Dance of the SNAREs: assembly and rearrangements detected with FRET at neuronal synapses.
Dance of the SNAREs: assembly and rearrangements detected with FRET at neuronal synapses.
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DOI：
10.1523/jneurosci.2337-12.2013
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发表时间：
2013-03-27
期刊：
影响因子：
--
通讯作者：
Zucker RS
中科院分区：
文献类型：
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作者：
Degtyar V;Hafez IM;Bray C;Zucker RS
可溶性NSF附着蛋白受体（SNARE）介导囊泡与质膜的融合，通过钙结合突触结合蛋白激活。我们使用荧光共振能量转移（FRET）和荧光寿命成像显微镜（FLIM）之间的荧光标记的SNARE蛋白表达在培养的大鼠海马神经元检测静息SNARE复合物，其构象重排的胞吐作用，其拆卸之前的囊泡蛋白的内吞作用，和SNARE组装在新停靠的囊泡。组装的SNARE不仅存在于停靠的囊泡中;意外的残留“孤儿SNARE复合物”也存在于副活性区区域中。实时变化的N-末端标记的SNAP-25和VAMP之间的FRET报告的SNARE基序的重新取向后，胞吐，SNARE解体的活动区周边，和SNARE重新组装在新停靠的囊泡。随着VAMP标记的C-末端，减少荧光的C-末端标记的突触融合蛋白（细胞外）报告的反式-顺式构象变化的SNARE囊泡融合。融合后，SNAP-25和突触融合蛋白与VAMP以及FRET信号本身一起沿着分散，表明囊泡融合后但在其外周分解之前完整SNARE的扩散。我们对SNARE构象变化和运动的时空动力学的测量完善了SNARE功能的模型。技术进步所需的检测微小的变化，在标记的蛋白质在突触前扣的时间尺度上的秒允许检测快速的小比例的蛋白质伴侣之间的分子间相互作用的细胞亚室。
Soluble NSF Attachment Protein Receptors (SNAREs) mediate vesicle fusion with the plasma membrane on activation by calcium binding to synaptotagmin. We used fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) between fluorescently-labeled SNARE proteins expressed in cultured rat hippocampal neurons to detect resting SNARE complexes, their conformational rearrangement on exocytosis, their disassembly prior to endocytosis of vesicular proteins, and SNARE assembly at newly docked vesicles. Assembled SNAREs are not only in docked vesicles; unexpected residual “orphan SNARE complexes” also reside in para-active zone regions. Real-time changes in FRET between N-terminally labeled SNAP-25 and VAMP reported a reorientation of the SNARE motif upon exocytosis, SNARE disassembly in the active zone periphery, and SNARE reassembly in newly docked vesicles. With VAMP labeled C-terminally, decreased fluorescence in C-terminally labeled syntaxin (extracellular) reported trans-cis conformational changes in SNAREs on vesicle fusion. After fusion SNAP-25 and syntaxin disperse along with VAMP, as well as the FRET signal itself, indicating diffusion of intact SNAREs after vesicle fusion but before their peripheral disassembly. Our measurements of spatio-temporal dynamics of SNARE conformational changes and movements refine models of SNARE function. Technical advances required to detect tiny changes in fluorescence in small fractions of labeled proteins in presynaptic boutons on a time scale of seconds permit the detection of rapid inter-molecular interactions between small proportions of protein partners in cellular sub-compartments.