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.

项目摘要/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 末端或内分泌细胞与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 孔和Ca2+。 Ca2+触发融合孔的开口和演变；融合孔是水通道 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 位于对分泌的精心调整。我们使用生物物理技术来探测融合孔 单盘级，跟踪其过渡并监视其对生物信号的反应。融合孔的研究 已经使我们对特定蛋白质在控制胞吐作用中的作用的价值见解。我们证明了圈套 蛋白质跨膜结构域通过内分泌和突触胞吐作用中的初始融合孔改变通量。我们 在理解内分泌胞胞菌病的新生融合孔方面取得了重要的进步，但是进步已经 理解内分泌融合孔的扩展以及融合孔如何影响突触传播的速度缓慢。 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，因为蔬菜失去了儿茶酚胺，并导致了新的发现，即 融合孔顺序扩展，合同并定位为亚稳态。我们将使用晚期的测量 阶段融合孔解决有关分泌生物学控制的长期问题。我们将晚期探测 通过脂质双层弹性，Ca2+，Synaptotagmins和Synaptophysin/Dynamin进行对照进行融合孔。项目2。 为了研究合成融合孔，我们与表达神经元和HEK293细胞开发了一个共培养系统4 突触后蛋白，Neuroligin 1，Glua2，Stargazin和PSD95。这些HEK细胞是突触释放的传感器， 产生融合孔贡献更清楚的特殊质量的微型突触电流数据 解决。与项目1同行，我们将使用HEK细胞神经元共培养来确定合成融合孔的方式 由双层弹性，Ca2+，突触蛋白和突触素/元素控制。内分泌和 突触融合孔将合成为调节分泌的综合框架。然后我们会适应 这种共同培养系统，用于研究突触可塑性的突触接吻和突触前的贡献。项目 3。我们将将HEK细胞突触传感器调整为从人类茎衍生的神经元的突触释放的研究 细胞。已招募合作者来提供神经元，我们将用来评估突触释放和融合 唐氏综合症的毛孔，脆弱的X智力低下，衰老，帕金森氏病和结节性硬化症复合体。这 工作将洞悉胞吐作用的分子机制，阐明其分子对照，并向我们展示 突触释放如何在疾病中出现问题。