Characterization of RAB-10 in PLM dendrite termination of C. elegans

To better understand how the brain works, it is necessary to study how axons and dendrites (neurites) regulate their outgrowth and maintain their shape. Directed membrane trafficking is crucial to neural development and morphogenesis. RAB-10, a small GTPase, has been shown to mediate polarized secretion and recycling of transport vesicles at the plasma membrane in both neuronal and non-neuronal cells. However, little is known about RAB-10 function in neural morphogenesis. Using the in vivo model, C. elegans, we focus on dendrite termination of the posterior mechanosensory neuron, PLM (posterior lateral microtubule). The conserved molecular pathway involved in termination includes two conserved genes, sax-1 and sax- 2. sax-2 encodes a large scaffold-like protein that functions with sax-1, a protein kinase. sax-1 and sax-2 single mutants, as well as the sax-1/sax-2 double mutant, lead to similar levels of PLM overextension, suggesting they function together to terminate dendrite outgrowth. Moreover, single mutant analysis of F09A5.4, a known SAX-1 cofactor, also leads to PLM overextension. Few downstream effectors of the sax-1/sax-2 pathway have been identified. In yeast, genetic and biochemical studies with Cbk1p, an ortholog of SAX-1, displays an interaction with Sec2p, an exchange factor of Sec4p, the founding member of the Rab family of GTPases. The closest animal homologs of Sec4p include RAB-8 and RAB-10. We find that loss of rab-10, but not rab-8, disrupts PLM dendrite termination comparably to sax-1. Rab GTPases function primarily in vesicle trafficking. Our finding that RAB-10 functions in PLM dendrite termination would be consistent with the observation that SAX-2 localizes to small puncta resembling transport vesicles. To determine if rab-10 functions in the same pathway as sax-1 and sax-2, double mutant strains, involving sax-1 and sax-2 were examined. Findings suggest that RAB-10 may utilize the SAX-2 scaffolding protein, but does not function in the same pathway as SAX-1. Next, transgenic animals expressing a rab-10::mCherry fusion in select worm neurons including PLM were built. Importantly, rab-10(+)::mCherry rescued the rab-10 loss-of-function mutant indicating that RAB-10::mCherry is functional. Moreover, this rescuing rab-10::mCherry fusion was used to observe the subcellular localization of RAB-10 in the PLM mechanosensory neuron, where RAB-10 was seen at the growth cone and various points along the PLM dendrite in L1 larval stage animals.