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First published online 25 February 2009
doi: 10.1242/dev.026666
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1 Molecular and Cell Biology, University of California, Berkeley, CA 94720,
USA.
2 Howard Hughes Medical Institute and Department of Biology, Massachusetts
Institute of Technology, Cambridge, MA 02139, USA.
3 Massachusetts General Hospital Cancer Center, Department of Medicine,
Massachusetts General Hospital, Boston, MA 02114, USA.
4 Helen Wills Neuroscience Institute, University of California, Berkeley, CA
94720, USA.
Author for correspondence (e-mail:
garriga{at}berkeley.edu)
Accepted 2 February 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Cell migration, Axon guidance, Rac GTPase, CARMIL (Lrrc16a), Trio, Robo
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Rho
family GTPases and the Ena/VASP family of actin-binding proteins appear to
mediate the effects of Slit and Netrin receptors
(Guan and Rao, 2003). Rho
GTPases cycle between active GTP-bound and inactive GDP-bound states. Guanine
nucleotide exchange factors (GEFs) catalyze the exchange of GDP for GTP to
activate the GTPase, whereas GTPase-activating proteins (GAPs) stimulate GTP
hydrolysis. In Drosophila, mutations in genes encoding Rac GTPases
(members of the Rho superfamily), their GEFs and GAPs, and Enabled (Ena), all
result in misguided axons (Gertler et al.,
1995; Hakeda-Suzuki et al.,
2002; Hu et al.,
2005; Lundstrom et al.,
2004; Ng et al.,
2002; Yang and Bashaw,
2006).
Rac signaling appears to regulate events not only downstream but also
upstream of guidance receptor signaling. Two recent studies suggest that Rac
signaling regulates the function of both Netrin and Slit receptors in C.
elegans (Levy-Strumpf and Culotti,
2007; Watari-Goshima et al.,
2007). The kinesin-like molecule VAB-8L and UNC-73, the homolog of
the Rac and Rho GEF Trio, increase the levels of SAX-3 (a homolog of Robo)
receptors to promote their function.
We sought to identify genes that regulate the Rac pathway. Since UNC-34/Ena
and CED-10/Rac are thought to mediate the effects of UNC-40/DCC independently
(Gitai et al., 2003), we
reasoned that isolating mutations that suppressed the defects of
unc-34 mutants might lead to the identification of genes that inhibit
Rac signaling. Here we show that loss of functional CRML-1, the C.
elegans homolog of the CARMIL (also known as Lrrc16a) actin-uncapping
protein (Jung et al., 2001;
Remmert et al., 2004;
Uruno et al., 2006;
Xu et al., 1997;
Yang et al., 2005), suppresses
the cell and growth cone migration defects of unc-34 mutants but not
the defects of unc-73 mutants. We provide evidence that CRML-1 acts
through the Rac GEF of UNC-73. We also find that CRML-1 lowers the levels of
the SAX-3 guidance receptor, a feature consistent with the role of CRML-1 as a
negative regulator of Rac signaling.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). N2 Bristol was
the wild-type strain, and experiments were performed at 20°C unless stated
otherwise. The following mutations and transgenic arrays were used: LG
I: unc-73(e936), unc-73(rh40), bli-4(e937), dpy-5(e61),
crml-1(gm326), crml-1(gm331), crml-1(n1960), crml-1(n1962); LG
II: rrf-3(pk1426), juIs76 [Punc-25:gfp, lin-15(+)];
LG IV: eri-1(mg366), gmIs14 [Pmec-7::vab-8L::gfp,
rol-6(su1006)]; LG V: unc-34(gm114), unc-34(gm104),
unc-34(e566). Other strains used: CB4856 Hawaiian isolate,
gmIs28 [Pmec-7::sax-3::gfp, Pttx-3::gfp] (LG
unknown).
Isolation of crml-1 mutations
The crml-1 alleles gm326, gm331, n1960 and n1962
were isolated in two separate screens for mutations that suppressed the
uncoordinated (Unc) phenotype of unc-34 mutants. Mutagenesis was
performed as described (Brenner,
1974). n1960 and n1962 were isolated by
screening the F2 progeny of mutagenized hermaphrodites, as previously
described (Bloom, 1993).
gm326 and gm331 were isolated in an F1 clonal screen, in
which we screened the F2 progeny from individual F1s for a non-Unc phenotype.
We screened the progeny of 5476 F1 hermaphrodites.
Cloning of crml-1
crml-1(gm326) mapped to the interval between 1.66 (snp_uCE1-921)
and 1.86 (snp_uCE1-924) (Wicks et al.,
2001). We fed bacteria expressing dsRNA complementary to each ORF
in this interval as described (Kamath et
al., 2001; Timmons and Fire,
1998) to the RNAi-sensitized strains eri-1(mg366);
unc-34(gm114) and rrf-3(pk1426); unc-34(gm114) and screened the
F1 progeny for suppression of both the Unc and CAN migration phenotypes. We
used primers PJV84-PJV91 to determine the sequences of crml-1 in
gm326, gm331, n1960 and n1962 mutant animals. The sequences
of primers used in this study are shown in
Table 1. The sequences obtained
covered all exons, splice junctions, 5' and 3' UTRs and most
introns. All mutations identified were confirmed using a new sample of genomic
DNA.
