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unc-53 controls longitudinal migration in C. elegans

Eve Stringham^1,*,, Nathalie Pujol^1,,,¶, Joel Vandekerckhove¹ and Thierry Bogaert^1,2,

¹ Department of Biochemistry, Ghent University – Flanders Interuniversity Institute for Biotechnology (VIB09), Gent 9000, Belgium
² Singapore Institute of Molecular and Cell Biology, Kent Ridge Crescent, Singapore, and Medical Research Council Laboratory of Molecular Biology, Cambridge, CB2 2QH, UK
^* Present address: Department of Biology, Trinity Western University, 7600 Glover Road, Langley, BC V2Y 1Y1, Canada
Present address: Centre d’Immunologie de Marseille-Luminy, CNRS/INSERM/Université de la Méditerranée, Luminy Case 906, 13288 Marseille Cedex 9, France
Present address: Devgen N.V., Technologiepark 9, Blok DF1.60.14, 9052 Zwijnaarde, Belgium
These two authors contributed equally to this work

View larger version (97K): [in a new window]   Fig. 1. unc-53 sex muscle phenotypes. (A,B) Male sex muscles; lateral view. (A) Wild type with a pAB::GFP reporter revealing the diagonal muscles. (B) In the unc-53(e2432) mutant, which is stained with phalloidin-FITC, one diagonal muscle is shorter than normal and is attached in a dorsal position (arrow); the spicule retractor muscles are also abnormally short (arrowhead). (C-F) Hermaphrodite sex muscles. (C,D) Lateral view of wild-type (C) or unc-53 (D) vulval muscles (vms) with phalloidin-FITC staining. The distance between the anterior and posterior vm1 cells is reduced in the mutant as indicated by the double-headed arrows. (E) A schematic ventral view of the wild-type vulval muscles, bodywall cells are in red, seam cells in blue. The feet-like structures extending from the base of the four vm1 and four vm2 muscles are shown (arrowhead). (F) Ventral view of the four vm1 in unc-53(n152) revealed with a pAB::GFP reporter. No feet-like structures are visible, and the vm do not attach at their normal position. (G,H) Ventral view of the sex myoblasts during their division and migration in the wild-type (G) and unc-53(n152) (H) worms. Top, idealised schema; bottom, fluorescent images of worms expressing the pAB::GFP reporter. In the mutant, no longitudinal migration is observed and the distance (indicated by the arrows) between the anterior and posterior set of sex myoblasts is eliminated; they are all grouped around the future vulva (dotted circle) in the mutant. The bright round cells are motoneurones (m) in a higher plane of focus. Scale bars: 10 µm.

View larger version (63K): [in a new window]   Fig. 2. Longitudinal migration defects in unc-53 mutants. (A) The wild-type excretory canal. Two posterior canals extend from the excretory cell on each side along the lateral cord from the head to the tail. The boxes show the regions represented in B,C. These are DIC photomicrographs of adult worms showing the trajectory of the excretory canal (arrowheads). (B) In the wild type, the canal stops at the level of the anus (arrow). (C) An unc-53(n152) mutant, with a canal that stops at the level of the vulva (arrow). (D) The wild-type ALN and PLN projections. ALN and PLN send anteriorly directed axons up to the head along the sublateral dorsal and ventral cords, respectively. The box shows the region represented in E-H. These are fluorescence photomicrographs of adult wild-type (E,G) and unc-53(n152) mutant (F,H) worms expressing a pA::GFP reporter construct, showing the trajectory of an ALN (E,F) or PLN (G,H) axon (arrows). (F) The ALN axon has stopped its anterior outgrowth before the mid-body and sends several dorsal branches directly to the dorsal cord (arrowhead). (H) The PLN axon has stopped its anterior outgrowth before the mid-body and sends several dorsal branches to the ventral cord (arrowhead). c, cell body; pag, pre-anal ganglion. Scale bars: 10 µm.

