Fusion pores in endocrine and synaptic exocytosis

项目摘要/摘要 神经元和内分泌细胞通过钙离子触发的胞吐作用释放信号分子。钙离子进入神经 终末或内分泌细胞，与钙离子感受器蛋白结合，并触发囊泡和质膜的融合，以 排出神经递质和荷尔蒙。为了研究胞吐的机制，我们的研究集中在融合 毛孔和钙离子。Ca~(2+)触发融合孔的开放和演化，融合孔为水通道 在囊泡内部和细胞外部之间。所有分泌的分子都通过一个融合孔，这在战略上是 处于对分泌物施加微调控制的位置。我们使用生物物理技术探测融合毛孔 单孔水平，跟踪它们的转变，并监测它们对生物信号的反应。融合孔的研究 使我们对特定蛋白质在胞吐作用控制中的作用有了有价值的见解。我们展示了那个陷阱 在内分泌和突触胞吐作用中，蛋白质跨膜结构域改变通过初始融合孔的通量。我们 在理解内分泌胞吐作用的新生融合毛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。 该实验室的创新创造了应对这些新挑战的机会。项目1.我们有 开发了一种分析电流记录以探索晚期内分泌融合动力学的新方法 毛孔。这种方法跟踪囊泡失去儿茶酚胺时融合孔的渗透性，并导致了新的发现 熔合孔依次膨胀、收缩，并稳定在亚稳态。我们将使用晚些时候的测量- 阶段融合毛孔，以解决长期存在的关于分泌物的生物控制的问题。我们将在晚些时候进行调查- 通过脂质双层弹性、钙离子、突触素和突触素/动力蛋白调控的分期融合毛孔。项目2. 为了研究突触融合孔，我们建立了一个神经元和HEK293细胞共培养系统，表达4 突触后蛋白，神经连接素1，GluA2，Stargazin和PSD95。这些HEK细胞充当突触释放的传感器， 产生极高质量的微型突触电流数据，其中融合孔的贡献更清楚 解决了。与项目1并行，我们将使用HEK细胞-神经元共培养来确定突触融合毛孔是如何 受双层弹性、钙离子、突触素和突触素/动力蛋白控制。关于内分泌和内分泌的结果 突触融合毛孔将被合成成一个全面的框架，用于调节分泌。然后我们将适应 这种共培养系统有助于研究突触接吻逃逸和突触前对突触可塑性的贡献。项目 3.我们将使用HEK细胞突触传感器来研究人类干细胞来源的神经元的突触释放 细胞。已经招募了合作者来提供神经元，我们将用它来评估突触的释放和融合 唐氏综合征、脆性X智力低下、衰老、帕金森氏症和结节性硬化症的毛孔。这 这项工作将深入了解胞吐作用的分子机制，阐明其分子控制，并向我们展示 突触释放在疾病中是如何出错的。

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.