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Scoring DD processes
We scored the amount of outgrowth of processes DD2-6 in L1 hermaphrodites
using juIs76 [Punc-25::gfp]
(Jin et al., 1999). Animals
were examined by fluorescence microscopy. We could not score DD1 because GFP
expression in the processes of head neurons (RMEs, AVL, RIS) overlapped the
DD1 processes. We classified the outgrowth defects by determining whether or
not the DD process reached the dorsal nerve cord (DNC). We determined the
percentage of DD processes that failed to reach the DNC and used this number
for comparison between strains. Statistical significance between two strains
was determined as described above.
Scoring ALM processes
We scored the process of ALM in L4 or young adult animals using the GFP
signal from the gmIs14 [Pmec-7::vab-8L::gfp] transgene. Processes
were scored as rerouted if they were bipolar or posterior. We defined a
process as posterior if it extended at least five ALM cell body lengths
towards the tail. We scored ALM as bipolar if the two processes extended at
least five cell body lengths in each direction. Bipolar and posterior
processes were pooled into one group for statistical analysis. Statistical
significance between two strains was determined as described above.
gmIs28 scoring
Embryos at the 2-fold stage were identified using Nomarski optics. Embryos
were scored for the presence or absence of a GFP signal in ALM and BDU. All
strains to be compared in a given experiment were scored on the same day to
ensure similar fluorescence bulb intensity. The ALM and BDU borders were
traced using a Region-of-Interest tool and epifluorescence intensities were
quantified by iVision software (BioVision Technologies). A two-tailed
Z-test was conducted to determine statistical significance between
two strains
(www.statcrunch.com).
Plasmid construction and transgenic strains
We isolated a full-length cDNA of crml-1 by PCR amplification
using an oligo(dT)-primed embryonic cDNA library as template. Determining the
sequence of this 3171 bp cDNA revealed ten exons and nine introns (see
Fig. 1A).
To generate the crml-1 transcriptional fusion (Pcrml-1::gfp), we amplified the intergenic region between crml-1 and the nearest upstream gene and cloned this region into the GFP Fire vector pPD95.75 (A. Fire, personal communication) to generate pPV33. gmEx504, gmEx505 and gmEx506 were generated by injecting pPV33 (50 ng/µL) and the co-injection marker pCL8 [Pmec-7::mcherry] (25 ng/µL) into wild-type hermaphrodites.
To generate the crml-1 translational fusion (Pcrml-1::crml-1cDNA::gfp), we inserted a full-length crml-1 cDNA between the crml-1 promoter and GFP coding sequences of pPV33 to create pPV35. gmEx374 and gmEx375 were generated by injecting pPV35 into wild-type hermaphrodites at 50 ng/µL with 25 ng/µL pRF4 [rol-6(su1006)].
To generate the crml-1 fusion to mCherry (Pmec-7::crml-1cDNA::mCherry), we inserted the full-length crml-1 cDNA between the mec-7 promoter and mCherry coding sequences of pCL8 to create pPV92. gmEx527 and gmEx529 were generated by injecting pPV92 into wild-type hermaphrodites at 50 ng/µL with 6 ng/µL Pmyo-2::gfp.
To integrate gmEx374, we UV-irradiated array-bearing worms using a Stratalinker 2400 (Stratagene) and selected F2 progeny that stably expressed both CRML-1::GFP and the co-injection marker rol-6(su1006). Multiple independent integrants were isolated.
Co-immunoprecipitation and immunoblotting
Co-immunoprecipitation experiments were conducted as described
(Chu et al., 2002). We prepared
embryo lysates from wild-type and gmIs30 (integrated version of
gmEx374) animals. For co-immunoprecipitation experiments, we used 300
µg total protein and added 2 µg of rabbit polyclonal anti-GFP antibody
(Abcam, #6556). For immunoblot analysis, 10 mL of immunoprecipitated samples
were separated on an SDS-PAGE gel by electrophoresis. Protein was transferred
onto a nitrocellulose membrane (Schleicher and Schuell), and the blot was
incubated with rabbit anti-UNC-73 antibodies at 1:6000 dilution
(Steven et al., 1998).
Following hybridization, blots were washed and then incubated with goat
anti-rabbit HRP-conjugated secondary antibody (1:10,000, BioRad). The antibody
complex was visualized using an ECL Kit (GE Healthcare Amersham) according to
the manufacturer's instructions.
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) using
the following antibodies: chicken anti-GFP (Upstate Biotechnology, 1:1000) and
rabbit anti-UNC-73 (1:1000). Secondary antibodies were Alexa488-conjugated
goat anti-chicken antibody (Molecular Probes) and Cy3-conjugated donkey
anti-rabbit antibody (Jackson ImmunoResearch Laboratories). |
RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). These
proteins can bind to Robo guidance receptors and to F-actin, suggesting that
Ena/VASP family members provide a link between guidance receptors and the
actin cytoskeleton (Lebrand et al.,
2004). Mutations in unc-34 cause an uncoordinated
locomotion phenotype (Unc) (Brenner,
1974), presumably resulting from cell migration and axon guidance
defects (Forrester and Garriga,
1997; Hedgecock et al.,
1985; McIntire et al.,
1992). Gitai et al. (Gitai et
al., 2003) provided genetic evidence that UNC-34 and CED-10/Rac
signaling mediates the effects of the Netrin receptor UNC-40/DCC. We reasoned
that loss of molecules that inhibit Rac signaling would increase Rac activity
and might bypass the need for UNC-34. To identify such molecules, we screened
for mutations that suppressed the Unc phenotype of unc-34 mutants.
