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

已经证实，突触小泡与突触前膜的融合是由迫使两层膜结合的SNARE蛋白介导的。使囊泡融合与神经元尖峰同步的关键人物是突触前钙传感器--突触集聚蛋白。目前的观点是，停靠的囊泡上的SNARE复合体处于部分组装状态，接近突触凝集素分子触发融合点的位置。通过突触凝集素的钙结合，移除了灯泡，并允许完全组装触发快速囊泡胞吐的圈套。SNARE-突触素复合体目前的晶体结构催生了几个机制模型，可以解释具有不同分子特性的突触素亚型如何调节神经递质释放的动力学和可塑性[3]。这些模型引起了激烈的争论，需要系统的测试[4]。目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]来获得对不同突触(特别是Syt1和Syt7)如何影响中枢突触中神经递质释放的时间和可塑性的缺失的机械性见解。(I)学生将结合Syt1和Syt7的遗传操作与单个突触终末中量子谷氨酸释放和突触前钙动力学的成像(伦敦大学学院的K.Volynski和D.Kullmann实验室)。关键的是，在成像囊泡释放之后，学生将对相同的突触进行组织后免疫荧光分析(参见上面的旋转项目)。这将允许他们将功能性突触前特性与不同突触集聚蛋白和囊泡释放机制的其他组件的表达水平联系起来。(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，结合突触前钙动力学和突触蛋白公认的钙、膜和陷阱结合特性，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].