Regulation of the Small GTPase RAB-3 at the C. elegans Neuromuscular Junction

The small GTPase RAB-3 associates with synaptic vesicle (SV) membranes in a GTP-dependent manner; promotes SV fusion; regulates SV release probability; and has been implicated in synaptic plasticity and the regulation of neurotransmitter release by a retrograde synaptic signal. Although the regulation of RAB-3 could provide a biochemical mechanism for altering SV release, it remains poorly understood.

The activity of RAB-3 depends on three parameters: its nucleotide state, the association/dissociation from SVs and its interaction with effectors. To dissect the mechanisms that regulate RAB-3 activity we have focused on using fluorescently-tagged wild type, GTP-locked and GDP-locked versions of RAB-3 to analyze the diffusion and membrane association of RAB-3 in C. elegans NMJ synapses. Preliminary data shows that RAB-3 is relatively immobile, suggesting that most of it is associated to SVs. Meanwhile, GTP- and GDP-locked versions of RAB-3 show a primarily diffuse distribution, suggesting they may be stranded in the plasma membrane and may be disrupting RAB-3 interaction with its effector UNC-10/RIM and thus RAB-3 function. We are currently analyzing how the disruption of the function of the RAB-3 regulatory proteins, namely its guanine-nucleotide exchange factor, its GTPase activating protein, and the RAB-3 effector UNC-10/RIM affect the diffusion and distribution of RAB-3 proteins. Finally, we are looking into how the nucleotide state of RAB-3 regulates SV secretion. In mice and flies, overexpression of GTP-locked RAB-3 leads to a decrease in secretion. We hypothesize that overexpression has this effect on secretion because it may alter the distribution of the RAB-3 effector UNC-10/RIM.