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Cellular compartments that cannot be biochemically isolated are challenging to characterize. Here we demonstrate the proteomic characterization of the synaptic clefts that exist at both excitatory and inhibitory synapses. Normal brain function relies on the careful balance of these opposing neural connections, and understanding how this balance is achieved relies on knowledge of their protein compositions. Using a spatially restricted enzymatic tagging strategy, we mapped the proteomes of two of the most common excitatory and inhibitory synaptic clefts in living neurons. These proteomes reveal dozens of synaptic candidates and assign numerous known synaptic proteins to a specific cleft type. The molecular differentiation of each cleft allowed us to identify Mdga2 as a potential specificity factor influencing Neuroligin-2's recruitment of presynaptic neurotransmitters at inhibitory synapses.
Pubmed ID: 27565350 RIS Download
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Biomedical technology research center that develops computer-aided, advanced microscopy for the acquisition of structural and functional data in the dimensional range of 1 nm to 100 um, a range encompassing macromolecules, subcellular structures and cells. Novel specimen-staining methods, imaging instrumentsincluding intermediate high-voltage transmission electron microscopes (IVEMs) and high-speed, large-format laser-scanning light microscopesand computational capabilities are available for addressing mesoscale biological microscopy of proteins and macromolecular complexes in their cellular and tissue environments. These technologies are developed to bridge understanding of biological systems between the gross anatomical and molecular scales and to make these technologies broadly available to biomedical researchers. NCMIR provides expertise, infrastructure, technological development, and an environment in which new information about the 3D ultrastructure of tissues, cells, and macromolecular complexes may be accurately and easily obtained and analyzed. NCMIR fulfills its mission through technology development, collaboration, service, training, and dissemination. It aims to develop preparative methods and analytical approaches to 3D microscopy applicable to neurobiology and cell biology, incorporating equipment and implementing software that expand the analysis of 3D structure. The core research activities in the areas of specimen development, instrument development, and software infrastructures maximize the advantages of higher voltage electron microscopy and correlated light microscopies to make ambitious imaging studies across scales routine, and to facilitate the use of resources by biomedical researchers. NCMIR actively recruits outside users who will not only make use of these resources, but who also will drive technology development and receive training.
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