Four of the suppressor mutations, gm326, gm331, n1960 and
n1962, mapped to chromosome I. Through a combination of genetic
mapping, RNAi and DNA sequencing, we identified these mutations as alleles of
K07G5.1, a conserved gene that we named crml-1.
Fig. 1B shows the domain
structure of CRML-1. Homologs of CRML-1 in Acanthamoeba,
Dictyostelium, mouse and human (24, 25, 32 and 33% identical to CRML-1,
respectively) have been described (Jung et
al., 2001; Xu et al.,
1997; Yang et al.,
2005). These homologs are known as CARMILs (Capping, Arp2/3,
Myosin I Linker), and were first described in Acanthamoeba and
Dictyostelium as proteins that bind to the SH3 domain of myosin I
(Jung et al., 2001;
Xu et al., 1997;
Xu et al., 1995). Components
of the Arp2/3 complex, which nucleates F-actin branching, also interact with
CARMILs (Jung et al., 2001).
The loosely defined Arp2/3-binding domain might not be conserved in metazoan
CARMILs, as mouse CARMIL failed to activate the Arp2/3 complex
(Yang et al., 2005).
Acanthamoeba and mammalian CARMILs can uncap actin filaments in vitro
(Uruno et al., 2006;
Yang et al., 2005). A domain
in the C-terminal portion of the protein binds capping protein with high
affinity and can remove it from the barbed ends of F-actin. This capping
protein-binding domain (CPBD) is conserved in all eukaryotic CARMIL homologs
(Fig. 1C).
All four mutations that mapped to chromosome I behave as semi-dominant suppressors, suggesting that they are likely to be different alleles of the same gene. We determined the sequence of the entire crml-1 gene from strains containing the suppressors and identified the mutation(s) in each strain. Two mutations were present in gm326: an opal nonsense mutation in the eighth exon and a missense mutation 80 amino acids further downstream. The crml-1 gene contained an ochre nonsense mutation in the fourth and ninth exons of n1962 and gm331 animals, respectively. The crml-1 gene in n1960 animals was found to contain a splice donor mutation after exon 6 (Fig. 1A,B).
Several observations indicate that these mutations reduce or eliminate
crml-1 functions. First, RNAi of crml-1 suppressed the Unc
and CAN defects of unc-34 mutants
(Fig. 2D; see Fig. S2 in the
supplementary material). Second, the mutant proteins should be severely
truncated by the three nonsense mutations, and nonsense-mediated decay should
degrade the crml-1 transcript produced by these mutants
(Pulak and Anderson, 1993).
Third, quantitative real-time PCR analysis of the three nonsense mutants
indicated that crml-1 mRNA levels are reduced by 50-70% in each
mutant (data not shown). Thus, the semi-dominant suppression caused by these
alleles could result from haploinsufficiency of crml-1.
Alternatively, CRML-1 fragments produced by the mutants could have antimorphic
activity. In either case, crml-1 function is reduced in the mutants.
At this point, we do not know whether the mutations eliminate all
crml-1 functions.
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; Canton et al.,
2006). The C. elegans genome contains a homolog of Cin85
but not of CKIP-1. In the absence of CRML-1 function, perhaps Y44E3A.4 could
serve in the same capacity, partially masking the loss of CRML-1 uncapping
function. However, RNAi of Y44E3A.4 in eri-1; crml-1(gm326) animals
did not result in locomotion or neuronal migration defects (data not
shown).
Suppression of ALM, CAN and DD defects
crml-1 mutants were identified by their ability to suppress the
Unc phenotype of unc-34 mutants. If the cell migration and axon
guidance defects of unc-34 mutants contribute to the Unc phenotype,
then crml-1 mutations should also suppress these defects.
ALMs are a pair of bilaterally symmetric neurons that are generated near
the head and migrate during embryogenesis
(Sulston et al., 1983). In
wild-type larvae, ALMs stop between the V2 and V3 hypodermal cells
(Fig. 2A; see Fig. S1 in the
supplementary material). In unc-34 mutants, ALMs often stopped
prematurely at various positions along their migratory route anterior to V2
(Fig. 2A,B; see Fig. S1 in the
supplementary material). We also scored the positions of ALMs in crml-1;
unc-34 mutants and found that mutations in crml-1 suppressed the
ALM migration defect of unc-34 mutants
(Fig. 2A,B; see Fig. S1 in the
supplementary material).
We observed a similar suppression of CAN migration. CANs are a pair of
bilaterally symmetric neurons that are generated in the head and undergo a
posterior migration (Fig. 2C)
(Sulston et al., 1983). In
unc-34 mutants, CANs were often defective in migration, and
crml-1 mutations suppressed this defect
(Fig. 2C,D; see Fig. S2 in the
supplementary material). In addition, the longer migrations of CANs revealed a
semi-dominant effect of crml-1 mutations. Mutating one copy of
crml-1 in an unc-34 background weakly suppressed the CAN
defect caused by the three alleles (gm326, gm331 and n1962)
of crml-1 tested (see Fig. S2 in the supplementary material).
Mutating the second copy of crml-1 suppressed the CAN defect further
(Fig. 2D; see Fig. S2 in the
supplementary material).
