Structural dynamics of the proteins involved in calcium-triggered exocytosis

神经元之间通过胞吐释放神经递质、多肽和小分子进行交流。含有神经递质的囊泡与质膜的融合是由一组被称为SNARS的核心蛋白质以及几个调节因子驱动的。虽然这些蛋白中的许多及其在钙触发的胞吐中的需求已经被证明，但SNARs和SNARE相关蛋白发挥作用的潜在分子机制仍然不清楚。一些根本性的问题仍然存在：核心陷阱融合机制是如何激活的，以驱动膜融合？构成融合机器的蛋白质的取向、结构和化学计量是什么？与膜融合相关的蛋白质的潜在结构重排是什么？为了解决这些和相关的问题，我们将使用新的荧光技术、生物化学和活细胞成像的组合。我们将集中精力进行两个层面的调查，研究分离的膜片和完整的活细胞中陷阱的结构和动力学。在这项研究中提出了两个具体的目标：1)用膜片钳荧光法定位质膜上驻留的t-SNARE合成素和合成素结合蛋白相对于彼此和质膜的方向；2)用单个胞外小泡和荧光标记蛋白的全内反射荧光(TIRF)显微镜表征体内SNARs和SNARE相关酶的化学计量、动态联系和功能作用。这些研究将绘制激活SNARS和驱动钙触发的神经元和内分泌细胞膜融合所需的精确分子步骤。详细了解这些酶在原子水平上的功能将为控制和调节突触传递、其故障如何导致疾病以及如何将其调制作为新疗法的靶点提供见解。

Neurons communicate with one another by releasing neurotransmitters, peptides, and small molecules by exocytosis. The fusion of neurotransmitter-containing vesicles with the plasma membrane is driven by a core set of proteins known as SNAREs along with several regulatory factors. While the identity of many of these proteins and their requirement in calcium-triggered exocytosis has been demonstrated, the underlying molecular mechanism by which SNAREs and SNARE-associated proteins function is still unknown. A number of fundamental questions remain: How is the core SNARE fusion machinery activated to drive membrane fusion? What are the orientations, architectures, and stoichiometries of the proteins that constitute the fusion machine? What are the underlying structural rearrangements of the proteins associated with membrane fusion? To address these and related questions, we will employ a combination of novel fluorescence techniques, biochemistry, and live cell imaging. We will focus our efforts at two levels of investigation, studying the structure and dynamics of SNAREs in isolated membrane patches as well as in intact living cells. In this grant two specific aims are proposed: 1) map the orientation of the plasma membrane resident t-SNARE Syntaxin and Syntaxin binding proteins relative to each other and to the plasma membrane with patch-clamp fluorometry; and 2) characterize the stoichiometry, dynamic associations, and functional roles of SNAREs and SNARE-associated enzymes in vivo with total internal reflection fluorescence (TIRF) microscopy of single exocytic vesicles and fluorescently-tagged proteins. These studies will map the precise molecular steps required to activate SNAREs and drive calcium-triggered membrane fusion in neurons and endocrine cells. A detailed understanding of how these enzymes function at the atomic level will provide insights into the control and regulation of synaptic transmission, how its malfunction leads to disease, and how its modulation might be targeted for novel therapies.