REGULATION OF EXOCYTOSIS STUDIES WITH FLIPPED SNARES

描述（由申请人提供）：膜融合是细胞器形成、营养吸收、激素和神经递质的分泌的基本生物学过程。这些过程的不平衡会导致重要的疾病，如糖尿病、肥胖和情绪障碍。该项目在前三年取得了重大进展，发现在胞外作用中，络合蛋白（CPX）和Synaptotagmin1 （SYT1）通过普遍存在的SNARE融合机制将钙离子偶联到其他自发融合中。通过将胞外SNAREs翻转到细胞外，并测量它们在融合整个细胞中的活性，我们创造了一个成分原始的环境，在这个环境中，CPX和SYT1等因子可以被添加到培养基中或从细胞中表达，一次一个或组合一个，以清楚地揭示它们的作用。在目前的资助期（从2004年7月1日开始），我们建议使用翻转SNARE测定法调查广泛的已知分子机制未知的体内调节因子。因此，我们现在正在缩小焦点，在未来五年内利用我们发现的用于钙触发胞吐的最小机器。具体来说，我们将1)使用翻转SNARE实验定义Complexin和Synaptotagmin对SNARE依赖融合的最小钙调节的结构要求，并测试这些发现的体内相关性；2)利用表面力仪（Surface Forces Apparatus， SFA）确定络合素/Synaptotagmin钳的分子机制；通过确定哪些成员有能力控制哪些SNARE蛋白的融合，全面和高通量地探索广泛的Synaptotagmin和Complexin家族的功能生物学。

DESCRIPTION (provided by applicant): Membrane fusion is a fundamental biological process for organelle formation, nutrient uptake, and the secretion of hormones and neurotransmitters. Imbalances in these processes give rise to important diseases, such as diabetes, obesity, and mood disorders. A major advance has been made in the first three years of this project with the discovery that in exocytosis, Complexin (CPX) and Synaptotagmin1 (SYT1) couple calcium ion to otherwise spontaneous fusion by the ubiquitous SNARE fusion machinery. By flipping exocytic SNAREs to the outside of cells, and measuring their activity in fusing whole cells, we create a compositionally virgin environment in which factors such as CPX and SYT1 can be added to the medium or expressed from the cells, one at a time or in combination, to reveal their roles with clarity. For the current grant period (commencing 7/1/04), we proposed to survey a broad range of known in vivo regulators with unknown molecular mechanism using the flipped SNARE assay. As a result, we are now narrowing the focus to capitalize on our discovery of a minimal machine for calcium-triggered exocytosis over the next five years. Specifically, we will 1) define the structural requirements for minimal calcium regulation of SNARE-dependent fusion by Complexin and Synaptotagmin using the flipped SNARE assay, and test the in vivo relevance of these findings; 2) define the molecular mechanism of the Complexin/Synaptotagmin clamp using the Surface Forces Apparatus (SFA); and comprehensively and in high throughput explore the functional biology of the broad Synaptotagmin and Complexin families by establishing which members have the capacity to clamp control fusion by which SNARE proteins. PUBLIC HEALTH RELEVANCE: Membrane fusion is a fundamental biological process for organelle formation, nutrient uptake, and the secretion of hormones and neurotransmitters. It is central to vesicular transport, storage, and release in many areas of endocrine and exocrine physiology, and imbalances in these processes give rise to important diseases, such as diabetes. Membrane fusion is the result of a highly orchestrated series of protein-protein interactions [5] which work together to activate and regulate the membrane fusion machinery known as SNARE proteins. Transport vesicles dock closely and firmly - within molecular contact distance of the target bilayer - as the cognate SNAREs zip-up to form a four helix bundle between the two membranes (termed a trans-SNARE complex or SNAREpin). When the SNARE complex fully zips up the bilayers are merged and the SNARE complex now emanates from the single, combined membrane. In regulated exocytosis, there are additional proteins added to this "core" machinery that appear to freeze the process at intermediate stages to allow rapid mobilization from these intermediates (for example, upon calcium entry) to allow a prompt bolus of secretion (for example, of insulin). How such proteins accomplish this, or potentially contribute directly to the bilayer fusion event itself, has been difficult to discern with any precision. The long-term goal of this project is to bridge the gap, utilizing functional studies to validate whether and how regulatory proteins influence exocytosis through their interactions with SNARE proteins.