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

摘要 在神经电路中，对传输信号变化的自适应反应是通过强度的微妙调制来传递的 突触连接。这种突触的短期可塑性涉及神经递质释放概率的调节。 位于突触前区域的突触小泡(SVS)称为活动区(AZ)。AZ分子的一个关键功能 机械是精确定位扩散膜电压门控钙通道(VGCC)，与Primed对准 从而建立局部钙浓度梯度，最终启动SV融合和神经递质释放。 以往的研究表明，活动区细胞骨架(CAZ)蛋白是决定细胞间空间耦合的因素。 VGCC和SVS，并且VGCC的移动性可以通过设置本地信道密度和 钙离子浓度。虽然这表明CAZ蛋白对VGCC动力学的调节可能是突触前的基础 可塑性是突触生物学中一个基本但仍未回答的问题，即Caz蛋白如何调节 VGCC，并首先在AZ内精确定位数百纳米大小的VGCC。 解决这一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 遗传和互补激活光学显微镜(Calm)，一种活体单分子(SM)成像技术， 我们最近介绍，我们已经开始定义AZ的分子结构如何调节纳米级的流动性 利用线虫秀丽线虫(C.elegans)作为活体动物模型的VGCC。我们的初步数据显示 神经元VGCC在体内具有不同的扩散行为，并且它们的纳米级迁移率有效 由关键的Caz蛋白控制。 在这里，我们在这项初步工作的基础上，进一步剖析了不同的caz蛋白 具体调控VGCC的突触前膜动力学，以保证精确的神经传递。 具体地说，我们将确定VGCC动力学是如何通过(I)CAZ蛋白RIM/UNC-10，(Ii)耦合到 SVS和(Iii)与其他CAZ调节子的耦合(目标1)，在AZ的SVS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射如何调制 扩散的VGCC。(目标3)。 总之，拟议的研究将促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。它还将为神经传递的调节提供新的模型 以及整合了VGCC的纳米动力学和AZ的结构组织的短期突触可塑性。

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.