Fusion pores in endocrine and synaptic exocytosis

内分泌和突触胞吐作用中的融合孔

• 批准号: 10615868
• 负责人: MEYER B. JACKSON
• 金额: $ 100.24万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2030-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10615868
• 关键词: Address; Aging; Binding; Biological; Catecholamines; Cell membrane; Cells; Coculture Techniques; Communication; Complex; Contracts; DLG4 gene; Data; Disease; Down Syndrome; Dynamin; Elasticity; Endocrine; Endocrine System Diseases; Endocrine system; Epilepsy; Evolution; Exocytosis; Fragile X Syndrome; Hormones; Human; Impairment; Integral Membrane Protein; Laboratories; Learning; Learning Disorders; Lipid Bilayers; Liquid substance; Measurement; Medical; Mental disorders; Methods; Molecular; Monitor; Nerve; Nervous System; Neurons; Neurotransmitters; Parkinson Disease; Permeability; Physiological; Process; Proteins; Research; Role; Running; SNAP receptor; Sensory Process; Signal Transduction; Signaling Molecule; Synapses; Synaptic Transmission; Synaptic plasticity; Synaptophysin; System; Transmembrane Domain; Tuberous Sclerosis; Vesicle; Work; aqueous; biophysical techniques; chemical release; human stem cells; innovation; insight; nervous system disorder; neuroligin 1; novel; postsynaptic; presynaptic; recruit; response; sensor; sensory input; stargazin; synaptotagmin; virtual

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.

项目概要/摘要 神经元和内分泌细胞通过 Ca2+ 触发的胞吐作用释放信号分子。 Ca2+进入神经 终末细胞或内分泌细胞，与 Ca2+ 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，我们的研究重点是融合 毛孔和Ca2+。 Ca2+触发融合孔的开放和演化；融合孔是水通道 位于囊泡内部和细胞外部之间。所有分泌的分子都通过融合孔，这是战略性的 位于对分泌进行精细控制的位置。我们使用生物物理技术来探测融合孔 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 为我们提供了关于特定蛋白质在控制胞吐作用中的作用的宝贵见解。我们展示了 SNARE 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通量。我们 在理解内分泌胞吐作用的新生融合孔方面取得了重要进展，但进展还不够 在理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的新方法，以探测晚期内分泌融合的动态 毛孔。当囊泡失去儿茶酚胺时，该方法跟踪融合孔渗透性，并得出了新的发现： 融合孔依次膨胀、收缩并进入亚稳态。我们将使用后期的测量 阶段融合孔解决了有关分泌生物控制的长期存在的问题。我们将稍后进行调查- 阶段融合孔由脂质双层弹性、Ca2+、突触结合蛋白和突触素/动力蛋白控制。项目2。 为了研究突触融合孔，我们开发了一种神经元和表达 4 的 HEK293 细胞的共培养系统 突触后蛋白、neuroligin 1、GluA2、stargazin 和 PSD95。这些 HEK 细胞充当突触释放的传感器， 产生高质量的微型突触电流数据，其中融合孔的贡献更加清晰 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孔的情况 由双层弹性、Ca2+、突触结合蛋白和突触素/动力控制。内分泌及相关结果 突触融合孔将被合成为调节分泌的综合框架。然后我们会进行调整 这种共培养系统用于研究突触接吻运行和突触前对突触可塑性的贡献。项目 3. 我们将采用HEK细胞突触传感器来研究源自人类干细胞的神经元的突触释放 细胞。已招募合作者来提供神经元，我们将用其来评估突触释放和融合 唐氏综合症、脆性X智障、衰老、帕金森病和结节性硬化症中的毛孔。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 疾病中突触释放如何出错。