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

现已确定突触囊泡与突触前膜的融合是由SNARE蛋白介导的，它迫使两个膜结合在一起。同步囊泡融合到神经元尖峰的关键参与者是突触前钙传感器-突触塔敏蛋白。目前的观点是，停靠囊泡上的SNARE复合物被“夹住”在靠近触发突触联合蛋白分子融合点的部分组装状态。钙与突触蛋白的结合消除了夹子，并允许SNAREs的完全组装，从而触发快速囊泡胞外分泌。最近的SNARE-synaptotagmin复合物的晶体结构提示了几种机制模型，这些模型可以解释具有不同分子特性的synaptotagmin亚型如何调节神经递质释放[3]的动力学和可塑性。这些模型引起了激烈的争论，需要进行系统的测试。当前博士项目的主要目标将是利用我们最近在方法学上的突破[1,2]，以获得缺失的机制见解，了解不同的突触tagmins（特别是Syt1和Syt7）如何塑造中枢突触中神经递质释放的时间和可塑性。(i)学生将把Syt1和Syt7的基因操作与量子谷氨酸释放和单个突触末端突触前钙动力学的成像结合起来（伦敦大学学院K. Volynski和D.Kullmann实验室）。至关重要的是，在成像囊泡释放后，学生将对相同的突触进行事后免疫荧光分析（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塔敏蛋白的表达水平和囊泡释放机制的其他成分联系起来。（ii）与华威大学计算机科学系（Yulia Timoffeva教授）合作，他们将开发一个计算模型，将突触前钙动力学和突触tagmins的钙、膜和snr结合特性结合起来，并将其与实验数据联系起来。

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].