We also assessed the morphology of the DD neuronal projections, hereafter
referred to as processes. We use the term `process' instead of axon or
dendrite because DDs are not polarized with an axon emerging from one part of
the cell and dendrites from another. Rather, the branched process of each DD
neuron has both presynaptic and postsynaptic domains. During embryogenesis,
DDs are generated in the ventral nerve cord (VNC) and extend long anterior
processes along the VNC. The anterior processes branch dorsally, reaching the
dorsal nerve cord (DNC). In the DNC, the processes branch anteriorly and
posteriorly, forming a ladder-like pattern (see Fig. S3A in the supplementary
material) (White et al.,
1986).
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). These processes either failed to exit the VNC or
extended dorsally but failed to reach the DNC, often branching along the
anterior-posterior axis in lateral positions. We quantified the effect of
losing one or two copies of crml-1 in unc-34 mutant animals.
Lowering crml-1 activity decreased the percentage of processes that
failed to reach the DNC in an unc-34 mutant (see Fig. S3B in the
supplementary material). Together with the CAN migration data, these findings
demonstrate that cell and growth cone migrations are affected in a
dose-dependent manner by mutations in crml-1. The suppression affects
migrations occurring along both the anterior-posterior and dorsal-ventral
axes, indicating that these effects are not specific to a particular direction
or cellular environment.
CRML-1 is expressed in the developing nervous system
We examined the expression of crml-1 by constructing two
reporters. One was a transcriptional reporter, for which we fused GFP to
sequences from the end of the gene upstream to crml-1 to the
initiator ATG of crml-1. We also created a translational reporter by
fusing a full-length crml-1 cDNA to GFP and driving expression of
this fusion from the same crml-1 promoter. Animals bearing the
transcriptional and translational reporters had similar GFP expression
patterns. As shown in Fig. 3A,
L1 animals carrying the translation reporter expressed GFP in many neurons,
including CANs, DD-type motoneurons and ALMs. Expression in the nervous system
began early in comma-stage embryos and peaked in intensity around the 3-fold
stage of embryogenesis (data not shown). Although neuronal expression was much
fainter at later larval stages, it persisted in some head and tail neurons
through adulthood. Non-neuronal cells that also expressed CRML-1::GFP included
the migrating distal tip cells, the pharynx, some vulval epithelial cells,
rectal epithelial cells and the excretory canal (data not shown).
Excess CRML-1 causes CAN migration defects
Expression of high levels of the crml-1 cDNA from its native
promoter caused a CAN cell migration defect similar to that seen in
unc-34 mutants. The transgenic animal shown in
Fig. 3A, for example, had a CAN
neuron displaced anterior to its normal position. We scored CAN cell body
position in animals carrying the extrachromosomal array gmEx374.
Extrachromosomal arrays are mitotically unstable, so animals carrying these
arrays frequently lose the array in one or more cell lineages. We scored both
the position of CAN in transgenic animals and whether or not CAN expressed
CRML-1::GFP, which should indicate the presence or absence of the
extrachromosomal array in CAN. CANs were usually defective in migration in
gmEx374 animals when a GFP signal was present in the CAN (CAN+)
(Fig. 3B). If an animal
expressed GFP in other cells, but not in the CAN, CAN was in its wild-type
position (CAN-) (Fig. 3B).
These observations indicate that when expressed in CANs, CRML-1 inhibited
their migrations. The migration defect caused by the integrated version of
this transgene, gmIs32, became less severe in a crml-1
mutant background (Fig. 3B),
consistent with the hypothesis that excess CRML-1 disrupts CAN migration and
that the crml-1 mutations reduce gene activity. CANs were in
wild-type positions in animals carrying the transcriptional reporter
gmEx505, confirming that neither the presence of the crml-1
promoter nor expression of GFP in CANs caused the migration defect seen in
gmEx374 or gmIs32 animals
(Fig. 3B).
crml-1 and the Rac pathway
The crml-1 alleles identified in our screens bypass the
requirement for UNC-34 protein as they suppress not only missense mutations
such as gm114, but also nonsense mutations such as gm104
(see Fig. S2 in the supplementary material). One explanation for this type of
suppression is that crml-1 inhibits a pathway that acts in parallel
to UNC-34. Since Gitai et al. (Gitai et
al., 2003) showed that the Rac pathway and the UNC-34 pathway
function in parallel to mediate the UNC-40 attractive signaling that guides
the AVM process ventrally, we asked whether crml-1 functions in the
Rac pathway. All three C. elegans Rac GTPases (ced-10, mig-2
and rac-2/3) function in CAN migration, and loss of each can enhance
the cell and growth cone migrations of unc-34 mutants
(Lundquist et al., 2001;
Shakir et al., 2006).
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). UNC-73 acts as a GEF for at least two of the C.
