Pathways of Synaptic Vesicle Recycling.
R.B. Kelley. Dept of Biochemistry & Biophysics, University of California, San Francisco, CA 94143-0448.
Neurons and endocrine cells use a specialized form of endocytosis to recycle the membranes of their regulated secretory vesicles after exocytosis. This compensatory endocytosis uses the basic cellular machinery of endocytosis adapted to suit its special needs. For example, many cells have an intracellular store of plasma membrane proteins in small vesicles that are generated from endosomes. Neuroendocrine cells recycle their secretory granule membranes into a related population of synaptic-like microvesicles that are generated from endosomes by a coating mechanism that requires the heterotetrameric adaptor complex AP3, a small GTPase of the ARF family, the v-SNARE VAMP/synaptobrevin and a casein-kinase 1alpha-like activity. Sorting into this storage pathway, and away from lysosomal destruction requires a dileucine motif on cargo proteins. After exocytosis synaptic vesicle membranes are internalized directly from the plasma membrane by a mechanism that utilizes AP2 and clathrin, but does not overlap with that for transferrin receptor internalization. It appears to require some elements unique to neuroendocrine cells. One unique feature of nerve terminals is the enrichment of the endocytotic machinery on the plasma membrane as a 'honeycomb' structure in the holes of which sites of exocytosis are embedded. One of the components of the honeycomb is a dynamin-binding protein, DAP160, which resembles a second dynamin-binding protein, syndapin, in resembling a regulator of the actin cytoskeleton. Thus syndapin and DAP160 provide molecular clues to how the endocytotic machinery interfaces with the actin cytoskeleton during neurotransmitter release.
New elements of synaptic vesicle internalization identified by genetic screens.
Mani Ramaswami, Ph.D. University of Arizona, Tucson, AZ
By molecular, functional and anatomical analyses of Drosophila behavioral mutants isolated in classical genetic screens, we have identified molecular components and mechanisms missed by traditionally employed biochemical methods. The fine localization of dynamin, encoded by the Drosophila shibire gene, first revealed a fundamental organization of nerve terminals into active zones for transmitter release, and flanking "periactive" zones where endocytosis, and adhesion-mediated signaling may occur. Mutations that enhance the temperature-sensitive paralysis phenotype of Drosophila shibirets mutants have identified proteins required for synaptic-vesicle formation in vivo. Two of these proteins, stonedA and stonedB, products of an unusual discistronic mRNA, function as essential escort, or adaptor, proteins during synaptic vesicle internalization after exocytosis. Conserved stoned orthologs in C. elegans, mouse and human genomes may perform similar functions. Another enhancer of shibire gene (awd) is the Drosophila ortholog of the human tumor-suppressor gene, nm23. Awd/nm23 encodes a nucleoside diphosphate kinase (NDK) required to generate GTP from GDP. High levels of Awd/nm23 activity are essential for synaptic transmission and endocytosis in vivo. Our data indicate that NDK functions as a novel GEF (Guanosine nucleotide Exchange Factor) for the GTPase, dynamin, an activity that may also contribute to the tumor suppressor function of nm23. Together these studies, which originate from classical genetic analyses, contribute new insight into mechanisms that regulate synaptic vesicle recycling in vivo.
