Dissecting pre- vs postsynaptic actin dynamics in synapse structure and strength

剖析突触结构和强度方面的突触前和突触后肌动蛋白动力学

• 批准号: 9755095
• 负责人: Aaron Donald Levy
• 金额: $ 6.12万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9755095
• 关键词: 3-Dimensional; AMPA Receptors; Actins; Acute; Affect; Area; Biochemical; Biological; Biological Factors; Biology; Brain; Cells; Cellular biology; Color; Complex; Data; Dendritic Spines; Diagnosis; Disease; Electrophysiology (science); Faculty; Future; Glutamate Receptor; Goals; Hippocampus (Brain); Image; Imaging Techniques; In Vitro; Light; Long-Term Potentiation; Measures; Mediator of activation protein; Memory; Mental disorders; Microfilaments; Modeling; Molecular; Monomeric GTP-Binding Proteins; N-Methyl-D-Aspartate Receptors; Nanostructures; Neurons; Neurosciences; Optics; Pathology; Physiology; Positioning Attribute; Presynaptic Terminals; Probability; Process; Proteins; Publishing; Reagent; Recombinants; Regulation; Role; Science; Side; Skeleton; Structure; Synapses; Synaptic Vesicles; Testing; Time; Ultraviolet Rays; Vertebral column; Work; actin depolymerizing proteins; career; depolymerization; design; dimer; experience; experimental study; improved; in vivo; insight; nanocluster; nanoscale; neurotransmitter release; optical imaging; optogenetics; polymerization; postsynaptic; presynaptic; receptor; receptor function; scaffold; spatiotemporal; tool; tool development; treatment strategy; vesicular release

Bidirectional, spatially restricted actin filament dynamics modify synapses by controlling the structure and function of each cell creating the synaptic contact. Dynamic actin filaments are disrupted in psychiatric disease, so understanding their role in regulating pre- vs postsynaptic structure and function will be key for elucidating molecular mechanisms of memory formation and developing precise disease treatment strategies. Unfortunately, most existing tools to experimentally manipulate actin lack control of one or more of these biological factors, making it difficult to parse specific contributions of pre- vs. postsynaptic actin dynamics to synapse strength. The overarching goal of this proposal is to clarify the particular roles of actin at each side of the synapse. To facilitate this goal, I propose to develop new tools to spatiotemporally, bidirectionally, and synapse-specifically manipulate actin dynamics. The tools will be valuable additions to the arsenal of reagents in diverse fields of neuroscience and in other areas of cell biology. In my project, I will use these tools to answer two critical questions of how pre- vs postsynaptic actin dynamics regulate synapse strength. My first aim is to validate tools for precise, bidirectional control of actin dynamics. To drive actin depolymerization, I will develop photoactivateable (PA) DeActs by caging these published, genetically encodable actin depolymerizing proteins with photo-dimerizable pdDronpa. To drive actin polymerization, I will optimize an existing PA-Rac1 probe, which drives actin branching via Arp2/3. This set of tools will be highly useful to broad areas of science, and I will make new transfectable and recombinant AAV versions publicly available. My second aim is to elucidate how pre- vs postsynaptic actin regulates subsynaptic nanoorganization. Pre- and postsynaptic proteins form subsynaptic, nanoscale clusters that align across the synapse, a newly discovered organization expected to influence synaptic strength. However, we do not understand the biology that forms and maintains synaptic nanoorganization. My preliminary data suggest alignment requires actin dynamics, consistent with actin being a key regulator of nanostructure. I will combine my tools and 2-color 3D dSTORM to determine how actin in each synaptic compartment controls nanoorganization. My third aim is to interrogate the role of acute actin dynamics in synapse strength. The precise and independent roles of pre- and postsynaptic actin in synapse strength have been clouded by non-specific manipulations. I will use the tools developed in Aim 1 in conjunction with in vivo electrophysiology and in vitro imaging techniques to answer how bidirectional perturbation of presynaptic actin dynamics changes aspects of vesicle release and whether postsynaptic actin polymerization is sufficient to drive receptor plasticity. These aims synthesize my biochemical background, my sponsor’s synaptic optical imaging expertise, and the experience of the diverse UMB faculty to improve our understanding of how actin controls the basic cellular and molecular physiology of synapses, and provide a strong platform to launch my independent career in science.

双向、空间受限的肌动蛋白细丝动力学通过控制突触的结构和 产生突触接触的每个细胞的功能。在精神疾病中，动态肌动蛋白细丝被破坏， 因此，了解它们在调节突触前和突触后的结构和功能中的作用将是阐明 记忆形成的分子机制和开发精确的疾病治疗策略。 不幸的是，大多数现有的实验操作肌动蛋白的工具缺乏对其中一个或多个的控制。 生物学因素，使得很难分析突触前和突触后肌动蛋白动力学对 突触的力量。这项建议的首要目标是澄清肌动蛋白在每个 突触的一侧。为了促进这一目标，我建议开发新的工具来时空、双向、 和突触--特定地操纵肌动蛋白动力学。这些工具将是对 神经科学的不同领域和细胞生物学的其他领域的试剂。在我的项目中，我将使用这些工具 为了回答两个关键问题，即突触前和突触后肌动蛋白动力学如何调节突触强度。 我的第一个目标是验证对肌动蛋白动力学进行精确、双向控制的工具。驱动肌动蛋白 解聚，我将开发光激活(PA)DeActs通过将这些已发表的、可遗传编码的 肌动蛋白解聚蛋白与光二聚化pdDronpa。为了推动肌动蛋白聚合，我将优化一个 现有的PA-rac1探针，它通过Arp2/3驱动肌动蛋白分支。这套工具将对BREAD非常有用 科学领域，我将公开提供新的可转基因和重组AAV版本。 我的第二个目标是阐明突触前和突触后肌动蛋白如何调节突触下的纳米结构。 突触前和突触后蛋白形成突触下的纳米级簇，在突触上排列，这是一种新的 已发现的组织有望影响突触强度。然而，我们并不了解生物学 形成并维持突触的纳米组织。我的初步数据显示，对齐需要肌动蛋白 动力学，与肌动蛋白是纳米结构的关键调节器一致。我会把我的工具和双色3D结合起来 DSTORM来确定每个突触间隔中的肌动蛋白如何控制纳米组织。 我的第三个目标是询问急性肌动蛋白动力学在突触强度中的作用。精准的和 突触前和突触后肌动蛋白在突触强度中的独立作用被非特异性遮蔽 操纵。我将结合体内电生理学和体外实验使用在AIM 1中开发的工具 用成像技术回答突触前肌动蛋白动力学的双向扰动如何改变 囊泡释放和突触后肌动蛋白聚合是否足以驱动受体的可塑性。 这些目标综合了我的生化背景，我的赞助人的突触光学成像专业知识，以及 不同的UMB教员的经验，以提高我们对肌动蛋白如何控制基本细胞和 突触的分子生理学，并为我开始独立的科学生涯提供了一个强大的平台。