Rac GTPase Plays an Essential Role in Exocytosis by Controlling the Fusion Competence of Release Sites

Abstract

The role of small GTPases of the Rho family in synaptic functions has been addressed by analyzing the effects of lethal toxin (LT) from Clostridium sordellii strain IP82 (LT82) on neurotransmitter release at evoked identified synapses in the buccal ganglion of Aplysia. LT82 is a large monoglucosyltranferase that uses UDP-glucose as cofactor and glucosylates Rac (a small GTPase related to Rho), and Ras, Ral, and Rap (three GTPases of the Ras family). Intraneuronal application of LT (50 nm) rapidly inhibits evoked acetylcholine (ACh) release as monitored electrophysiologically. Injection of the catalytic domain of the toxin similarly blocked ACh release, but not when key amino acids needed for glucosylation were mutated. Intraneuronal application of competitive nucleotide sugars that differentially prevent glucosylation of Rac- and Ras-related GTPases, and the use of a toxin variant that affects a different spectrum of small GTPases, established that glucosylation of Rac is responsible for the reduction in ACh release. To determine the quantal release parameters affected by Rac glucosylation, we developed a nonstationary analysis of the fluctuations in postsynaptic response amplitudes that was performed before and after the toxin had acted or during toxin action. The results indicate that neither the quantal size nor the average probability for release were affected by lethal toxin action. ACh release blockage by LT82 was only caused by a reduction in the number of functional release sites. This reveals that after docking of synaptic vesicles, vesicular Rac stimulates a membrane effector (or effectors) essential for the fusion competence of the exocytotic sites.

Adaptation of Granule Cell to Purkinje Cell Synapses to High-Frequency Transmission

“…Variance-mean analysis was developed and described in detail in previous studies (Silver et al, 1998;Reid and Clements, 1999;Clements and Silver, 2000;Humeau et al, 2001Humeau et al, , 2002Humeau et al, , 2007Clements, 2003;Silver, 2003;Saviane and Silver, 2007;Doussau et al, 2010). Because of the long stable recordings required for these analyses, they were applied to recordings in which compound GC responses were elicited in PCs by extracellular stimulation in the GC layer.…”

“…Thus, N does not take into account long-lasting silent sites. In practical terms, only sites containing a readily-releasable vesicle (i.e., docked and fully primed) would contribute to the observed N (Humeau et al, 2001(Humeau et al, , 2002(Humeau et al, , 2007. Individual responses are assumed to sum linearly (a significant approximation in the presence of multivesicular release).…”

“…Because paired GC-PC recordings do not allow the long protocols required for extensive direct characterization of Pr site (Silver et al, 1998(Silver et al, , 2003Oleskevich et al, 2000;Humeau et al, 2002), we sought to determine the probability of failures of transmission, Pf syn (see definitions in Materials and Methods). We first used the approach of Isope and Barbour (2002), based on the comparison of the histogram of EPSC amplitudes and that of the noise.…”

Distinct roles of Rac1/Cdc42 and Rho/Rock for axon outgrowth and nucleokinesis of precerebellar neurons toward netrin 1

“…Lethal toxin from Clostridium sordellii strain VPI9048 [LT-9048 (Humeau et al, 2002)] was used at a final concentration of 1 ng/ml to inhibit Rac1 and Cdc42. TAT-C3 [a kind gift from Jacques Bertoglio (Sauzeau et al, 2001)] was used at a final concentration of 20 µg/ml to inhibit RhoA, RhoB and RhoC signaling.…”

βPIX-activated Rac1 stimulates the activation of phospholipase D, which is associated with exocytosis in neuroendocrine cells

“…Earlier experiments based on the use of clostridial toxins, which differentially impair the function of Rho GTPases, suggest that Rac might also play a role in secretion, although in an actin-independent manner (Gasman et al, 1999). Hence, the participation of Rac1 in Ca 2+ -regulated exocytosis has subsequently been demonstrated in neurons (Humeau et al, 2007;Humeau et al, 2002) and in various endocrine cells, including chromaffin cells and pancreatic β-cells and acini (Amin et al, 2003;Bi and Williams, 2005). The upstream events leading to the activation of Rac1 and the downstream signalling pathway by which Rac1 controls the exocytotic response, however, remains unexplored.…”