The small GTP-binding protein Rab3A regulates a late step in synaptic vesicle fusion

Max Planck Institute for Experimental Medicine, Gottingen, Germany.
Nature (Impact Factor: 41.46). 07/1997; 387(6635):810-4. DOI: 10.1038/42954
Source: PubMed


The Rab family of low-molecular-mass GTP-binding proteins are thought to guide membrane fusion between a transport vesicle and the target membrane, and to determine the specificity of docking. The docking and fusion of vesicles is, however, a complex multistep reaction, and the precise point at which Rab proteins act in these sequential processes is unknown. In brain, the Rab protein Rab3A is specific to synaptic vesicles, whose exocytosis can be monitored with submillisecond resolution by following synaptic transmission. We have now determined the precise point at which Rab3A acts in the sequence of synaptic vesicle docking and fusion by using electrophysiological analysis of neurotransmitter release in Rab3A-deficient mice. Unexpectedly, the size of the readily releasable pool of vesicles is normal, whereas Ca2+-triggered fusion is altered in the absence of Rab3A in that a more-than-usual number of exocytic events occur within a brief time after arrival of the nerve impulse.

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    • "Synaptotagmin (syt) 7 complexes with CaM to accelerate replenishment in hippocampal neurons (Liu et al., 2014), but there is no evidence for syt 7 at retinal ribbon synapses (Kantardzhieva et al., 2012). Another potential CaM target is Rab3a, a vesicle-associated protein involved in both long-and short-term plasticity (Castillo et al., 1997; Geppert et al., 1997; Nonet et al., 1997; Leenders et al., 2001; Schlüter et al., 2004, 2006; accelerate replenishment by quickening the transition of vesicles to release sites at the ribbon base. (d) Vesicles spend only 47 ms at the membrane surface before fusion (membrane dwell time; Fig. S2), far shorter than the time constant for replenishment. "
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    ABSTRACT: At the first synapse in the vertebrate visual pathway, light-evoked changes in photoreceptor membrane potential alter the rate of glutamate release onto second-order retinal neurons. This process depends on the synaptic ribbon, a specialized structure found at various sensory synapses, to provide a supply of primed vesicles for release. Calcium (Ca(2+)) accelerates the replenishment of vesicles at cone ribbon synapses, but the mechanisms underlying this acceleration and its functional implications for vision are unknown. We studied vesicle replenishment using paired whole-cell recordings of cones and postsynaptic neurons in tiger salamander retinas and found that it involves two kinetic mechanisms, the faster of which was diminished by calmodulin (CaM) inhibitors. We developed an analytical model that can be applied to both conventional and ribbon synapses and showed that vesicle resupply is limited by a simple time constant, τ = 1/(Dρδs), where D is the vesicle diffusion coefficient, δ is the vesicle diameter, ρ is the vesicle density, and s is the probability of vesicle attachment. The combination of electrophysiological measurements, modeling, and total internal reflection fluorescence microscopy of single synaptic vesicles suggested that CaM speeds replenishment by enhancing vesicle attachment to the ribbon. Using electroretinogram and whole-cell recordings of light responses, we found that enhanced replenishment improves the ability of cone synapses to signal darkness after brief flashes of light and enhances the amplitude of responses to higher-frequency stimuli. By accelerating the resupply of vesicles to the ribbon, CaM extends the temporal range of synaptic transmission, allowing cones to transmit higher-frequency visual information to downstream neurons. Thus, the ability of the visual system to encode time-varying stimuli is shaped by the dynamics of vesicle replenishment at photoreceptor synaptic ribbons.
    The Journal of General Physiology 10/2014; 144(5). DOI:10.1085/jgp.201411229 · 4.79 Impact Factor
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    • "Thus, despite a wealth of genetic, overexpression, electrophysiological, and biochemical data regarding Rab3s (A, B, C, and D), it is difficult to formulate a coherent hypothesis about the functions of these proteins in membrane fusion. Rab3A has been claimed to maintain the supply of vesicles into the releasable pool (Coleman and Bykhovskaia, 2009) and to be unnecessary for refilling this pool (Geppert et al., 1997; Leenders et al., 2001); to increase the docking of vesicles to the plasma membrane (Tsuboi and Fukuda, 2006; Coleman et al., 2007; van Weering et al., 2007) and not to be involved in docking but in the recruitment of vesicles (Leenders et al., 2001; Tian et al., 2012); to regulate the number of vesicles that fuse during stimulation (Wang et al., 2008) and the amount of transmitter in a quantum (Geppert et al., 1997; Coleman and Bykhovskaia, 2009); to be unnecessary for fusion itself (Johannes et al., 1994) but to control exocytosis of vesicles through direct interaction with fusion pores of unusual characteristics (Wang et al., 2008); to limit evoked release to a single quantum for each release site during a single impulse (Johannes et al., 1998); to participate in priming (Dulubova et al., 2005; Huang et al., 2011), etc. Perhaps the only notion all studies agree on is that Rab3A modulates exocytosis. "
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    ABSTRACT: Exocytosis of mammalian sperm dense-core secretory granule relies on the same fusion molecules as all other secretory cells; one such molecule is the small GTPase Rab3A. Here, we report an in-depth biochemical characterization of the role of Rab3A in secretion by scrutinizing the exocytotic response of streptolysin O-permeabilized human sperm to the acute application of a number of Rab3A-containing constructs and correlating the findings with those gathered with the endogenous protein. Full length, geranylgeranylated, and active Rab3A elicited human sperm exocytosis per se. With Rab3A/Rab22A chimeric proteins, we demonstrated that the carboxy-terminal domain of the Rab3A molecule was necessary and sufficient to promote exocytosis, whereas its amino-terminus prevented calcium-triggered secretion. Interestingly, full length Rab3A halted secretion when added after the docking of the acrosome to the plasma membrane. This effect depended on the inability of Rab3A to hydrolyze GTP. We combined modified immunofluorescence and acrosomal staining protocols to detect membrane fusion and the activation status of endogenous Rab3 simultaneously in individual cells, and found that GTP hydrolysis on endogenous Rab3 was mandatory for fusion pores to open. Our findings contribute to establishing that Rab3 modulates regulated exocytosis differently depending on the nucleotide bound and the exocytosis stage under study.
    Journal of Molecular Cell Biology 08/2014; 6(4):286-98. DOI:10.1093/jmcb/mju021 · 6.77 Impact Factor
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    • "The levels of many other synaptic proteins, including GDI – known to bind RAB3A – were found not altered. In a later work, (Geppert et al., 1997) further characterized Rab3a-null mice and reported that the readily releasable pool of vesicles is normal in Rab3a-null mice, whereas the Ca 2+ triggered fusion is altered in the absence of RAB3A. In another work it was shown that in hippocampal CA3 mossy fiber synapses LTP and LTD were abolished in Rab3a-null mutant mice (Castillo et al., 1997). "
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    ABSTRACT: A RAS-related class of small monomeric G proteins, the RAB GTPases, is emerging as of key biological importance in compartment specific directional control of vesicles formation, transport and fusion. Thanks to human genetic observation and to the consequent dedicated biochemical work, substantial progress has been made on the understanding of the role played by RAB GTPases and their effector proteins on neuronal development and the shaping of cognitive functions. This review is highlighting these initial elements to broaden the current scope of research on developmental cognitive deficits and take the point of view of RAB GTPases control on membrane transport in neurons and astrocytes.
    Neuroscience & Biobehavioral Reviews 01/2014; 46. DOI:10.1016/j.neubiorev.2013.12.009 · 8.80 Impact Factor
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