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First published online January 23, 2009
doi: 10.1242/10.1242/dev.016816

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The cell migration molecule UNC-53/NAV2 is linked to the ARP2/3 complex by ABI-1

¹ Department of Biology, Trinity Western University, 7600 Glover Road, Langley, BC V2Y 1Y1, Canada.
² Department of Molecular Biology and Biochemistry, 8888 University Drive, Simon Fraser University, Burnaby, BC V5A 1S6, Canada.

View larger version (42K): [in this window] [in a new window]   Fig. 1. Characterization of the long isoforms (UNC-53L) of unc-53. (A) Structure of the unc-53 gene. The start of the various UNC-53L and UNC-53S isoforms are indicated by arrows. The promoter for UNC-53SA is between exons 5 and 8, and the promoter for UNC-53SB is located between exons 8 and 13 (Choi and Newman, 2006; Stringham et al., 2002). 2.9 kb of DNA upstream of the transcriptional start site of UNC-53LA was used to construct punc-53L::gfp. Alternatively spliced exons are shown in pink. unc-53(n152) is a 319-bp deletion removing parts of exons 18 and 19, producing a stop codon in exon 20 (Stringham et al., 2002), and n166 is a single nucleotide C to T transition in exon 19 that introduces a premature stop codon. (B) The longest polypeptide, UNC-53LA, is 1654 amino acids and contains a calponin homology domain (CH, red; amino acids 11-109), two LKK motifs (LKK, purple; 114-133 and 1097-1116), two proline-rich SH3-binding motifs (SH3b, green; 487-495 and 537-545), two coiled-coil regions (CC, blue; 890-923 and 1078-1113) and an AAA domain (yellow; 1292-1425). n166 introduces a premature stop codon at amino acid 949. Both n152 and n166 remove the coiled-coil, LKK and AAA domains from all isoforms. The first five exons of UNC-53 (UNC-53N; amino acids 1-139) were used for the production of PAB-UNC-53N antisera and the GAL4 DNA-binding domain (GAL4DBD) in pVA200 for yeast two-hybrid studies. (C,D) Expression pattern using punc-53L::gfp. (C) Adult hermaphrodite (anterior is left), showing GFP expression in head (HNeu) and tail (TNeu) neurons, the excretory cell (EXc), and the ventral nerve cord (VC). (D) Midbody, showing expression in the sex myoblasts (SM) and the ventral cord (VC). (E,F) Expression pattern using PAb-UNC-53N antisera. (E) Expression of UNC-53L throughout the excretory cell and canals (EXc). (F) UNC-53L expression in a pair of coelomocytes (CC). Scale bars: 100 µm.

View larger version (26K): [in this window] [in a new window]   Fig. 2. ABI-1 physically interacts with the N terminus of UNC-53. (A) ABI-1::GAL4AD interacts with UNC-53N::GAL4DBD by yeast two-hybrid assay. Yeast harboring both bait (pVA200) and prey (pVA305) plasmids grow in triple drop-out media (-Trp-Leu-His) and are positive for β-galactosidase, in contrast to yeast transformed with empty vectors (pAS2 and pSE1107), or with either the bait or prey vector alone. (B) GST pull-down assay. Equal amounts of ABI-1(12-427)-GST and GST alone were expressed in E. coli and bound to glutathione-conjugated beads (Bound). Soluble UNC-53N::6His lysates (left lane) were incubated with protein-bound beads and were observed to bind ABI-1(12-427)-GST (middle lane) but not GST alone (right lane), as detected by PAb-UNC-53N.

View larger version (49K): [in this window] [in a new window]   Fig. 3. Molecular organization of C. elegans ABI-1 gene and protein. (A) Gene structure of abi-1. The boundaries of introns, exons and the abi-1(tm494) deletion are indicated. (B) Protein structure of ABI-1. ABI-1 contains a Q-SNARE domain (Q-SNARE, green; amino acids 56-110) (Echarri et al., 2004), an Abl-interactor homeodomain homologous region (ABL-HHR, purple; 95-173), a serine-rich region (Ser-rich, orange; 243-259), three proline-rich SH3-binding motifs (SH3b, red; 296-306, 347-351 and 371-374) and an SH3 domain (SH3, blue; 416-469). ABL-HHR and SH3 domains were predicted using the Simple Modular Architecture Research Tool (SMART, http://smart.emblheidelberg.de). (C) Comparison of C. elegans ABI-1 with C. briggsae, human, mouse and Drosophila orthologs. Multiple alignments were performed using Clustal W 1.83 (http://align.genome.jp) with ABI-1 proteins from C. elegans (CE29545), C. briggsae (CAE64316), H. sapiens (NP_005461), M. musculus (Q3TJ64) and D. melanogaster (NP_477263), and were drawn using BOXSHADE (http://www.ch.embnet.org). The peptide sequence used to generate ABI-1 antibody PAb-ABI-1 is indicated in blue and the start site of the tm494 deletion is indicated by an asterisk.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Excretory canal morphology in wild-type, unc-53 and abi-1 animals. (A-G) Fluorescence micrographs of hermaphrodites carrying the ppgp-12::gfp transgene, allowing for the visualization of the excretory cell body and canals (anterior and posterior termini marked by arrows, A-E). Anterior is to the left and animals are displayed laterally with the exception of B and E, which are shown ventrally. (A) Morphology of the wild-type excretory cell body and processes. The excretory cell body is positioned on the ventral side of the posterior pharyngeal bulb and extends two canals towards the anterior of the animal to the tip of the head and two canals posteriorly to the tail. (B) unc-53(n166). (C) unc-53(rnai). (D) abi-1(rnai). (E) abi-1(tm494). (F,G) Lateral view of wild-type excretory canal (F) and abi-1(rnai) canal (G), showing numerous small cysts (arrows). (H) Quantification of posterior excretory canal outgrowth defects. The outgrowth of the posterior canals was divided into three regions (1-3) between the vulva and the tail as shown. The stop point of canals was determined by fluorescence microscopy for wild type (n=72), abi-1(rnai) (n=141), abi-1(tm494) (n=116), unc-53(rnai) (n=87), unc-53(n152) (n=37), unc-53(n166) (n=55), unc-53(n166)/mnDf87 (n=107), unc-53(n166); abi-1(rnai) (n=55), unc-53(n166); abi-1(tm494) (n=55), abi-1(tm494); ppgp-12::abi-1 (n=51) and unc-53(n152); ppgp-12::unc-53L (n=28). Scale bars: 100 µm.

