann, C. I. (1998). UNC-115, a conserved protein with predicted LIM and actin-binding domains, mediates axon guidance in C. elegans.Neuron 21,385 -392.[Medline]
Lundquist, E. A., Reddien, P. W., Hartwieg, E., Horvitz, H. R.
and Bargmann, C. I. (2001). Three C. elegans Rac
proteins and several alternative Rac regulators control axon guidance, cell
migration and apoptotic cell phagocytosis. Development
128,4475
-4488.
Luo, L. (2000). Rho GTPases in neuronal morphogenesis. Nat. Rev. Neurosci. 1, 173-180.[CrossRef][Medline]
Luo, L., Hensch, T. K., Ackerman, L., Barbel, S., Jan, L. Y. and Jan, Y. N. (1996). Differential effects of the Rac GTPase on Purkinje cell axons and dendritic trunks and spines. Nature 379,837 -840.[CrossRef][Medline]
Luo, L., Liao, Y. J., Jan, L. Y. and Jan, Y. N. (1994). Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion. Genes Dev. 8,1787 -1802.[Abstract]
Mello, C. and Fire, A. (1995). DNA transformation. In Caenorhabdidtis elegans: Modern Biological Analysis of an Organism (Methods in Cell Biology), Vol.48 (ed. H. F. Epstein and D. C. Shakes), pp.451 -482. San Diego, CA: Academic Press.[Medline]
Miller, K. G., Fields, C. M., Alberts, B. M. and Kellog, D. R. (1991). Use of actin filament and microtubule affinity chromatography to identify proteins that bind to the cytoskeleton. In Methods in Enzymology (ed. R. B. Vallee), pp.303 -319. San Diego, CA: Academic Press.
Newsome, T. P., Schmidt, S., Dietzl, G., Keleman, K., Asling, B., Debant, A. and Dickson, B. J. (2000). Trio combines with dock to regulate Pak activity during photoreceptor axon pathfinding in Drosophila. Cell 101,283 -294.[Medline]
Ng, J., Nardine, T., Harms, M., Tzu, J., Goldstein, A., Sun, Y., Dietzl, G., Dickson, B. J. and Luo, L. (2002). Rac GTPases control axon growth, guidance and branching. Nature 416,442 -447.[CrossRef][Medline]
Reddien, P. W. and Horvitz, H. R. (2000). CED-2/CrkII and CED-10/Rac control phagocytosis and cell migration in Caenorhabditis elegans. Nat. Cell Biol. 2, 131-136.[CrossRef][Medline]
Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D. and Hall, A. (1992). The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70,401 -410.[Medline]
Roberts, A. W., Kim, C., Zhen, L., Lowe, J. B., Kapur, R., Petryniak, B., Spaetti, A., Pollock, J. D., Borneo, J. B., Bradford, G. B. et al. (1999). Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense. Immunity 10,183 -196.[Medline]
Roof, D. J., Hayes, A., Adamian, M., Chishti, A. H. and Li,
T. (1997). Molecular characterization of abLIM, a novel
actin-binding and double zinc finger protein. J. Cell
Biol. 138,575
-588.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Soto, M. C., Qadota, H., Kasuya, K., Inoue, M., Tsuboi, D.,
Mello, C. C. and Kaibuchi, K. (2002). The GEX-2 and GEX-3
proteins are required for tissue morphogenesis and cell migrations in C.elegans. Genes Dev. 16,620
-632.
Spencer, A. G., Orita, S., Malone, C. J. and Han, M.
(2001). A RHO GTPase-mediated pathway is required during P cell
migration in Caenorhabditis elegans. Proc. Natl. Acad. Sci.
USA 98,13132
-13137.
Steven, R., Kubiseski, T. J., Zheng, H., Kulkarni, S., Mancillas, J., Ruiz Morales, A., Hogue, C. W., Pawson, T. and Culotti, J. (1998). UNC-73 activates the Rac GTPase and is required for cell and growth cone migrations in C. elegans. Cell 92,785 -795.[Medline]
Sugihara, K., Nakatsuji, N., Nakamura, K., Nakao, K., Hashimoto, R., Otani, H., Sakagami, H., Kondo, H., Nozawa, S., Aiba, A. and Katsuki, M. (1998). Rac1 is required for the formation of three germ layers during gastrulation. Oncogene 17,3427 -3433.[CrossRef][Medline]
Sulston, J. and Hodgkin, J. (1988). Methods. InThe Nematode Caenorhabditis elegans (ed. W. B. Wood), pp. 587-606. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Takenawa, T. and Miki, H. (2001). WASP and WAVE
family proteins: key molecules for rapid rearrangement of cortical actin
filaments and cell movement. J. Cell Sci.
114,1801
-1809.
Tessier-Lavigne, M. and Goodman, C. S. (1996).
The molecular biology of axon guidance. Science
274,1123
-1133.
The C. elegans Genome Sequencing Consortium
(1998). Genome sequence of the nematode C. elegans: a
platform for investigating biology. Science
282,2012
-2018.
Vardar, D., Chishti, A. H., Frank, B. S., Luna, E. J., Noegel, A. A., Oh, S. W., Schleicher, M. and McKnight, C. J. (2002). Villin-type headpiece domains show a wide range of F-actin-binding affinities. Cell Motil. Cytoskel. 52, 9-21.[CrossRef][Medline]
Weaver, A. M., Karginov, A. V., Kinley, A. W., Weed, S. A., Li, Y., Parsons, J. T. and Cooper, J. A. (2001) Cortactin promotes and stabilizes Arp2/3-induced actin filament network formation. Curr. Biol. 11,370 -374.[CrossRef][Medline]
Weed, S. A., Du, Y. and Parsons, J. T. (1998).
Translocation of cortactin to the cell periphery is mediated by the small
GTPase Rac1. J. Cell Sci.
111,2433
-2443.
White, J. G., Southgate, E., Thomson, J. N. and Brenner, S. (1986). The structure of the nervous system of the nematode Caenorhabditis elegans. Philos. Trans. R. Soc. Lond. 314, 1-340.
Wu, Y., Cheng, T., Lee, M. and Weng, N. (2002). Distinct Rac activation pathways control Caenorhabditis elegans cell migration and axon outgrowth. Dev. Biol. 250,145 -155.[CrossRef][Medline]
Xiao, G. H., Beeser, A., Chernoff, J. and Testa, J. R.
(2002). p21-activated kinase links Rac/Cdc42 signaling to merlin.
J. Biol. Chem. 277,883
-886.
Zhang, F. L. and Casey, P. J. (1996). Protein prenylation: molecular mechanisms and functional consequences. Annu. Rev. Biochem. 65,241 -269.[CrossRef][Medline]
Zigmond, S. H. (1996). Signal transduction and actin filament organization. Curr. Opin. Cell Biol. 8, 66-73.[CrossRef][Medline]
Zipkin, I. D., Kindt, R. M. and Kenyon, C. J. (1997). Role of a new Rho family member in cell migration and axon guidance in C. elegans. Cell 90,883 -894.[Medline]