Regulation of ER to Golgi Transport byLuminal Calcium

项目总结(此部分与与父拨款一起提交的部分相同) 分泌途径利用囊泡运输为细胞产物的出口提供线性途径。 细胞膜和细胞器成分在细胞质中的分布。很多疾病， 包括神经退行性变，涉及生物合成分泌途径的中断和未解决 分泌压力--使我们有必要了解在不同的环境下，分泌是如何上调和下调的 生理条件。而囊泡萌发、对接和融合的基本引擎 对于它们是如何对生理条件和压力做出反应的，人们知之甚少。 在哺乳动物中，内质网到高尔基体的运输，代表着分泌途径中的限速步骤和 与运输相关疾病最相关的步骤，已经在体内和 在体外重组。从广义上讲，ER到Golgi的运输是在ER出口地点(ERE)启动的，并已 已被证明包括：1)由COPII囊泡外壳介导的货物分拣和囊泡萌发； 同型COPII囊泡拴系融合形成高尔基体前细胞器 称为泡状管状团(VTC)；以及3)VTC介导的货物分拣和沿 导致与高尔基体融合的微管。关于这些过程是如何调整的，我们知之甚少 动态匹配分泌物产出率与加强分泌物质量控制的需要，避免ER 应激，并与分泌蛋白的生物发生和细胞生长保持同步。 近年来，调节内质网到高尔基体运输的一个关键方面是 呃，流明的钙。当钙通过通道蛋白从内质网释放出来时，似乎与 胞浆中的五肽-EF手蛋白(PEF)与内质网出口部位的COPII涂层结合并调节 它的组装和货物从急救室流出的速度。然而，这些蛋白质的作用机制是 产生不同的分泌结果，以及它们如何与内质网钙稳态和 钙离子通道尚不清楚。该项目将采用内质网到高尔基体转运的动力学分析。 完整的哺乳动物细胞系、活细胞钙测量和内质网出口处蛋白质动力学的显微镜观察 网站了解钙通道、PEF蛋白和COPII成分是如何动态整合的 调节分泌速度。这些研究将具有广泛的意义，因为适当的分泌调节是 在生理应激状态下，细胞功能和细胞存活的基础。

PROJECT SUMMARY (this section remains identical to that submitted with the parent grant) The secretory pathway employs vesicle transport to provide a linear pathway for export of cellular products and distribution of membrane and organelle components throughout the cytoplasm. Many diseases, including neurodegeneration, involve disruption of the biosynthetic secretory pathway and unresolved secretory stress--making it essential to understand how secretion is up- and down-regulated under different physiological conditions. While the basic engines of vesicle budding, docking and fusion have been identified, little is known of how they are tuned to respond to physiological conditions and stresses. In mammals, ER-to-Golgi transport, which represents the rate-limiting step in the secretory pathway and the step most relevant to transport-related diseases, has been extensively characterized in vivo and reconstituted in vitro. In broad terms, ER-to-Golgi transport is initiated at ER exit sites (ERES) and has been shown to comprise: 1) cargo sorting and vesicle budding mediated by the COPII vesicle coat; 2) homotypic COPII vesicle tethering and fusion mediated by tethers and SNAREs to form pre-Golgi organelles called vesicular tubular clusters (VTCs); and 3) VTC-mediated cargo sorting and transport along microtubules leading to fusion with the Golgi. Little is known about how these processes are adjusted dynamically to match secretory output rates with the needs to enforce secretory quality control, avoid ER stress, and keep pace with secretory protein biogenesis and cell growth. One key aspect to regulation of ER-to-Golgi transport that has become apparent in recent years is the role of ER luminal calcium. Calcium, when released from the ER by channel proteins appears to interact with penta-EF hand proteins (PEFs) in the cytoplasm that bind to the COPII coat at ER exit sites and modulate its assembly and the rate of cargo egress from the ER. However, the mechanisms of these proteins to produce different secretory outcomes and how they are integrated with ER calcium homeostasis and calcium channels are not understood. This project will employ kinetic assays of ER-to-Golgi transport in intact mammalian cell lines, live-cell calcium measurements, and microscopy of protein dynamics at ER exit sites to learn how calcium channels, PEF proteins, and COPII components are integrated to dynamically regulate secretion rate. These studies will have wide significance because proper regulation of secretion is fundamental to cell function and cell survival during physiological stresses.