QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/dev.00300

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Struckhoff, E. C. Articles by Lundquist, E. A. Search for Related Content PubMed PubMed Citation Articles by Struckhoff, E. C. Articles by Lundquist, E. A.

The actin-binding protein UNC-115 is an effector of Rac signaling during axon pathfinding in C. elegans

Department of Molecular Biosciences, University of Kansas, 1200 Sunnyside Avenue, Lawrence, KS 66045-7534, USA

View larger version (102K): [in a new window]   Fig. 1. rac double mutants display defects in PDE axon pathfinding. Fluorescence micrographs of PDE neurons visualized using the integrated osm-6::gfp transgene lqIs3 are shown. Anterior is towards the left. In A-D,G, dorsal is upwards. (E-F) Ventral aspects showing PDE axons in the ventral nerve cord (VNC). (A,B) PDE neurons in wild-type animals. (A) The left PDE cell body is indicated with an arrow. The VNC is indicated with arrowheads. The amphid and phasmid ganglia of the head and tail, respectively, are shown. The V indicates the position of the vulva. The out-of-focus spot to the left of the PDE is the out-of-focus PDE cell body from the right side of the animal. (B) A single, unbranched PDE axon extended ventrally in the post-deirid commisure to the VNC, where the axon bifurcated and extended anteriorly and posteriorly in the VNC (arrowheads). A single, unbranched dendrite with an exposed, ciliated tip extended dorsally from the PDE cell body. The broken line indicates the out-of-focus PDE axons in the VNC. (C,D) The PDE axons of ced-10(M+); mig-2 animals. (C) The PDE axon failed to reach the VNC (arrowheads) and wandered along the lateral body wall. (D) The PDE axon bifurcated prematurely before reaching the VNC (arrowheads) and the axons extended anteriorly and posteriorly along the lateral body wall. (E) Wild-type PDE axons formed a tight bundle in the VNC as they extended anteriorly and posteriorly. (F) The PDE axons of a ced-10(M+); mig-2 animal were defasciculated and terminated prematurely in the ventral cord. (G) The PDE axon displayed an ectopic branch (arrow). Scale bar: 20 µm in A; 10 µm in B-G.

View larger version (149K): [in a new window]   Fig. 2. Constitutively-active Racs induce ectopic morphogenetic structures in the PDE neuron. Fluorescence micrographs of PDE neurons from adult animals harboring an osm-6::gfp transgene. Anterior is towards the left and dorsal is upwards. The PDE cell bodies are indicated by arrowheads. (A) A wild-type PDE neuron displayed a single, unbranched axon extending to the ventral nerve cord (image is the same as in Fig. 1B). (B-F) PDE neurons of animals harboring a rac-2(G12V) transgene. (B) A rac-2(G12V) PDE neuron displayed ectopic axon branching and ectopic axons extending from the cell body. The ciliated dendrite (cilium marked by an arrow) also displayed ectopic branches. (C) A PDE neuron displayed a large, sheet-like plasma membrane extension (arrow). (D) A PDE neuron exhibited numerous thin, finger-like plasma membrane extensions (arrow). (E,F) A PDE neuron displayed two ectopic lamellipodia-like extensions (a and b) that were dynamic over time. (F) After 2 hours, one sheet-like extension (a) had ramified into two thinner neurite-like structures and another (b) had narrowed and elongated. New filopodia-like projections from the cell body not seen in E are indicated (c). Scale bars: 5 µm.

View larger version (87K): [in a new window]   Fig. 3. GFP::RAC-2 is anchored to the plasma membrane. (A) A diagram of the unc-115::gfp::rac-2 transgene and the putative GFP::RAC-2 molecule produced from this transgene are shown. The neuron-specific unc-115 promoter was used to drive expression of gfp fused in-frame to the entire wild-type rac-2-coding region, including introns. This transgene is predicted to produce a RAC-2 molecule with an N-terminal GFP tag. The CAAX motif at the C terminus of RAC-2 might direct covalent addition of a prenyl group and subsequent anchorage of the molecule to the plasma membrane. The position of the frame shift mutation in unc-115::gfp::(FS)::rac-2 is indicated. (B,D) Wild-type animals harboring the unc-115::gfp::rac-2 transgene. GFP::RAC-2 accumulated at the cell margins of neuroblasts of the 1.5-fold embryo in (B) and of neurons in the L1 larva in C. The animal in C displayed strong GFP::RAC-2 accumulation in the nerve ring. (D) A magnified image of the area around the nerve ring of the animal in (C). Neuron cell bodies with GFP::RAC-2 at their periphery are evident. (E,G) Wild-type animals of the same age as those in B,C harboring the unc-115::gfp::(FS)rac-2 transgene that contains a frameshift mutation between gfp and rac-2 sequences. This transgene is predicted to encode a full-length GFP without RAC-2 sequences. GFP accumulation was no longer observed at cell margins of neuroblasts and neurons and was observed throughout the cytoplasm, and nerve ring accumulation was abolished. (G) A magnified view of the area around the nerve ring of the animal in F. Scale bars: in B 10 µm for B,C,E,F; in D 2 µm for D,G.

View larger version (28K): [in a new window]   Fig. 4. The villin headpiece domain of UNC-115 binds to actin filaments. (A) The 639-residue UNC-115 polypeptide. The N terminus consists of three LIM domains and the C-terminus is similar to the headpiece domain of villin (VHD). The region of UNC-115 used in actin-binding assays is indicated by a bar below the diagram. (B) An alignment of the UNC-115 VHD and chicken villin VHD (Gg HP67). Basic residues in the UNC-115 VHD are in blue, and those that were changed to glutamic acid residues to produce the VHD mutant protein are shown with asterisks. The leucine and tryptophan residues that form a hydrophobic `cap' (see below) are boxed. (C) A structural model of the UNC-115 VHD based upon the NMR structure of Gg HP67 (Varder et al., 2002) (see Materials and Methods). Blue, basic groups; red, acidic groups; white, neutral groups. Shown are the `cap' formed by the leucine and tryptophan boxed in B, the charged `crown' (broken line), and the `positive patch' formed by basic residues (Vardar et al., 2002). The locations of basic residues that were changed to glutamic acid residues in UNC-115 VHD mutant protein are indicated by asterisks. (D) A western blot showing that the UNC-115 VHD bound to actin. Equimolar amounts (5 µM) of wild-type and VHD mutant 6HIS::DHFR::UNC-115 (46kD) were mixed with actin filaments, which were then sedimented (see Materials and Methods). Wild-type 6HIS::DHFR::UNC-115 co-sedimented in the presence but not in the absence of actin filaments. Mutant 6HIS::DHFR::UNC-115 (see B) failed to co-sediment with actin filaments.

View larger version (14K): [in a new window]   Fig. 5. UNC-115 acts downstream of Rac signaling in CAN and PDE axon pathfinding. The genetic relationships between the three racs, unc-73 and unc-115 are shown. Arrows indicate that the genes act in the same pathway. Three rac genes ced-10, mig-2 and rac-2/3, define three parallel pathways with overlapping function. unc-73 acts in all three pathways, and unc-115 acts downstream of rac-2 and possibly ced-10. unc-73 might have rac-independent roles in axon pathfinding possibly mediated by the rho-1 gene that encodes the single C. elegans Rho GTPase. GEF, GTP exchange factor; abp, actin-binding protein.