A RHO GTPase-mediated pathway is required during P cell migration in Caenorhabditis elegans

Abstract

The Rho family of guanine triphosphate hydrolases controls various cellular processes, including cell migration. We describe here the demonstration of a role for a RhoA GTPase homologue during cell migration in Caenorhabditis elegans. We show that eliminating or reducing rho-1 gene function by using a dominant-negative transgene or dsRNA interference results in a severe defect in migration of hypodermal P cells to a ventral position. Biochemical and genetic data also suggest that unc-73, which encodes a Trio-like guanine nucleotide exchange factor, may act as an activator of rho-1 in the migration process. Mutations in let-502 ROCK, a homologue of a RhoA effector in mammals, also cause defects in P cell migration, suggesting that it may be one of several effectors acting downstream of rho-1 during P cell migration. Finally, we provide evidence to support the idea that other small Rac subfamily small GTPases act redundantly and in parallel to RHO-1 in this specific cell migration event.

Figure 1

Phenotype of rho-1(dn) animals. (A) Bristol N2 strain during the second larval stage with P cells (arrowheads) and a full ventral cord; (B) rho-1(dn) animals lacking P cells and many descendants thereof in the ventral cord. The ventral cord is very thin in this animal because of missing P cells. (C) Lateral surface of a rho-1(dn) animal during L2 animal showing ectopic P cell neuronal descendants (arrows). (Bar = 10 μm.)

Figure 2

rho-1(dn) can cause a cytokinesis defect. (A) Abnormally large neurons (flanked by arrows) in a living rho-1(dn) animal during the second larval stage. (B) Fluorescent visualization of the unc-47∷GFP-positive cells (flanked by arrows) in the animal shown in A. (C) Fluorescent visualization of unc-47∷GFP-positive neurons in a fixed rho-1(dn) animal. (D) DAPI visualization of the multiple nuclei observed in the unc-47∷GFP-positive cells shown in C. Arrowheads in C and D denote the positions of nuclei as determined by DAPI staining. Arrows in C and D show an unc-47∷GFP-positive cell without the cytokinesis defect. (Bar = 7 μm.) In wild-type animals (see Fig. 1A), multiple neurons are clearly visible in the same region of a multinuclear cell in the mutant.

Figure 3

unc-47∷GFP visualization of P cell-derived neurons in strains with P cell migration defects. (A) Nomarski and (B) fluorescent image of unc-73(e936) in the unc-47∷GFP background. (C) Nomarski and (D) fluorescent image of a let-502(RNAi) larva in the unc-47∷GFP background. Mislocalized GABA neurons are indicated with white arrows.

Figure 4

GDP/GTP exchange assays. HeLa cell extracts expressing GEF1 or GEF2 of UNC-73 were assayed for their ability to catalyze GDP release from RhoA and Rac1, as described in Materials and Methods. (A) Relative amounts of bound [³H]-GDP after incubation of GEF1 or GEF2 with Rac1 or RhoA. (B) Schematic diagram of UNC-73.

Figure 5

Model for rho-1-mediated P cell migration.