elegans Rac proteins (Wu et al.,
2002). Although it does not appear to act downstream of
unc-40, UNC-73 is required for CAN migration
(Forrester and Garriga, 1997;
Lundquist et al., 2001;
Steven et al., 1998). UNC-73
contains two GEF domains: a Rac GEF required for neuronal cell migrations and
axon guidance, and a Rho GEF required for pharyngeal development and synaptic
activity (Fig. 4A)
(Steven et al., 1998;
Steven et al., 2005). At least
eight different UNC-73 isoforms are produced. Some isoforms contain the Rac
GEF, whereas others contain the Rho GEF, and one isoform contains both. We
examined two mutations predicted to disrupt Rac GEF function: e936 is
a splice donor mutation that disrupts the two transcripts that encode the Rac
GEF-containing isoforms, and rh40 is a missense mutation in the Rac
GEF domain. This latter mutation eliminates GDP-GTP exchange activity of the
Rac GEF in vitro (Steven et al.,
1998). Because rh40 eliminates the Rac GEF function of
UNC-73, we assume any effects of this allele reflect a specific defect in Rac
pathway function. Both mutations frequently resulted in defective ALM and CAN
migration (Fig. 4B,C). To test whether unc-34 and unc-73 act in parallel, we attempted to construct the double mutant. However, double mutants died as embryos, preventing an analysis of their migration defects. RNAi of unc-73 into unc-34(gm114) mutants also resulted in embryonic lethality. If CRML-1 negatively regulates Rac signaling, then loss of crml-1 should not affect the ALM or CAN defects of unc-73 mutants. Unlike the unc-34 interaction, loss of crml-1 did not suppress the ALM or CAN defects of either unc-73 allele (Fig. 4B,C). Similarly, we observed no difference in the DD processes of unc-73 crml-1 and unc-73 mutants (see Fig. S3B in the supplementary material). Although it is possible that crml-1 functions independently of unc-73, our findings are consistent with the hypothesis that CRML-1 negatively regulates the UNC-73 pathway.
CRML-1 and UNC-73 form a complex in vivo
To provide further evidence that CRML-1 acts in the Rac pathway, we tested
whether CRML-1 and UNC-73 physically interact in vivo. Using anti-GFP
antibodies, we immunoprecipitated CRML-1::GFP from extracts of embryos
containing gmIs30, an integrated version of the crml-1
translational reporter, and probed immunoblots of the precipitated proteins
with an anti-UNC-73 antiserum. We detected the UNC-73 isoforms predicted to
react with this antibody (Fig.
5A) (Steven et al.,
2005), showing that the two proteins form a complex in vivo.
Consistent with this hypothesis, we found that UNC-73 and CRML-1 were
co-expressed in CANs (and their axons) during embryogenesis and partially
co-localize (Fig. 5B).
CRML-1 is a negative regulator of SAX-3
After migrating, each ALM extends a single anterior projection that
branches in the nerve ring, the main C. elegans nerve bundle
(Fig. 2A and data not shown)
(Chalfie and Sulston, 1981;
White et al., 1986). Because
the anterior projections and branches have both dendritic and axonal
properties, we refer to them as processes. Recent work demonstrated that in
ALMs, UNC-73 acts with VAB-8L, a kinesin-like molecule, as a positive
regulator of guidance receptors including SAX-3
(Levy-Strumpf and Culotti,
2007; Watari-Goshima et al.,
2007). VAB-8L expression in ALMs produces two phenotypes: ALMs
migrate beyond their normal destination and reverse the polarity of their
process, causing it to extend toward the tail
(Wolf et al., 1998). We call
this latter defect ALM rerouting. As with expression of VAB-8L, increased
SAX-3 receptor activity leads to ALM rerouting. Furthermore, altering the
levels of VAB-8L in ALMs results in corresponding changes in the levels of
SAX-3 (Watari-Goshima et al.,
2007). These and other observations suggest that VAB-8L signals
through UNC-73 to regulate SAX-3 trafficking
(Watari-Goshima et al.,
2007).
We reasoned that if CRML-1 negatively regulates the UNC-73 pathway, CRML-1 loss should enhance ALM cell migration and rerouting defects caused by VAB-8L misexpression. Using the gmIs14 transgene (Pmec-7::vab-8L::gfp), we found that crml-1(gm326) enhanced both of the defects caused by VAB-8L misexpression (Fig. 6A,B). Using the unc-73(rh40) crml-1(gm326) double-mutant background, we also conducted the rerouting assay to see whether the effect of the crml-1 mutation is dependent on the presence of UNC-73 Rac GEF activity. If crml-1 acts by inhibiting unc-73, then unc-73(rh40) crml-1(gm326); gmIs14 and unc-73(rh40); gmIs14 animals should exhibit similar frequencies of ALM rerouting. We indeed observed such an effect (Fig. 6B), demonstrating that unc-73 functions downstream of crml-1 in this assay. However, when we examined ALM cell migration, we found that ALMs more frequently migrated too far posterior in crml-1 unc-73; gmIs14 animals as compared with unc-73; gmIs14 animals (Fig. 6A). Therefore, the relationship between VAB-8L, UNC-73 and CRML-1 might be more complex in ALM migration than in ALM process rerouting.
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We also asked whether loss of CRML-1 would change the levels of SAX-3 in ALMs. If CRML-1 negatively regulates UNC-73, loss of crml-1 would be predicted to cause an increase in the expression of SAX-3 in ALMs. To test this prediction, we used the gmIs28 transgene, which results in the expression of SAX-3::GFP in both ALMs and in their lineal sisters, the BDUs. We quantified the number of embryos in which we could detect the SAX-3::GFP signal in ALMs and BDUs at the 2-fold stage and found that crml-1(gm326) enhanced the percentage of cells in which we saw GFP (Fig. 6C-G). Quantification of the amount of SAX-3::GFP in these cells revealed that the levels appeared to be slightly higher in embryos that contained the crml-1 mutation and lower in embryos that contained the unc-73 mutation (Fig. 6H). The unc-73(rh40) crml-1(gm326) double mutant appeared identical to the unc-73 single mutant, suggesting that CRML-1 signals through UNC-73 to alter SAX-3::GFP levels (Fig. 6G,H). Crossing the crml-1 and unc-73 mutations out of the gmIs28 transgenic background and rescoring SAX-3::GFP levels in the original and newly isolated gmIs28 strains showed that the gmIs28 transgene was not altered during construction of the transgenic strains containing the mutations.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Uruno et al., 2006;
Xu et al., 1997;
Yang et al., 2005). Our
genetic analysis of crml-1; unc-34 mutants demonstrates that CRML-1
acts in parallel to UNC-34 as an inhibitor of cell and axon migrations. This
inhibitory function contrasts with the positive role CARMIL homologs are
thought to play in promoting glioblastoma cell migrations
(Jung et al., 2001;
Yang et al., 2005). Our
genetic and biochemical analyses indicate a previously unreported role for a
CARMIL homolog: inhibiting migration through the Rac pathway.
