Synergistic regulation of neurotransmitter release and short-term synaptic plasticity by different members of the synaptotagmin family

已经确定的是，突触囊泡与突触前膜的融合是由SNARE蛋白介导的，SNARE蛋白迫使两个膜在一起。使囊泡融合与神经元棘波同步的关键参与者是突触前钙感受器-突触结合蛋白。目前的观点是，对接囊泡上的SNARE复合物被突触结合蛋白分子“夹住”在接近融合点的部分组装状态。突触结合蛋白与钙离子的结合消除了突触钳，并允许SNARE完全组装，触发快速囊泡胞吐。最近SNARE-突触结合蛋白复合物的晶体结构提出了几种机制模型，这些模型可以解释具有不同分子特性的突触结合蛋白亚型如何调节神经递质释放的动力学和可塑性[3]。这些模型存在激烈的争论，需要进行系统的测试[4]。当前博士项目的主要目标将是利用我们最近的方法学突破[1，2]，以获得缺失的关于不同突触结合蛋白（特别是Syt 1和Syt 7）如何塑造中枢突触中神经递质释放的时间和可塑性的机制见解。(i)学生将结合联合收割机基因操作Syt 1和Syt 7与成像的量子谷氨酸释放和突触前钙动力学在个别突触终端（K。Volynski和D.Kullmann实验室在UCL。关键的是，在对囊泡释放成像后，研究人员对相同的突触进行了事后免疫荧光分析（见上面的旋转项目）。这将使他们能够将功能性突触前特性与不同突触结合蛋白和囊泡释放机制的其他成分的表达水平联系起来。(ii)他们将与沃里克大学计算机科学系（Yulia Ziffeva教授）合作开发一个计算模型，该模型将突触前钙动力学和突触结合蛋白的公认的钙、膜和SNARE结合特性结合起来，并将其与实验数据联系起来[5]。

It is well established that the synaptic vesicle fusion with the presynaptic membrane ismediated by the SNARE proteins that force the two membranes together. The keyplayers that synchronise vesicle fusion to neuronal spikes are presynaptic calciumsensors - synaptotagmins. The current view is that SNARE complexes on dockedvesicles are 'clamped' in a partially assembled state close to the point of triggeringfusion by synaptotagmin molecules. Calcium binding by synaptotagmins removes theclamp and allows full assembly of SNAREs that triggers fast vesicle exocytosis. Therecent crystal structure of the SNARE-synaptotagmin complex prompted severalmechanistic models that can explain how synaptotagmin isoforms with distinctmolecular properties regulate kinetics and plasticity of neurotransmitter release [3].These models are intensely debated and call for systematic testing [4]. The main goalof the current PhD project will be to capitalise on our recent methodologicalbreakthroughs [1,2] to obtain missing mechanistic insights into how differentsynaptotagmins (specifically Syt1 and Syt7) shape the timing and the plasticity ofneurotransmitter release in central synapses. (i) The student will combine geneticmanipulation of Syt1 and Syt7 with imaging of quantal glutamate release and ofpresynaptic calcium dynamics in individual synaptic terminals (K. Volynski and D.Kullmann laboratories at UCL. Critically, after imaging vesicular release, the studentwill perform a post-hoc immunofluorescence analysis of the same synapses (see therotation project above). This will allow them to relate the functional presynapticproperties to the expression levels of different synaptotagmins and the othercomponents of vesicular release machinery. (ii) In collaboration with the Departmentof Computer Science at the University of Warwick (Prof. Yulia Timoffeva) they willdevelop a computational model incorporating the presynaptic calcium dynamics andthe well-established calcium, membrane- and SNARE-binding properties ofsynaptotagmins, and relate it to the experimental data [5].