View larger version (35K): [in a new window]   Fig. 3. Molecular organisation of the unc-53 gene. (A) The exon-intron structure of the unc-53 gene. The different SL1 transpliced sites are indicated, as well as the regions deleted in alleles n152 and e2432. Alternatively spliced exons are in pink. The extent of the phage and several cosmid clones are shown. The structure of the different GFP and minigenes constructs is also presented. (B) Structure of UNC-53. UNC-53 contains a calponin homology domain (CH, light green; amino acids 11-109), two proline-rich SH3-binding motifs, (SH3 b, dark green; 487-495 and 537-545), two coiled-coil regions (CC, red; 890-923 and 1078-1113; predicted by COILS at http://www.ch.embnet.org) and an ATPases associated with diverse cellular activities domain (AAA, yellow; 1292-1425) that contains a NTP-binding motif (1300-1307). The positions of the two potential LKK motifs (green, 114-133; 1097-1116) are indicated. Arrowheads mark the positions corresponding to the start of the e2432 and n152 deletions. (C) Alignment of the CH domain (PF00307), SH3-, LKK and NTP-binding motifs (PS00017). The Genbank Accession numbers for all the sequences used are: CeUNC-53, AF504312; HsNAV2, AX009326; Hs-actinin, P12814; GgDystrophin, P11533; MmSOS, Q62245; DmSOS, P26675; MmDynamin, P39053; RnPI3K-p85, Q63787; HsVillin, XP_010866; HsDematin, Q08495; CeThymosin, T32473.

View larger version (119K): [in a new window]   Fig. 4. punc-53::GFP expression pattern. DIC (A) and fluorescence images of pAB::GFP expression in transgenic animals (B-H). (A-F) Embryos of successive stages. (A,B) 1.5-fold stage embryo showing a pioneering neurone growing in the head (B, arrow). (C,D) A view between the end of the pharynx and the mid-body of a three-fold stage embryo, showing expression in DA motoneurones pioneering the dorsal cord; C and D are two different focal planes. (E,F) Late three-fold stage embryo: (E) shows the ventral side, (F) the dorsal side, revealing expression in all DA motoneurones and some neurones in head and tail ganglia. (G-L) Adult stage; anterior is towards the left. (G,H) Adult tail, ventral (G) and mid-plane (H) views showing expression in a phasmid socket (PHso), sphincter muscle (sph) and neurones in the preanal ganglion (pag). (I) Adult head showing expression in the amphid socket (AMso), ALA and several neurones in the dorsal, ventral and retrovesicular ganglion (rvg), as well as pharyngeal neurones, including M5. nr, nerve ring. (J) Ventral view of the mid-body showing vulval sex muscles (vm1 and vm2) of an adult hermaphrodite. One of the four foot-like attachments is highlighted. Ventral (K) and dorsal (L) views of the male tail, showing specific expression in diagonal (diag) and spicule retractor (sp) muscles. Scale bars: 10 µm.