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It was recently reported that Rac signaling regulates SAX-3 and Netrin
receptors in C. elegans. According to this hypothesis, the
kinesin-like molecule VAB-8L acts through UNC-73 to increase guidance receptor
levels and promote posterior migrations in C. elegans
(Levy-Strumpf and Culotti,
2007; Watari-Goshima et al.,
2007). Our results indicate that UNC-73 is not only positively
regulated by VAB-8L, but is also negatively regulated by CRML-1, suggesting
that UNC-73 might integrate opposing signals to regulate neuronal migration
and process outgrowth (Fig.
7).
Most models place Rac GTPase signaling downstream of guidance receptors.
Our data support a less commonly held view that Rac signaling can also
regulate receptor levels, presumably through membrane trafficking. As a
negative regulator of the receptor SAX-3 and the Rac GEF UNC-73, CRML-1 could
act directly on SAX-3, making it refractory to UNC-73 activity. This type of
inhibition might be similar to the role of vertebrate Rig1 (also known as
Robo3.1) on Robo1 at the ventral midline of the spinal cord. Rig1 inhibits the
ability of Robo1 to respond to Slit, but the precise mechanism of inhibition
is unknown (Chen et al., 2008;
Sabatier et al., 2004).
Alternatively, CRML-1 could act indirectly on SAX-3 by inhibiting UNC-73. Our
ability to detect UNC-73 in a complex with CRML-1 supports this latter
hypothesis.
Rac signaling can modulate cell surface receptor levels. Rac signaling has
been implicated in clathrin-mediated endocytosis of both transferrin and EGF
receptors (Jou et al., 2000;
Lamaze et al., 1996). These
studies demonstrated that activated Rac inhibits receptor endocytosis in cell
culture. Our results are consistent with a model in which Rac signaling
regulates endocytic events in C. elegans. Controlling the levels of
the receptor itself is one way to control the amount of repulsive signaling
occurring through the SLT-1/Slit-SAX-3/Robo pathway. Based on our genetic and
biochemical data, we favor a model in which CRML-1 and VAB-8L signaling
converges on UNC-73, the role of which is to integrate these signals and
determine the amount of SAX-3 endocytosis during neuronal development.
|
;
Keleman et al., 2005). By
downregulating Robo, Comm blinds these growth cones to the effects of Slit at
the CNS midline. Subsequent loss of Comm expression after crossing allows Robo
to reach the growth cone and respond to Slit, ensuring that the growth cone
does not recross the midline (Keleman et
al., 2002; Keleman et al.,
2005). Although only insects have comm homologs, similar
mechanisms might control Robo levels in other organisms. The inhibition by
CRML-1 of Rac signaling could prevent SAX-3 from reaching the cell
surface.
The types of receptors and signaling pathways expressed define how
migrating cells and growth cones respond to their environment. Our
observations are consistent with an emerging view that it is not only
signaling downstream of guidance receptors that is regulated, but that
receptor function and expression are also regulated. VAB-8L and UNC-73 can
positively regulate the Robo and Netrin receptors to control the directional
migration of cells and growth cones
(Levy-Strumpf and Culotti,
2007; Watari-Goshima et al.,
2007). Like CRML-1, Drosophila Comm and vertebrate Rig1
inhibit the function of Robo homologs
(Keleman et al., 2005;
Sabatier et al., 2004). As new
regulators of guidance receptors are identified, the challenge will be to
understand how they collaborate to coordinate cell and axon migrations.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/7/1201/DC1
|
Footnotes |
|---|
* Present address: University of Chicago, Department of Molecular Genetics
and Cell Biology, 901 E. 58th St, Chicago, IL 60637, USA 
Present address: Wyeth Pharmaceuticals, Biological Technologies Department,
Cambridge, MA 02140, USA
|
REFERENCES |
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|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Bloom, L. (1993). Genetic and molecular
analysis of genes required for axon outgrowth in Caenorhabditis
elegans. In Biology, p.410
. Cambridge, MA: Massachusetts Institute of
Technology.
Brenner, S. (1974). The genetics of
Caenorhabditis elegans. Genetics
77, 71-94.
Bruck, S., Huber, T. B., Ingham, R. J., Kim, K., Niederstrasser,
H., Allen, P. M., Pawson, T., Cooper, J. A. and Shaw, A. S.
(2006). Identification of a novel inhibitory actin-capping
protein binding motif in CD2-associated protein. J. Biol.
Chem. 281,19196
-19203.
Canton, D. A., Olsten, M. E., Niederstrasser, H., Cooper, J. A.
and Litchfield, D. W. (2006). The role of CKIP-1 in cell
morphology depends on its interaction with actin-capping protein.
