Fusion pores in endocrine and synaptic exocytosis

项目总结/摘要 神经元和内分泌细胞通过Ca 2+触发的胞吐作用释放信号分子。Ca 2+进入神经 终末或内分泌细胞，与Ca 2+传感器蛋白结合，并触发囊泡和质膜的融合， 排出神经递质和激素。为了研究胞吐作用的机制，我们的研究集中在融合 孔隙和Ca 2+。Ca 2+触发融合孔的开放和演化，融合孔是水通道 在囊泡内部和细胞外部之间。所有分泌的分子通过一个融合孔，这是战略性的 能够精确控制分泌物的位置。我们使用生物物理技术探测融合孔在 单孔水平，跟踪它们的转换，并监测它们对生物信号的反应。融合孔的研究 给了我们关于控制胞吐作用的特定蛋白质的重要见解。我们发现， 蛋白质跨膜结构域改变内分泌和突触胞吐作用中通过初始融合孔的流量。我们 在了解内分泌胞吐作用的新生融合孔方面取得了重要进展，但进展 在了解内分泌融合孔扩张以及融合孔如何影响突触传递方面进展缓慢。 该实验室的创新为应对这些新挑战创造了机会。项目1。我们有 开发了一种分析安培记录的新方法，以探测晚期内分泌融合的动力学 毛孔这种方法跟踪融合孔的渗透性，因为囊泡失去儿茶酚胺，并导致新的发现， 融合孔依次膨胀、收缩并进入亚稳态。我们将使用最近的测量结果- 阶段融合孔，以解决长期存在的问题，生物控制的分泌。我们会晚些时候调查 通过脂质双层弹性、Ca 2+、突触结合蛋白和突触素/发动蛋白控制分期融合孔。项目2. 为了研究突触融合孔，我们开发了神经元和表达4 突触后蛋白，神经连接蛋白1，GluA 2，stargazin和PSD 95。这些HEK细胞充当突触释放的传感器， 从而产生具有优异质量的微型突触电流数据，其中融合孔贡献更清楚 解决了与项目1平行，我们将使用HEK细胞神经元共培养物来确定突触融合孔是如何形成的。 由双层弹性、Ca 2+、突触结合蛋白和突触素/发动蛋白控制。内分泌和 突触融合孔将被合成为用于调节分泌的综合框架。然后我们会适应 这种共培养系统的研究突触吻和运行和突触前突触可塑性的贡献。项目 3.我们将HEK细胞突触传感器应用于人类干细胞神经元突触释放的研究 细胞合作者已经招募来提供神经元，我们将用它们来评估突触释放和融合 唐氏综合症、脆性X染色体智力低下、衰老、帕金森病和结节性硬化症。这 这项工作将提供对胞吐作用的分子机制的深入了解，阐明其分子控制，并向我们展示 疾病中突触释放是如何出错的

PROJECT SUMMARY/ABSTRACT Neurons and endocrine cells release signaling molecules through Ca2+‐triggered exocytosis. Ca2+ enters a nerve terminal or endocrine cell, binds to a Ca2+ sensor protein, and triggers the fusion of vesicle and plasma membranes to expel neurotransmitters and hormones. To investigate the mechanisms of exocytosis our research focuses on fusion pores and Ca2+. Ca2+ triggers the opening and evolution of the fusion pore; the fusion pore is an aqueous passage between the vesicle interior and cell exterior. All secreted molecules pass through a fusion pore, which is strategically situated to exert finely tuned control over secretion. We use biophysical techniques to probe fusion pores at the single‐pore level, track their transitions, and monitor their responses to biological signals. Studies of the fusion pore have given us valuable insights into the roles of specific proteins in the control of exocytosis. We showed that SNARE protein transmembrane domains alter flux through initial fusion pores in both endocrine and synaptic exocytosis. We have made important advances in understanding the nascent fusion pores of endocrine exocytosis, but progress has been slow in understanding endocrine fusion pore expansion, and how fusion pores impact synaptic transmission. Innovations from this laboratory have created opportunities to take on these new challenges. Project 1. We have developed a new method for analyzing amperometric recordings to probe the dynamics of late‐stage endocrine fusion pores. This method tracks fusion pore permeability as vesicles lose catecholamine, and led to the novel findings that a fusion pore sequentially expands, contracts, and settles into a metastable state. We will use measurements of late‐ stage fusion pores to address long‐standing questions about the biological control of secretion. We will probe late‐ stage fusion pores for control by lipid bilayer elasticity, Ca2+, synaptotagmins, and synaptophysin/dynamin. Project 2. To study synaptic fusion pores we developed a co‐culture system with neurons and HEK293 cells expressing 4 postsynaptic proteins, neuroligin 1, GluA2, stargazin, and PSD95. These HEK cells serve as sensors of synaptic release, yielding miniature synaptic current data of exceptional quality in which fusion pore contributions are more clearly resolved. In parallel with Project 1, we will use HEK cell‐neuron co‐cultures to determine how synaptic fusion pores are controlled by bilayer elasticity, Ca2+, synaptotagmins, and synaptophysin/dynamin. The results on endocrine and synaptic fusion pores will be synthesized into a comprehensive framework for regulated secretion. We will then adapt this co‐culture system to the study of synaptic kiss‐and‐run and presynaptic contributions to synaptic plasticity. Project 3. We will adapt HEK cell synaptic sensors to the study of synaptic release from neurons derived from human stem cells. Collaborators have been recruited to provide neurons, which we will use to evaluate synaptic release and fusion pores in Down syndrome, fragile X mental retardation, aging, Parkinson's disease, and tuberous sclerosis complex. This work will provide insight into the molecular mechanisms of exocytosis, illuminate its molecular control, and show us how synaptic release goes awry in diseases.