View larger version (48K): [in this window] [in a new window]   Fig. 5. Mechanosensory neuron phenotype in wild-type, unc-53 and abi-1 animals. (A) Fluorescence micrograph showing a lateral view of a wild-type hermaphrodite carrying the pmec-4::gfp transgene. The PLM, PVM and ALM neuronal cell bodies and axons are shown. The stop point of the PLM axon was scored with respect to the wild-type position near the ALM cell body (arrowheads indicate final position of the PLM axon). (B) unc-53(n166) animal with truncated PLM and PVM axons. (C) unc-53(rnai) animal with truncated PLM and PVM axons. (D) abi-1(rnai) animal showing truncated PLM axon stopping short of the PVM cell body. (E) AVM axon in a wild-type animal. AVM axons were considered wild type if they projected anteriorly past the nerve ring (NR) and terminated at the tip of the animal. (F) abi-1(tm494) animal showing an AVM axon misdirected dorsally (arrow), followed by an abnormal posteriorly directed migration. Scale bars: 100 µm.

View larger version (63K): [in this window] [in a new window]   Fig. 6. ABI-1 expression and motoneuron migration defects. (A-D) Expression pattern of ABI-1. (A) Head of a larva expressing pabi-1::gfp in the AIYL/R neurons. (B,C) Expression of ABI-1 detected by immunostaining with PAb-ABI-1. (B) Head of a wild-type animal showing ABI-1 expression in the nerve ring and in the cell bodies of the RMEL/R neurons (arrow). (C) Ventral surface of the midbody of an adult worm showing ABI-1 in the cell bodies of motoneurons (arrowheads), in addition to the longitudinal tracts of the ventral cord (VC) and the dorsal commissures (arrow). (D) Expression of ABI-1 in coelomocyte (CC). (E-H) Fluorescence micrographs showing wild-type (E) and abi-1(rnai) (F-H) animals. Ventral is down in all cases except H. (E) Wild-type animal with motoneuron cell bodies located ventrally and dorsal commissures extending to the dorsal cord. (F) abi-1(rnai) animal showing a truncated and misguided dorsal commissure (arrowhead), and a truncated dorsal commissure with anterior and posterior ectopic lateral branches (arrow). (G) Misrouting and branching of dorsal commissures (arrows). (H) Ventral view of the ventral cord of an abi-1(rnai) animal showing marked defasciculation (arrows). Scale bars: 50 µm.

View larger version (36K): [in this window] [in a new window]   Fig. 7. RNAi of the long isoform of UNC-53 and overexpression of the UNC-53 CH domain generates excretory cell defects. (A) RNAi directed toward the long isoform of unc-53 results in posterior canal migration, with posterior excretory canals terminating at the vulva (arrow). Three percent of animals had canals terminating at the vulva and 24% failed to exit past the posterior gonad arm (n=92). (B) Adult animal expressing ppgp12::unc-53CH::gfp in the excretory cell body, as indicated by merge (yellow, arrowhead) with dsRED co-injection marker pDPY-30::NLS::DSRED2, displays a posterior excretory canal truncated at the midbody position near the vulva (arrow). Seven percent of canals terminate near the vulva and 22% failed to exit the posterior gonad arm (n=116). (C,D) Defects of the excretory canals in adult animals expressing ppgp-12::unc-53CH::gfp in the excretory canal. (C) Head of an adult animal showing cysts (arrow) and ectopic anterior branches (arrowhead). (D) Adult animal showing various excretory canal cysts (arrows). Scale bars: 100 µm.

View larger version (21K): [in this window] [in a new window]   Fig. 8. Excretory cell morphology in wild type, wve-1(rnai), nck-1(ok694) and arx-2(rnai) animals. (A-D) Fluorescence micrographs of hermaphrodites carrying the ppgp-12::gfp transgene. The final positions of the posterior excretory canals are marked by arrows and the anus is marked by arrowheads. Lateral views are shown in all cases except for B, which is a ventral view. Anterior is to the left. Scale bars: 100 µm. (A) Wild-type animal with posterior canals terminating near the anus. (B-D) RNAi-treated and mutant animals, as indicated have posterior excretory canals that stop anterior to their wild-type positions (arrows). (E) Quantification of posterior longitudinal migration defects. Wild type (n=72), wsp-1(gm324) (n=69), abl-1(ok171) (n=77), wve-1(rnai) (n=173), nck-1(ok694) (n=50), arx-2(rnai) (n=137), unc-53(n166) (n=55), wve-1(rnai); unc-53(n166) (n=90), arx-2(rnai); unc-53(n166) (n=91).

View larger version (24K): [in this window] [in a new window]   Fig. 9. Expression pattern of nck-1 using pnck-1::gfp. (A) Adult hemaphrodite showing expression in the excretory cell (Exc) and head neurons (HNeu). (B) View of the midbody of adult hemaphrodite showing expression in several motoneurons of the ventral cord (VC). Scale bars: 50 µm.

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