J. Biol. Chem. 281,36347
-36359.
Chalfie, M. and Sulston, J. (1981).
Developmental genetics of the mechanosensory neurons of Caenorhabditis
elegans. Dev. Biol. 82,358
-370.[CrossRef][Medline]
Chen, Z., Gore, B. B., Long, H., Ma, L. and Tessier-Lavigne,
M. (2008). Alternative splicing of the Robo3 axon guidance
receptor governs the midline switch from attraction to repulsion.
Neuron 58,325
-332.[CrossRef][Medline]
Chu, D. S., Dawes, H. E., Lieb, J. D., Chan, R. C., Kuo, A. F.
and Meyer, B. J. (2002). A molecular link between
gene-specific and chromosome-wide transcriptional repression. Genes
Dev. 16,796
-805.
Finney, M. and Ruvkun, G. (1990). The unc-86
gene product couples cell lineage and cell identity in C. elegans.
Cell 63,895
-905.[CrossRef][Medline]
Forrester, W. C. and Garriga, G. (1997). Genes
necessary for C. elegans cell and growth cone migrations.
Development 124,1831
-1843.[Abstract]
Gertler, F. B., Comer, A. R., Juang, J. L., Ahern, S. M., Clark,
M. J., Liebl, E. C. and Hoffmann, F. M. (1995). Enabled, a
dosage-sensitive suppressor of mutations in the Drosophila Abl tyrosine
kinase, encodes an Abl substrate with SH3 domain-binding properties.
Genes Dev. 9,521
-533.
Gitai, Z., Yu, T. W., Lundquist, E. A., Tessier-Lavigne, M. and
Bargmann, C. I. (2003). The netrin receptor UNC-40/DCC
stimulates axon attraction and outgrowth through enabled and, in parallel, Rac
and UNC-115/AbLIM. Neuron
37, 53-65.[CrossRef][Medline]
Guan, K. L. and Rao, Y. (2003). Signalling
mechanisms mediating neuronal responses to guidance cues. Nat. Rev.
Neurosci. 4,941
-956.[Medline]
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]
Hedgecock, E. M., Culotti, J. G., Thomson, J. N. and Perkins, L.
A. (1985). Axonal guidance mutants of Caenorhabditis elegans
identified by filling sensory neurons with fluorescein dyes. Dev.
Biol. 111,158
-170.[CrossRef][Medline]
Hu, H., Li, M., Labrador, J. P., McEwen, J., Lai, E. C.,
Goodman, C. S. and Bashaw, G. J. (2005). Cross
GTPase-activating protein (CrossGAP)/Vilse links the Roundabout receptor to
Rac to regulate midline repulsion. Proc. Natl. Acad. Sci.
USA 102,4613
-4618.
Jin, Y., Jorgensen, E., Hartwieg, E. and Horvitz, H. R.
(1999). The Caenorhabditis elegans gene unc-25 encodes glutamic
acid decarboxylase and is required for synaptic transmission but not synaptic
development. J. Neurosci.
19,539
-548.
Jou, T. S., Leung, S. M., Fung, L. M., Ruiz, W. G., Nelson, W.
J. and Apodaca, G. (2000). Selective alterations in
biosynthetic and endocytic protein traffic in Madin-Darby canine kidney
epithelial cells expressing mutants of the small GTPase Rac1. Mol.
Biol. Cell 11,287
-304.
Jung, G., Remmert, K., Wu, X., Volosky, J. M. and Hammer, J. A.,
3rd (2001). The Dictyostelium CARMIL protein links capping
protein and the Arp2/3 complex to type I myosins through their SH3 domains.
J. Cell Biol. 153,1479
-1497.
Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G.
and Ahringer, J. (2001). Effectiveness of specific
RNA-mediated interference through ingested double-stranded RNA in
Caenorhabditis elegans. Genome Biol.
2, RESEARCH0002.
Keleman, K., Rajagopalan, S., Cleppien, D., Teis, D., Paiha, K.,
Huber, L. A., Technau, G. M. and Dickson, B. J. (2002). Comm
sorts robo to control axon guidance at the Drosophila midline.
Cell 110,415
-427.[CrossRef][Medline]
Keleman, K., Ribeiro, C. and Dickson, B. J.
(2005). Comm function in commissural axon guidance:
cell-autonomous sorting of Robo in vivo. Nat.
Neurosci. 8,156
-163.[CrossRef][Medline]
Lamaze, C., Chuang, T. H., Terlecky, L. J., Bokoch, G. M. and
Schmid, S. L. (1996). Regulation of receptor-mediated
endocytosis by Rho and Rac. Nature
382,177
-179.[CrossRef][Medline]
Lebrand, C., Dent, E. W., Strasser, G. A., Lanier, L. M.,
Krause, M., Svitkina, T. M., Borisy, G. G. and Gertler, F. B.
(2004). Critical role of Ena/VASP proteins for filopodia
formation in neurons and in function downstream of netrin-1.
Neuron 42,37
-49.[CrossRef][Medline]
Levy-Strumpf, N. and Culotti, J. G. (2007).
VAB-8, UNC-73 and MIG-2 regulate axon polarity and cell migration functions of
UNC-40 in C. elegans. Nat. Neurosci.
10,161
-168.[CrossRef][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.
