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The GTPase Rab3a controls calcium-dependent exocytosis negatively
The Latest in Synaptic Transmission
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The GTPase Rab3a controls calcium-dependent exocytosis negatively Ludger Johannes*, Pierre-Marie Lledo§, Mich~le Roa#, Jean-Didier Vincent§, Jean-Pierre Henry* and Francois Darchen* *CNRS URA 1112, Institut de Biologie Physico-chimique, 13 rue P. et M. Curie, 75005 Paris, France; §CNRS, Institut A. Fessard, Avenue de la Terrasse, 91198 Gif sur Yvette Cedex, France; #CNRS URA 361, Institut Pasteur, 25 rue du Dr. Roux, 75724 Pads Cedex, France.
Abstract There is accumulating evidence that small GTPases of the rab family regulate intracellular vesicle traffic. It has been suggested that Rab3a, which is associated with synaptic vesicles in neurons and with secretory granules in adrenal chromaffin cells, might regulate exocytosis. To address the function of Rab3a we expressed in PC12 cells various Rab3a mutant proteins. Mutants either defective in GTP hydrolysis or in guanine nucleotide binding inhibited exocytosis, as measured by a double indirect immunofluorescence assay. Moreover, injection of the purified mutant proteins into bovine adrenal chromaffin cells also inhibited exocytosis, as monitored by membrane capacitance measurements. In contrast, a GDP-bound Rab3a mutant did not affect the secretory response. When bovine chromaffin cells were stimulated repetitively, desensitisation of the response was observed. However, cells which were intracellularly injected with antisense oligonucleotides targeted to the rab3a messenger exhibited an increasing potential to respond to repetitive stimulations. These results provide clear evidence that Rab3a is involved in regulated exocytosis and suggest that Rab3a is a regulatory factor that prevents exocytosis to occur unless secretion is triggered. It is proposed that Rab3a is involved in adaptive processes such as response habituation. Finally, the calcium sensitivity of the secretory response was investigated and found to be modified in antisense injected cells, suggesting a link between calcium signalling and Rab3a activity.
POSTER
403
39
The GTPase Rab3a controls calcium-dependent exocytosis negatively Ludger Johannes*, Pierre-Marie Lledo§, Mich~le Roa#, Jean-Didier Vincent§, Jean-Pierre Henry* and Francois Darchen* *CNRS URA 1112, Institut de Biologie Physico-chimique, 13 rue P. et M. Curie, 75005 Paris, France; §CNRS, Institut A. Fessard, Avenue de la Terrasse, 91198 Gif sur Yvette Cedex, France; #CNRS URA 361, Institut Pasteur, 25 rue du Dr. Roux, 75724 Pads Cedex, France.
Abstract There is accumulating evidence that small GTPases of the rab family regulate intracellular vesicle traffic. It has been suggested that Rab3a, which is associated with synaptic vesicles in neurons and with secretory granules in adrenal chromaffin cells, might regulate exocytosis. To address the function of Rab3a we expressed in PC12 cells various Rab3a mutant proteins. Mutants either defective in GTP hydrolysis or in guanine nucleotide binding inhibited exocytosis, as measured by a double indirect immunofluorescence assay. Moreover, injection of the purified mutant proteins into bovine adrenal chromaffin cells also inhibited exocytosis, as monitored by membrane capacitance measurements. In contrast, a GDP-bound Rab3a mutant did not affect the secretory response. When bovine chromaffin cells were stimulated repetitively, desensitisation of the response was observed. However, cells which were intracellularly injected with antisense oligonucleotides targeted to the rab3a messenger exhibited an increasing potential to respond to repetitive stimulations. These results provide clear evidence that Rab3a is involved in regulated exocytosis and suggest that Rab3a is a regulatory factor that prevents exocytosis to occur unless secretion is triggered. It is proposed that Rab3a is involved in adaptive processes such as response habituation. Finally, the calcium sensitivity of the secretory response was investigated and found to be modified in antisense injected cells, suggesting a link between calcium signalling and Rab3a activity.