期刊
EMBO JOURNAL
卷 33, 期 2, 页码 157-170出版社
WILEY
DOI: 10.1002/embj.201386120
关键词
proteomics; synaptosome; vesicular glutamate transporter; Fluorescence Activated Synaptosome Sorting; subcellular fractionation
资金
- European Union
- Max Planck Society
- German Research Foundation [GRK521, GSC 226/1]
- Agence Nationale de la Recherche [ANR-12-JSV4-0005-01 VGLUT-IQ, ANR-10-LABX-43 BRAIN]
Abstract For decades, neuroscientists have used enriched preparations of synaptic particles called synaptosomes to study synapse function. However, the interpretation of corresponding data is problematic as synaptosome preparations contain multiple types of synapses and non-synaptic neuronal and glial contaminants. We established a novel Fluorescence Activated Synaptosome Sorting (FASS) method that substantially improves conventional synaptosome enrichment protocols and enables high-resolution biochemical analyses of specific synapse subpopulations. Employing knock-in mice with fluorescent glutamatergic synapses, we show that FASS isolates intact ultrapure synaptosomes composed of a resealed presynaptic terminal and a postsynaptic density as assessed by light and electron microscopy. FASS synaptosomes contain bona fide glutamatergic synapse proteins but are almost devoid of other synapse types and extrasynaptic or glial contaminants. We identified 163 enriched proteins in FASS samples, of which FXYD6 and Tpd52 were validated as new synaptic proteins. FASS purification thus enables high-resolution biochemical analyses of specific synapse subpopulations in health and disease. Synopsis image This study describes the establishment, validation, and application of a new fluorescence-activated sorting method - Fluorescence Activated Synaptosome Sorting or FASS - that allows for purification of glutamatergic synaptosomes from knock-in mutant mice expressing the fluorescent synaptic marker protein VGLUT1-Venus. Fluorescence Activated Synaptosome Sorting from samples of VGLUT1-Venus knock-in mice enriches glutamatergic synaptosomes to near homogeneity. Glutamatergic synaptosomes purified by Fluorescence Activated Synaptosome Sorting contain bona fide glutamatergic synapse proteins but lack other synapse types and glia cell contaminants. Proteomic analysis of glutamatergic synaptosomes purified by Fluorescence Activated Synaptosome Sorting allows for identifying and cataloguing known and novel components of glutamatergic synapses. Proteomic analysis of glutamatergic synaptosomes purified by Fluorescence Activated Synaptosome Sorting identified FXYD6 and Tpd52 as novel components of glutamatergic synapses.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据