Lundstrom, A., Gallio, M., Englund, C., Steneberg, P., Hemphala,
J., Aspenstrom, P., Keleman, K., Falileeva, L., Dickson, B. J. and Samakovlis,
C. (2004). Vilse, a conserved Rac/Cdc42 GAP mediating Robo
repulsion in tracheal cells and axons. Genes Dev.
18,2161
-2171.
McIntire, S. L., Garriga, G., White, J., Jacobson, D. and
Horvitz, H. R. (1992). Genes necessary for directed axonal
elongation or fasciculation in C. elegans. Neuron
8, 307-322.[CrossRef][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]
Pulak, R. and Anderson, P. (1993). mRNA
surveillance by the Caenorhabditis elegans smg genes. Genes
Dev. 7,1885
-1897.
Remmert, K., Olszewski, T. E., Bowers, M. B., Dimitrova, M.,
Ginsburg, A. and Hammer, J. A., 3rd (2004). CARMIL is a bona
fide capping protein interactant. J. Biol. Chem.
279,3068
-3077.
Sabatier, C., Plump, A. S., Le M., Brose, K., Tamada, A.,
Murakami, F., Lee, E. Y. and Tessier-Lavigne, M. (2004). The
divergent Robo family protein rig-1/Robo3 is a negative regulator of slit
responsiveness required for midline crossing by commissural axons.
Cell 117,157
-169.[CrossRef][Medline]
Shakir, M. A., Gill, J. S. and Lundquist, E. A.
(2006). Interactions of UNC-34 Enabled with Rac GTPases and the
NIK kinase MIG-15 in Caenorhabditis elegans axon pathfinding and neuronal
migration. Genetics 172,893
-913.
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.[CrossRef][Medline]
Steven, R., Zhang, L., Culotti, J. and Pawson, T.
(2005). The UNC-73/Trio RhoGEF-2 domain is required in separate
isoforms for the regulation of pharynx pumping and normal neurotransmission in
C. elegans. Genes Dev.
19,2016
-2029.
Sulston, J. E., Schierenberg, E., White, J. G. and Thomson, J.
N. (1983). The embryonic cell lineage of the nematode
Caenorhabditis elegans. Dev. Biol.
100,64
-119.[CrossRef][Medline]
Timmons, L. and Fire, A. (1998). Specific
interference by ingested dsRNA. Nature
395, 854.[CrossRef][Medline]
Uruno, T., Remmert, K. and Hammer, J. A., 3rd
(2006). CARMIL is a potent capping protein antagonist:
identification of a conserved CARMIL domain that inhibits the activity of
capping protein and uncaps capped actin filaments. J. Biol.
Chem. 281,10635
-10650.
Watari-Goshima, N., Ogura, K., Wolf, F. W., Goshima, Y. and
Garriga, G. (2007). C. elegans VAB-8 and UNC-73 regulate the
SAX-3 receptor to direct cell and growth-cone migrations. Nat.
Neurosci. 10,169
-176.[CrossRef][Medline]
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. B Biol.
Sci. 314,1
-340.
Wicks, S. R., Yeh, R. T., Gish, W. R., Waterston, R. H. and
Plasterk, R. H. (2001). Rapid gene mapping in Caenorhabditis
elegans using a high density polymorphism map. Nat.
Genet. 28,160
-164.[CrossRef][Medline]
Wolf, F. W., Hung, M. S., Wightman, B., Way, J. and Garriga,
G. (1998). vab-8 is a key regulator of posteriorly directed
migrations in C. elegans and encodes a novel protein with kinesin motor
similarity. Neuron 20,655
-666.[CrossRef][Medline]
Wu, Y.-C., Cheng, T.-W., Lee, M.-C. and Weng, N.-Y.
(2002). Distinct Rac activation pathways control Caenorhabditis
elegans cell migration and axon outgrowth. Dev. Biol.
250,145
-155.[CrossRef][Medline]
Xu, P., Zot, A. S. and Zot, H. G. (1995).
Identification of Acan125 as a myosin-I-binding protein present with myosin-I
on cellular organelles of Acanthamoeba. J. Biol. Chem.
270,25316
-25319.
Xu, P., Mitchelhill, K. I., Kobe, B., Kemp, B. E. and Zot, H.
G. (1997). The myosin-I-binding protein Acan125 binds the SH3
domain and belongs to the superfamily of leucine-rich repeat proteins.
Proc. Natl. Acad. Sci. USA
94,3685
-3690.
Yang, C., Pring, M., Wear, M. A., Huang, M., Cooper, J. A.,
Svitkina, T. M. and Zigmond, S. H. (2005). Mammalian CARMIL
inhibits actin filament capping by capping protein. Dev.
Cell 9,209
-221.[CrossRef][Medline]
Yang, L. and Bashaw, G. J. (2006). Son of
sevenless directly links the robo receptor to Rac activation to control axon
repulsion at the midline. Neuron
52,595
-607.[Medline]
Yu, T. W. and Bargmann, C. I. (2001). Dynamic
regulation of axon guidance. Nat. Neurosci.
4 Suppl,1169
-1176.[Medline]
Yu, T. W., Hao, J. C., Lim, W., Tessier-Lavigne, M. and
Bargmann, C. I. (2002). Shared receptors in axon guidance:
SAX-3/Robo signals via UNC-34/Enabled and a Netrin-independent UNC-40/DCC
function. Nat. Neurosci.
5,1147
-1154.[CrossRef][Medline]
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