Neurotransmitter release by fusion of synaptic vesicles with the pre-synaptic plasma membrane upon transient increases in intracellular Ca2+ is essential for propagating action potentials between neurons. Synaptic vesicle (SV) fusion requires cooperative interactions between the lipids and proteins of both the pre-synaptic and SV membranes. Although the structure of many SV proteins have been solved and a prototypic structural model of an individual SV has been presented, an overall picture of how proteins are organized at the vesicle surface is still lacking. It is well established that the vertebrate and invertebrate nervous systems exhibit many similarities in terms of neuronal function. The squid nervous system in particular has been used to demonstrate the neuronal resting potential as well as to record electrical action potentials. The squid was also used to define the role of calcium in synaptic transmission. The squid optic lobe contains 50-80% of the neurons in the squid central nervous system and is therefore an excellent source of synaptic vesicles to study their biophysical and structural properties. Dowdall and Whittaker described the isolation of synaptic vesicle rich fractions from squid optic lobe obtained by osmotic shock. However, the purity of their final fraction was never critically evaluated either by biochemical or electron microscopy techniques (Dowdall and Whittaker, 1973). Chin and Goldman used the same method to purify synaptic vesicles from frozen squid optic lobe and added controlled-pore glass chromatography as a final purification step. Based on their detailed biochemical analysis, the vesicle fraction was approximately 60% pure. Using advances in the purification of synaptic vesicles from rat brain (Huttner et al., 1983), a SV isolation protocol for squid (Logilo pealei) optic lobe was optimized in this study to obtain a highly pure and intact SV population for biochemical and ultrastructural studies. The SV-enriched fractions were analyzed to evaluate their purity and size distribution by electron microscopy (EM), as well as the effects of different EM specimen preparation techniques on the average SV size. The distribution of SV size in SV-enriched fractions suggests that the SV we isolated are more than 95 percent pure. Finally, the purified vesicles were used to characterize the organization of the surface of individual SVs by tungstate-based negative stain EM tomography. Here we present a three-dimensional molecular rendering of the surface structure, presumably V-ATPase, reflecting an individual SV, rather than the average of many SV. Dowdall and Whittaker described the isolation of a synaptic vesicle rich fraction from squid optic lobe. However, the purity of the SV-rich fraction was not evaluated by either biochemical or electron microscopy techniques. Subsequently, SVs purified by this method from frozen squid optic lobe with the additional purification step of controlled-pore glass chromatography yielded a purity of approximately 60% (Chin and Goldman, 1992). Since rat brain SV can be purified to >95% purity (Huttner et al., 1983) we modified this purification scheme for rat brain to isolate highly pure SVs from fresh squid optic lobes. Determinations of SV size distributions in SV-enriched fractions suggests that this isolation protocol provides >95% pure synaptic vesicles from squid optic lobes. The results presented here clearly demonstrate that the estimates of SV size are dependent upon the method of preparation of the SV sample for EM. Section thickness is unlikely to be a source of variation because we only measured vesicles for which the delimiting edges of the membrane were visible in a single section and then computed the size based on ferrets diameter with a circularity value close to unity. Fixation and processing conditions can alter the absolute dimensions of organelles;glutamatergic vesicle diameter in the rapid frozen, freeze-substituted anteroventrocochlear nucleus is notably greater (7 nm) than in aldehyde-fixed hippocampus. Negative stained, non-fixed SV samples had the largest mean diameter relative to the other samples obtained by other preparation techniques. It should be kept in mind that the negative stain image is a projection of the whole vesicle while the sections are often only part of a vesicle and not every instance include the section from the equator. It is also known that when protein-containing lipid vesicles are negatively stained, these vesicles dry down and collapse by approaching the diameter of two discs with the same area, one on top, one below. Changes in vesicle shape from spheres to disk by negative staining would explain the significant increase in negative stained SV diameter that we observed. Indeed such flattening is directly demonstrated in the tomographic reconstructions here (Fig 3A, inset). With this method, SV membranes appear to be uniformly coated with structures big knobs and smaller hairs, ostensibly the knobs are the V- ATPase standing out because it is much larger than other SV proteins. Because the stain does not get inside the vesicle, its membrane is not enclosed on both sides, and it is poorly outlined by negative stain in XZ projections of tomograms...the membrane appears only as a faint boundary between stain and non-stain. The upper and lower boundaries of vesicles, due to the hair-like structures, are hard to see except where the boundary is decorated by knobs. Here, the knobs clearly delineate the position of the membrane at that point. Thus, knobs on vesicle membranes near the centers of vesicles appear to be on a flat surface suspended across the ends of the vesicles, like the top of a drum, rather than perched on domes. Thus the vesicles are flattened, and actually a little thicker at their edges where their membranes fold back on themselves. It remains to be determined the degree to which detergents used for vesicle isolation in previously published methods, and the specificity of metal-protein surface interactions implicit to the negative staining procedure, affect the organization of proteins on the SV surface. We demonstrate here three-dimensional molecular reconstructions based on tomograms from single intact synaptic vesicles isolated without detergents. The tomograms are obtained by negative stain based electron microscopy, which permits imaging free from the assumptions of symmetry, classification, and averaging. While further research is needed to determine if proteins of specific type may cluster with each other in restricted domains, below the structures noted above, negative staining tomography shows a continuous sheet of protein blanketing the surfaces of synaptic vesicles. The more our knowledge of both macromolecular crystal structures and the biochemical regulation of macromolecular structure in synapses grow, the more clear our need becomes for detailed structural analysis of synaptic membranes. Analysis of the spatial organization of these membranes helps us to understand the assembly of synaptic organelles and the interactions between them, crucial to understanding how these change in time with activity and plasticity, and how they traffic. Here, we have optimized the routine collection of relatively large quantities of highly purified synaptic vesicles and performed negative staining tomography, to investigate macromolecular distribution on the synaptic vesicle. Proteins on the surfaces of SV appear spread over their entire surface.

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21
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2011
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$1,163,820
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