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

摘要 在神经回路中，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达的。 突触连接的方式。这种短期突触可塑性涉及神经递质释放概率的调整 突触囊泡（SV）位于突触前区域，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）与预充的 SV，以便建立最终启动SV融合和神经递质释放的局部Ca 2+浓度梯度。 以前的研究表明，活性区细胞基质（CAZ）蛋白是空间耦合之间的决定因素， VGCC和SV的移动性，并且VGCC的移动性可以通过设置局部通道密度和 Ca 2+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，一个基本的，但仍然没有答案的问题，在突触生物学是如何CAZ蛋白调节的流动性， VGCC并将它们精确定位在AZ内，尺寸为几百纳米。 解决这一问题的一个重要挑战是缺乏允许直接可视化的方法， 在活体动物中完整突触的AZ内以纳米尺度定量VGCC动力学。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纳米级的流动性， 使用线虫秀丽隐杆线虫（C.elegans）作为活动物模型的VGCC。我们的初步数据显示 神经元VGCC在体内具有不均匀的扩散行为，并且它们的纳米级流动性有效地被抑制。 由关键的CAZ蛋白控制。 在此，我们在这项初步工作的基础上，进一步剖析了不同CAZ蛋白质在细胞内表达的分子机制。 特异性调节VGCC的突触前膜动力学，以保证精确的神经传递。 具体地说，我们将确定VGCC动力学如何通过（i）CAZ蛋白RIM/RIM-10，（ii）与 SV和（iii）与其他CAZ调节剂的偶联（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射如何调节VGCC的纳米限制区（目标2）。 扩散VGCC。(Aim 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， 活体动物中完整神经元内的突触AZ。它还将为调节神经传递提供新的模型 以及整合VGCC的纳米级动力学和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.