Nanoscale dynamics of voltage-gated calcium channels at presynaptic active zones in live C. elegans

活线虫突触前活动区电压门控钙通道的纳米级动力学

• 批准号: 10056911
• 负责人: Fabien Pinaud
• 金额: $ 43.43万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10056911
• 关键词: Action Potentials; Address; Animal Model; Animals; Architecture; Area; Behavior; Binding; Biology; Brain; CRISPR/Cas technology; Caenorhabditis elegans; Calcium; Calcium Channel; Cell membrane; Clustered Regularly Interspaced Short Palindromic Repeats; Co-Immunoprecipitations; Communication; Complement; Complex; Coupled; Coupling; Data; Diffuse; Docking; Electrons; Equilibrium; Exocytosis; Genetic; Imaging Techniques; Impairment; Individual; Knock-in; Lateral; Link; Mass Spectrum Analysis; Membrane; Methods; Modeling; Molecular; N-terminal; Names; Nematoda; Neurons; Population; Positioning Attribute; Presynaptic Terminals; Probability; Proteins; Registries; Regulation; SNAP receptor; Signal Transduction; Synapses; Synaptic Transmission; Synaptic Vesicles; Synaptic plasticity; Testing; Transgenic Organisms; Vesicle; Visualization; Work; cytomatrix; density; gain of function mutation; genome editing; imaging study; in vivo; light microscopy; loss of function; microscopic imaging; mutant; nanometer; nanoscale; neural circuit; neurotransmission; neurotransmitter release; organizational structure; presynaptic; response; single molecule; synaptic function; tool; vesicular release; voltage

ABSTRACT In neural circuits, adaptive responses to changes in transmitted signals are conveyed by subtle modulations in the strength of synaptic connections. This short-term synaptic plasticity involves adjustments of the neurotransmitter release probability of synaptic vesicles (SVs) positioned in presynaptic areas called active zones (AZ). A key function of the AZ molecular machinery is to precisely position diffusing membrane voltage-gated calcium channels (VGCCs) in registry with primed SVs, so as to establish the local Ca2+ concentration gradients that ultimately initiate SV fusion and neurotransmitter release. Previous studies have shown that active zone cytomatrix (CAZ) proteins are determinants of the spatial coupling between VGCCs and SVs and that the mobility of VGCCs can tune the SV release probability by setting local channel densities and Ca2+ concentrations. While this suggests that modulation of VGCC dynamics by CAZ proteins could underlie presynaptic plasticity, a fundamental, yet still unanswered question in synaptic biology is how CAZ proteins regulate the mobility of VGCCs and precisely positioned them within AZ a few hundreds of nanometer in size, in the first place. An important challenge in addressing this question has been the absence of methods that allow direct visualization and quantification of VGCC dynamics at the nanometer scale within AZ of intact synapses in live animals. Using CRISPR genetics and complementation activated light microscopy (CALM), an in vivo single molecule (SM) imaging technique that we introduced recently, we have started to define how the molecular machinery of AZ modulates the nanoscale mobility of VGCCs using the nematode Caenorhabditis elegans (C.elegans) as a live animal model. Our preliminary data demonstrate that neuronal VGCCs have heterogeneous diffusive behaviors in vivo, and that their nanoscale mobility is effectively controlled by key CAZ proteins. Here, we built on this preliminary work to further dissect the molecular mechanisms by which different CAZ proteins specifically regulate the presynaptic membrane dynamics of VGCCs in order to guaranty precise neurotransmission. Specifically, we will determine how VGCC dynamics are regulated by (i) the CAZ protein RIM/UNC-10, (ii) coupling to SVs and (iii) coupling to other CAZ regulators (Aim 1), how the priming levels of SVs at AZ influence the mobility of VGCCs (Aim 2), and how the presynaptic dense projection centered in the AZ modulates the nanoconfinement zones of diffusing VGCCs. (Aim 3). Together, the proposed studies will advance our fundamental understanding of the molecular organization and function of the synaptic AZ within intact neurons in live animals. It will also provide new models for regulation of neurotransmission and short-term synaptic plasticity that integrate the nanoscale dynamics of VGCCs and the structural organization of AZ.

抽象的 在神经回路中，对传输信号变化的自适应响应是通过强度的微妙调制来传达的 突触连接。这种短期突触可塑性涉及神经递质释放概率的调整 突触小泡 (SV) 位于称为活动区 (AZ) 的突触前区域。 AZ分子的关键功能 机器的作用是将扩散膜电压门控钙通道（VGCC）精确定位在已涂底漆的位置上 SV，从而建立局部 Ca2+ 浓度梯度，最终启动 SV 融合和神经递质释放。 先前的研究表明，活性区细胞基质（CAZ）蛋白是细胞间空间耦合的决定因素。 VGCC 和 SV 以及 VGCC 的移动性可以通过设置本地通道密度来调整 SV 释放概率 Ca2+ 浓度。虽然这表明 CAZ 蛋白对 VGCC 动力学的调节可能是突触前 可塑性是突触生物学中一个基本但尚未解答的问题，即 CAZ 蛋白如何调节突触的活动性 首先，VGCC 并将它们精确地定位在数百纳米大小的 AZ 内。 解决这个问题的一个重要挑战是缺乏允许直接可视化和 活体动物完整突触 AZ 内纳米级 VGCC 动力学的量化。使用CRISPR 遗传学和互补激活光学显微镜 (CALM)，一种体内单分子 (SM) 成像技术 我们最近介绍，我们已经开始定义 AZ 的分子机制如何调节 AZ 的纳米级迁移率 VGCC 使用线虫秀丽隐杆线虫 (C.elegans) 作为活体动物模型。我们的初步数据表明 神经元 VGCC 在体内具有异质扩散行为，并且它们的纳米级迁移性有效 由关键的 CAZ 蛋白控制。 在这里，我们在此基础上进一步剖析不同 CAZ 蛋白的分子机制 特异性调节 VGCC 的突触前膜动力学，以保证精确的神经传递。 具体来说，我们将确定 VGCC 动力学如何通过 (i) CAZ 蛋白 RIM/UNC-10，(ii) 耦合来调节 SV 和 (iii) 与其他 CAZ 调节器耦合（目标 1），AZ 处 SV 的启动水平如何影响 VGCC（目标 2），以及以 AZ 为中心的突触前致密投影如何调节 VGCC 的纳米限制区 扩散 VGCC。 （目标 3）。 总之，拟议的研究将增进我们对分子组织和功能的基本理解 活体动物完整神经元内的突触 AZ。它还将为神经传递调节提供新模型 以及短期突触可塑性，整合了 VGCC 的纳米级动力学和 AZ 的结构组织。