Mutational Analysis of Rab3 Function for Controlling Active Zone Protein Composition at the Drosophila Neuromuscular Junction

Abstract

At synapses, the release of neurotransmitter is regulated by molecular machinery that aggregates at specialized presynaptic release sites termed active zones. The complement of active zone proteins at each site is a determinant of release efficacy and can be remodeled to alter synapse function. The small GTPase Rab3 was previously identified as playing a novel role that controls the distribution of active zone proteins to individual release sites at the Drosophila neuromuscular junction. Rab3 has been extensively studied for its role in the synaptic vesicle cycle; however, the mechanism by which Rab3 controls active zone development remains unknown. To explore this mechanism, we conducted a mutational analysis to determine the molecular and structural requirements of Rab3 function at Drosophila synapses. We find that GTP-binding is required for Rab3 to traffick to synapses and distribute active zone components across release sites. Conversely, the hydrolytic activity of Rab3 is unnecessary for this function. Through a structure-function analysis we identify specific residues within the effector-binding switch regions that are required for Rab3 function and determine that membrane attachment is essential. Our findings suggest that Rab3 controls the distribution of active zone components via a vesicle docking mechanism that is consistent with standard Rab protein function.

Rab3-GEF Controls Active Zone Development at theDrosophilaNeuromuscular Junction

“…3 B ). Rab3-GEF and YFP-Rab3 also partially overlap, although the colocalization between YFP-Rab3 and Brp is more prominent, as described previously for endogenous Rab3 ( Graf et al, 2009 ; Chen et al, 2015 ). In addition, Rab3-GEF localization at the NMJ appears generally unaffected by rab3 disruption.…”

“…The following fly lines were obtained from the Bloomington Stock Center: the P-element line P{EPgy2}Rab3-GEF EY06511 , the deficiency lines Df(1)ED7289 and Df(2R)ED2076, the UAS-cacophony-GFP line P(UAS-cac1-EGFP)422A ( Kawasaki et al, 2004 ), and the UAS-YFP-rab3 line P{UASp-YFP.Rab3}4EHP 05b ( Zhang et al, 2007 ). The rab3 rup mutant ( Graf et al, 2009 ) and the lines containing the UAS-rab3 and UAS-rab3Q80L transgenes ( Chen et al, 2015 ) were described previously.…”

“…Similar to other small GTPases, Rab3 activity is controlled by the opposing actions of GTPase accelerating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) that determine its GDP- versus GTP-bound state ( Cherfils and Zeghouf, 2013 ). Rab3 function for controlling Brp distribution requires both GTP binding and membrane association ( Chen et al, 2015 ), but not the binding of the effector protein RIM (Rab3 interacting molecule; Graf et al, 2012 ). However, the mechanism by which Rab3 controls CAZ formation and the proteins with which Rab3 must interact are largely unknown.…”

MAGIC: Mosaic Analysis by gRNA-Induced Crossing-over

“…( Figure 1F). Since Rab3 is a non-essential gene expressed only in neurons [37], its disruption in the female germline should affect neither egg production nor embryonic development of the progeny. Due to the dominant effect of ovo D1 , restoration of egg production can only result from mitotic recombination proximal to ovo D1 (e.g.…”

Presynaptic morphogenesis, active zone organization and structural plasticity in Drosophila

“…With an elegant experimental setup allowing for temporally restricted re-expression of Rab3 in a rab3 mutant background, BRP misdistribution could be reverted within 6–9 hours, suggesting a dynamic reshuffling of scaffold material between pre-existing AZs. To execute this function, Rab3 requires GTP-binding and membrane attachment, but surprisingly not the GTP hydrolytic activity, potentially suggesting a Rab3-dependent vesicle docking mechanism in this context [102]. A more recent study also showed that the Rab3 GDP-GTP exchange factor (Rab3-GEF) acts in conjunction with Rab3 to control AZ protein composition [103].…”