View larger version (28K): [in a new window]   Fig. 5. Phenotypes of unc-53 and their promoter-specific rescue. (A) The Egl phenotype was scored by counting the number of progeny from individual worms of different genotypes and is represented as an average with standard deviation for each strain. For wild-type, n152 and e2432 homozygous, and n152/mnDf87 heterozygous worms, the progeny from 16, 29, 20 and 23 individual worms, respectively, were counted. Transgenic strains, all in an unc-53(n152) background, containing the cosmid T28D2 or the unc-53 minigene expressed under the control of the AB, A or B promoter are represented as n152;T28D2, n152;pAB::unc-53, n152;pA::unc-53 and n152;pB::unc-53, respectively. For each of these, the progeny of 12, 23, 23 and 19 individuals from two, five, two and five independent strains, respectively, were counted. Each independent strain of a given genotype gave similar results. Both unc-53 alleles show a strong Egl phenotype, indistinguishable from that of n152/mnDf87 heterozygotes. The unc-53(n152) phenotype was largely rescued by the cosmid T28D2, by pAB::unc-53 and by pB::unc-53, but not by pA::unc-53. (B) Quantification of ALN and PLN axonal outgrowth defects. The worm was divided into three regions (1-3) with reference to the vulva and the anus. The stop point of ALN axons was determined using a pA::GFP reporter carried by wild-type (n=100), n152 homozygous (n=50) and two independent strains of n152;pA::unc-53 (n=100) worms. The percentage of axons stopping at each of the three positions was then calculated. The PLN defect was scored similarly for 70, 44 and 80 worms, respectively. The two n152;pA::unc-53 strains gave comparable results. The unc-53(n152) phenotype was largely rescued by pA::unc-53. (C) Quantification of the excretory canal outgrowth defect. The worm was divided into four regions (1-4) between the vulva and the anus. The stop point of canals was determined by DIC microscopy for wild type (n=100), n152 (n=100), n152/mnDf87 (n=52), n152;T28D2 (n=48), n152;pAB::unc-53 (n=50), n152;pA::unc-53 (n=62) and n152;pB::unc-53 (n=57). For the transgenic strains, two independent lines for each were examined. The percentage of canals stopping at each of the four positions was then calculated. unc-53(n152) was associated with a stereotyped arrest of the canal at the level of the vulva, that was almost as penetrant as n152/mnDf87 heterozygotes. The phenotype was largely rescued by the cosmid T28D2. The pAB::unc-53 and pA::unc-53 constructs gave a degree of rescue, in contrast to pB::unc-53.

View larger version (125K): [in a new window]   Fig. 6. Overexpression of unc-53 in muscle cells. In body wall muscle cells, overexpression of unc-53 results in over-extension along the longitudinal axis. (A,C) Wild-type body wall muscle cells visualised with the punc-54::GFP reporter pPD93.48, at the comma (A) and three-fold (C) stages. (B,D) Transgenic embryos carrying the construct punc-54::unc-53 (pTB113) immunostained with the anti-UNC-53 monoclonal antibody MAb 16-48-2 at the comma (B) and three-fold stages (D). Arrows mark cell extremities. (E) Wild-type punc-54::GFP L1 larva and three-fold embryo visualised with DIC optics. (F) Mutant punc-54::unc-53 L1 larva showing severe morphological defects in the posterior body. Scale bars: 10 µm.

View larger version (42K): [in a new window]   Fig. 7. UNC-53 binds SEM-5/GRB2. (A) Immunoprecipitation of in vitro produced UNC-53. Radiolabelled UNC-5371C protein, transcribed and translated in vitro, was immunoprecipitated with the MAb 16-48-2 under denaturing conditions, then incubated with protein G sepharose. The beads were washed and the bound products analysed by SDS-PAGE and fluorography (lane + Ab). As a control (lane – Ab), a reaction without MAb 16-48-2 was treated identically. The lower bands most likely correspond to proteins initiated at internal methionines, or arising from premature termination or proteolytic degradation. (B) GST pull-down assays. The radiolabelled UNC-5371C protein was incubated with SEM-5-GST (left panel) or GST (right panel) coupled to sepharose beads. After four washes, the remaining proteins bound to the beads were analysed by SDS-PAGE and fluorography. U, unbound; W1-4, washes 1 to 4; B, bound protein. (C) Western blot overlay assays. Cell lysates from bacteria containing the UNC-5371C-encoding plasmid pTB61, from induced (+) or uninduced (–) cultures were denatured in Laemmli buffer and the proteins separated by 5-25% gradient SDS-PAGE. Total proteins were revealed by Coomassie Blue staining (left panel). Additional gels were blotted to nylon membrane, incubated with biotinylated GST (middle panel) or biotinylated GST-GRB2 protein (right panel), and bound protein complexes subsequently detected using an alkaline phosphatase-linked anti-streptavidin-antibody and chromogenic substrate. The positions of the molecular weight markers (in kDa) are shown.