Regulation of secretion by Synaptic Vesicle Protein 2 (SV2)
Sandra Bajjalieh, University of Washington, Seattle, WA
The secretion of neurotransmitters shares many features with other membrane trafficking in eucaryotic cells. However, exocytosis at the synapse is distinguished from other forms of membrane fusion by its relatively low probability and strict dependence on elevated calcium concentrations. This tight regulation suggests the presence of regulators specific to regulated secretion. The synaptic vesicle protein SV2 is required for normal neurotransmission but not for vesicle fusion suggesting that it plays a role in the regulation of exocytosis. Mice lacking the most prevalent form of SV2, SV2A, fail to grow, develop severe seizures and die within three weeks of birth. Although neuronal and synaptic morphology is normal, GABAergic neurotransmission is aberrant in the hippocampus of SV2A knockouts. Recent studies of vesicle fusion in adrenal chromaffin cells suggest that loss of SV2A results in decreased calcium-stimulated vesicle fusion but not a change in the calcium sensitivity of fusion. In brain there is a significant decrease in high molecular weight SDS-resistant protein complexes containing the protein syntaxin, a component of the basic membrane fusion machinery. Together these results suggest that SV2 plays a role in the regulation of protein complexes required for vesicle fusion. SV2 may perform this function through its interaction with the synaptic vesicle protein synaptotagmin. Like SV2, synaptotagmin is required for normal neurotransmission but not for vesicle fusion. It is hypothesized to impart calcium regulation to secretion by participating in molecular interactions vis its two calcium binding domains, termed C2 domains. The second C2 domain (C2B) mediates a number of interactions including synaptotagmin dimerization, an interaction hypothesized to organize fusion complexes bound to the first C2 domain with proteins of the basic membrane fusion machinery. The C2B domain also mediates the calcium-inhibited interaction with SV2. We have mapped the site of SV2 binding to the same region of the C2B domain as required for synaptotagmin dimerization, and the binding of inositol polyphosphates, the clathrin adaptor AP2, calcium channels and the t-SNARE SNAP-25. This overlap suggests that SV2 may regulate synaptotagmin function by competing and/or facilitating its binding to other synaptic proteins. To test this hypothesis we are assessing the effects of SV2 peptides on the binding of synaptic proteins to recombinant C2B fusion peptides and have examined whether manipulation of this interaction affects the regulation of exocytosis in situ. Injection of SV2 peptides into cultured superior cervical ganglion neurons reduced the amplitude of post-synaptic responses but did not abolish them. These results are consistent with a role for SV2 in the modulation of synaptic exocytosis and suggest that at least part of its action may be the regulation of synaptotagminÕs interactions.
Towards an understanding of NSF's role in vesicle docking and fusion
Phyllis Hanson, Ph.D., Washington University, St. Louis, MO
NSF (N-ethylmaleimide sensitive fusion protein) is an ATPase required for ongoing membrane trafficking. It acts, at least in part, by dissociating the SNARE complexes thought to catalyze membrane fusion, freeing individual SNAREs for recycling and assembly into new complexes. In exocytosis, current models place NSF's activity after the fusion event and before recycling; other data hint at a role directly preceding fusion. Our biochemical analyses of reactions catalyzed by NSF suggest that it has effects on a variety of SNARE-containing complexes that could explain pre- and post-fusion roles. NSF can also directly regulate the conformation of individual SNAREs. I will present data characterizing these transformations, and will argue that NSF's ability to dissociate otherwise stable membrane protein complexes provides an unique form of regulation to vesicle docking and fusion.
Using single molecule and vesicle resolution in synaptic transmission studies.
Gadi Peleg1, Thomas Perroudt1, Marc H. Levin1, Clyde F. Wilson1, Dan T. Chiu1, Richard C. Lin2, Jagath R. Junutula2, Richard H. Scheller2 & Richard N. Zare1
1The Department of Chemistry, and 2The Department of Molecular and Cellular Physiology, Stanford University.
Advances in the ability to measure the activity of single units in neurons - such as patch clamp single channel recording - carry the potential for new findings about the actual ways the synapse works. The nondestructive nature of optical techniques can be implemented to follow the motions of the molecular machines while doing their function, and to provide more clues for answering questions such as how the SNARE complex (the synaptic core complex) mediates synaptic fusion. Few copies of the SNARE are believed to be acting in this molecular cooperative effort involved in fusion, and thus, single molecule optical studies are in particular suitable for this task. By developing methodologies to manipulate single vesicles, a complementary tool is provided for the performance of a single unit fusion assay. An overview of the single molecule and vesicle analysis methods will be presented. Examples of the advantages of the approach in studying the machinery of fusion and analyzing single vesicles will be given.
Supported by NIDA.
Gadi Peleg would like to acknowledge the NIH for a postdoctoral fellowship.
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