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First published online 25 February 2009
doi: 10.1242/dev.026666

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C. elegans CARMIL negatively regulates UNC-73/Trio function during neuronal development

Pamela J. Vanderzalm^1,*, Amita Pandey¹, Michael E. Hurwitz^2,3, Laird Bloom^2,, H. Robert Horvitz² and Gian Garriga^1,4,

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.

View larger version (14K): [in this window] [in a new window]   Fig. 1. The crml-1 gene and CRML-1 protein. (A) The gene structure of C. elegans crml-1. The n1960 allele is a G-to-A splice donor mutation at the 5' end of intron 6. White boxes, exons; lines, introns; arrow, transcription start site. (B) CRML-1 contains 16 leucine-rich repeats, a capping protein-binding domain (CPBD), and three PxxP motifs that fit the consensus sequence for SH3-binding domains. The CPBDs of Acanthamoeba and mouse CARMIL can bind capping protein and uncap F-actin (Uruno et al., 2006; Yang et al., 2005). The n1962, gm326 and gm331 alleles are ochre, opal and ochre nonsense mutations in the second, twelfth and fourteenth leucine-rich repeats, respectively. (C) Sequence alignments of the CPBD region were performed using ClustalW. Conserved residues important for binding to capping protein are highlighted in gray, with dark gray indicating identity and light gray indicating similarity. A CARMIL consensus sequence is depicted below. GenBank accession numbers for CARMIL sequences used in phylogenetic analyses are: Caenorhabditis elegans (NP-492024), Homo sapiens (NP-060110), Mus musculus (AAR96060), Rattus norvegicus (XP-225336), Drosophila melanogaster (NP-610316), Acanthamoeba castellanii (AAB57739) and Dictyostelium discoideum (AAK72255). The sequences of three other proteins that contain a CPBD and act as actin-uncappers in vitro are aligned below the CARMIL consensus sequence. GenBank accession numbers for Homo sapiens homologs of CPBD-containing family members used in phylogenetic analyses are: CD2AP (Q9Y5K6), CIN85 (SH3KBP1) (Q96B97), CKIP-1 (PLEKHO1) (CAI14264).

View larger version (23K): [in this window] [in a new window]   Fig. 2. Loss of CRML-1 suppresses ALM and CAN migration defects of unc-34 mutants. (A,C) Positions of stationary P and V hypodermal nuclei used to score final ALM (A) or CAN (C) positions in newly hatched L1s. Anterior is to the left, dorsal is up. The gray dashed line in A shows the ALM migration path during embryogenesis, and the black dashed line in C shows the CAN migration path. Each box lists the percentage of cells found in the region indicated. The black vertical lines delineate the region that contains most wild-type ALMs or CANs. (B,D) Graphical representation of the cell positioning data shown in A and C. Any ALM that did not migrate posterior of V2, or any CAN that did not migrate posterior of V3, was scored as defective, and the percentage defective for each genotype examined is shown. Error bars, s.e.m. (B) Statistically significant differences between unc-34 and crml-1; unc-34 strains are marked (***P<0.0001, **P<0.003). For the distribution of ALMs relative to P and V cells for all genotypes and the number of ALMs scored, see Fig. S1 in the supplementary material. At least 45 animals were scored per genotype. (D) Statistically significant differences between unc-34 and crml-1; unc-34 strains and between control RNAi and crml-1 RNAi strains are marked (***P<0.0001). Error bars, s.e.m. For the distribution of CANs relative to P and V cells for all genotypes and the number of CANs scored, see Fig. S2 in the supplementary material. At least 45 animals were scored per genotype.

View larger version (38K): [in this window] [in a new window]   Fig. 3. Excess CRML-1 disrupts CAN migration. (A) Confocal projection of an L1 C. elegans expressing the extrachromosomal array gmEx374 [Pcrml-1::crml-1::gfp]. CRML-1::GFP is expressed in neurons of the head (HG) and tail (TG) ganglia, and in axons that form the nerve ring, the ventral nerve cord (VNC) and the dorsal nerve cord (DNC) (not shown). The position of a CAN is indicated (arrowhead). This CAN is displaced anterior to its normal position, which is indicated by the dashed arrow. A schematic of the animal is presented on the right. (B) CAN migration defects in CRML-1 transgenic animals. See Fig. 2C legend for details. Extrachromosomal arrays are lost during mitosis, which allowed us to determine differences between animals expressing the array in CAN and those that had lost the array in CAN. For animals carrying gmEx374, we scored CANs in which we detected CRML-1::GFP in the cells (CAN+) and CANs in which we could not (CAN-). Statistically significant differences between CAN+ and CAN-animals and between gmIs32 and crml-1; gmIs32 are marked (***P<0.0001). For the distribution of CANs relative to P and V cells and the number of CANs scored, see Fig. S2 in the supplementary material. Error bars, s.e.m. At least 34 animals were scored per genotype.

