| The small GTPase Rab3 regulates several aspects of neurotransmitter release, such as synaptic vesicle (SV) fusion and release probability, and has been implicated in synaptic plasticity and the regulation of neurotransmitter release by a retrograde synaptic signal. Like all Rab proteins, Rab3 cycles through GTP- and GDP-bound states. Membrane-associated Rab3 binds to its effectors (e.g. RIM) in the GTP-bound state and is extracted from membranes by GDI in the GDP-bound state. This Rab3 cycle occurs in parallel to the SV exo- and endocytosis cycle; however, relatively little is known about how the RAB-3 and SV cycles are coupled.;To address this issue, we used fluorescence r&barbelow;ecovery a&barbelow;fter p&barbelow;hotobleaching (FRAP) of GFP-tagged Rab3, to investigate how SV fusion regulates the dynamic exchange of RAB-3 at C. elegans neuromuscular synapses. Photobleaching experiments indicate that 40% of synaptic RAB-3 is mobile. Mobile RAB-3 molecules undergoing dynamic exchange at synapses consist of molecules associated with the plasma membrane (undergoing lateral diffusion), whereas SV-bound RAB-3 molecules are immobile. Mutations disrupting the GTP cycle decrease RAB-3 binding to SVs, thereby diminishing RAB-3’s ability to promote exocytosis. Synaptic RAB-3 molecules can exhibit fast or slow mobility. Mutations that block exocytosis dramatically reduce the rapidly exchanging RAB-3 pool but have little effect on the RAB-3 pool undergoing slow exchange. Interestingly, different exocytic proteins and different RAB-3 effectors promote kinetically distinct forms of RAB-3 exchange. Collectively, our results suggest that synapses contain readily and slowly exchanging pools of RAB-3 and that different modes of exocytosis produce distinct modes of RAB-3 mobility. Our results also suggest a mechanism for how changes in synaptic activity cause changes in the composition of active zones. |
