Analysis of the RAB family of GTPases in C. elegans and their role in regulating neuronal membrane trafficking

Rab GTPases are master regulators of intracellular trafficking. They are involved in every aspect of membrane transport from vesicle budding to vesicle fusion. Their functions are regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). The mammalian genome encodes over 60 different Rab proteins. Each of these Rabs localizes to specific compartments. The actions of multiple Rabs are coordinated through Rab cascades where a single Rab recruits the machinery to activate or inactivate a secondary Rab. Considering the large Rab family size, very little is known about the majority of its members and how they work together. As opposed to the human system, the C. elegans RAB family encodes 28 members with normally just one isoform per Rab member. The smaller Rab family size, together with the genetic tractability of C. elegans allowed for a broad-scale analysis of each RAB protein. For this analysis it was decided to initially determine the expression pattern and sub-cellular localization of all C. elegans RABs. Expression pattern analyses revealed that C. elegans rabs are differentially expressed with a pattern of preference for the nervous system. Additionally, the sub-cellular localization analysis of the Rabs showed that they localize to specific sub-cellular compartments with many displaying partial staining to the Golgi apparatus. The availability of mutants for 90% of the rabs in C. elegans further provided the opportunity to elucidate novel RAB functions. Since most RABs were neuronally expressed, all rab mutants were tested for changes in a set of nervous system mediated behaviors: movement, defecation motor program, and egg laying. Analysis revealed that several RABs are important regulators of these behaviors. Additionally all rab mutants were also tested for defects in synaptic transmission through sensitivity to aldicarb. Several new RABs were identified to modulate aldicarb sensitivity. Mutant analysis showed that although several rab mutants displayed phenotypes of nervous system dysfunction, the majority of animals were healthy. This suggested that many RABs function redundantly together in C. elegans. To elucidate which RABs cooperate together, a synthetic RNAi screen was conducted where each rab mutant was co-depleted with the remaining 27 Rabs. Co-depletion of multiple rabs has provided novel insights into the higher order RAB network. In the second chapter of this study, we aimed to identify novel RABs involved in dense core vesicle (DCV) signaling. Interestingly rab-5 and rab-10 mutants showed defects in DCV secretion. Additionally, two TBC (Tre-2/Cdc16/Bub2) domain-containing molecules, TBC-2 and TBC-4, were identified to function as potential GAPs for RAB-5 and RAB-10 respectively. Lastly we have identified an interaction between a RAB-5 effector, Rabaptin-5 (RABN-5), and TBC-4, which provides a link between RAB-5 and RAB-10 function. Taken together, these results suggest the existence of a novel Rab exclusion cascade in the regulation of DCV release, where active RAB-5 recruits TBC-4 for the local inactivation of RAB-10.