View larger version (12K): [in this window] [in a new window]   Fig. 4. crml-1 mutations fail to suppress the CAN and ALM migration defects of unc-73 mutants. (A) Schematic of the two Rac GEF-containing isoforms of C. elegans UNC-73. PH, Pleckstrin homology domain. UNC-73A contains a Rho GEF and a second PH domain that UNC-73B lacks. The rh40 missense mutation eliminates the Rac GEF activity of UNC-73 (Steven et al., 1998). (B) The ALM defects of unc-73 mutants are unaffected by mutation of crml-1. ALM positions were scored as described in Fig. 2A. Error bars indicate s.e.m. For the distribution of ALMs relative to P and V cells and the number of ALMs scored, see Fig. S1 in the supplementary material. At least 41 animals were scored per genotype. (C) The CAN defects of unc-73 mutants are unaffected by mutation of crml-1. CAN positions were scored as described in Fig. 2C. For the distribution of CANs relative to P and V cells and the number of CANs scored, see Fig. S2 in the supplementary material. At least 52 animals were scored per genotype.

View larger version (32K): [in this window] [in a new window]   Fig. 5. UNC-73 forms a complex in vivo with CRML-1. (A) Immunoprecipitation of CRML-1::GFP and UNC-73 from C. elegans embryo extracts. The GFP tag on the CRML-1 fusion protein was used to immunoprecipitate CRML-1 from embryo lysates. Immunoblots were probed with antibodies against UNC-73, which reliably detected four isoforms in the gmIs30 lane, but not in the wild-type lane: the two Rac GEF-containing isoforms A and B, and the C2 and F isoforms; the previously described non-specific (n-s) band (Steven et al., 1998; Steven et al., 2005) is also indicated. (B) Confocal images of a 3-fold stage gmIs30 embryo stained with antibodies against GFP to detect CRML-1::GFP (green) and UNC-73 (red). To the left is a schematic of the embryo orientation in the eggshell, showing the head of the animal folded over the body just posterior to the CANs, which are located near the middle of the animal. Next to the schematic is a projection of the z-stack showing the co-expression of CRML-1 (green) and UNC-73 (red) in CANs (CANs were identified by co-staining with CAN-specific markers, data not shown). The three images to the right are a single 0.9 µm section of the focal plane that includes the lower CAN (boxed, enlarged in insets) and its anteriorly projecting axon. CRML-1 and UNC-73 overlap in the CAN and its axon. Scale bars: 5 µm.

View larger version (48K): [in this window] [in a new window]   Fig. 6. CRML-1 regulates VAB-8L-induced ALM abnormalities and SAX-3 levels. (A) Excessive ALM migration caused by VAB-8L misexpression is enhanced by loss of CRML-1 and suppressed by loss of UNC-73 Rac GEF activity. The percentage of ALMs found posterior of wild-type positions is depicted in dark gray (above x-axis), and the percentage of ALMs found anterior of wild-type positions is depicted in light gray (beneath x-axis). Statistically significant differences between gmIs14 [Pmec-7::vab-8::gfp] and crml-1; gmIs14 or unc-73; gmIs14 are marked (***P<0.0001). Differences between unc-73 crml-1; gmIs14 and unc-73; gmIs14 are indicated (#, P<0.03). unc-73 crml-1; gmIs14 was compared with unc-73; gmIs14. Error bars, s.e.m. For the distribution of ALMs relative to P and V cells and the number scored, see Fig. S1 in the supplementary material. At least 40 animals were scored per genotype. Animals were raised at 25°C. (B) VAB-8L-dependent ALM process rerouting is enhanced by loss of CRML-1 and suppressed by both the reduction of UNC-73 and overexpression of CRML-1. Statistically significant differences between gmIs14 and other strains are marked (***P<0.0001, *P<0.05); n.s., not significant. The statistically significant difference between gmIs14; gmEx527 strains bearing the array in ALM compared with those bearing the array but not in ALM is marked (#, P<0.03). At least 125 animals were scored per genotype, except gmIs14; gmEx527, ALM+ where n=26. (C-F') Nomarski (C-F) and GFP (boxed regions enlarged in C'-F') images of 2-fold C. elegans embryos expressing gmIs28 [Pmec-7::sax-3::gfp] in ALM and BDU neurons (arrows) just prior to cell migration. Anterior is to the left, dorsal is up. Scale bars: 5 µm. (G) Percentage of embryos in which a SAX-3::GFP signal was detected in ALM and BDU neurons during the 2-fold stage of embryogenesis. Strains were scored on the same day to ensure that the GFP signal intensities could be directly compared. The statistically significant difference observed between gmIs28 and crml-1; gmIs28 is marked (, P<0.009); n.s., not significant. Error bars indicate s.e.m. At least 80 animals were scored per genotype. The increase observed in the crml-1 mutant background was confirmed through an independent experiment that was scored blindly (data not shown). (H) Relative fluorescence intensities of SAX-3::GFP signals in ALM and BDU neurons from G. Only animals with both cells clearly in focus were included; at least 30 animals were scored per genotype. Error bars indicate s.e.m. Statistically significant differences between gmIs28 alone and the two unc-73-containing strains are marked (#, P<0.03).

View larger version (9K): [in this window] [in a new window]   Fig. 7. Model of SAX-3/Robo regulation. VAB-8L signals through UNC-73/Trio to positively regulate SAX-3/Robo levels and promote neuronal cell and axon growth cone migration, whereas CRML-1 signals through UNC-73/Trio to negatively regulate SAX-3/Robo levels, reducing its ability to promote migration.

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