REGULATION OF EXOCYTOSIS STUDIES WITH FLIPPED SNARES

描述(申请人提供)：膜融合是细胞器形成、营养吸收以及激素和神经递质分泌的基本生物学过程。这些过程中的不平衡会导致重要的疾病，如糖尿病、肥胖症和情绪障碍。这个项目的前三年取得了重大进展，发现在胞吐作用中，复合素(CPX)和突触素(SYT1)结合钙离子，通过无处不在的SNARE融合机制进行自发融合。通过将胞外陷阱翻转到细胞外，并测量它们在融合整个细胞时的活性，我们创造了一个成分纯净的环境，在这个环境中，可以将CPX和SYT1等因子添加到培养基中或从细胞中表达，一次一个或联合表达，以清晰地揭示它们的作用。在目前的授权期(从2004年7月1日开始)，我们建议使用翻转陷阱实验来调查一系列分子机制未知的已知体内调节因子。因此，我们现在正在缩小重点，以利用我们在未来五年发现的钙触发胞吐的最小机器。具体地说，我们将1)利用翻转SNARE实验定义复杂蛋白和突触素对SNARE依赖融合的最低限度钙调节的结构要求，并检验这些发现的体内相关性；2)使用表面力仪器(SFA)定义复杂蛋白/突触素钳住的分子机制；以及通过建立哪些成员具有钳制控制融合的能力来高通量地探索广泛的突触素和复杂蛋白家族的功能生物学。 与公共卫生相关：膜融合是细胞器形成、营养吸收以及激素和神经递质分泌的基本生物学过程。在内分泌和外分泌生理的许多领域，它是囊泡运输、储存和释放的中心，这些过程中的不平衡会导致一些重要的疾病，如糖尿病。膜融合是一系列高度协调的蛋白质-蛋白质相互作用的结果，这些相互作用共同激活和调节被称为SNARE蛋白质的膜融合机制。运输囊泡紧密而牢固地对接-在目标双层的分子接触距离内-作为同源SNARs拉链，在两层膜之间形成一个四螺旋束(称为反式SNARE复合体或SNAREpin)。当诱捕复合体完全拉上拉链时，双层被合并，诱捕复合体现在从单一的组合膜发出。在受调控的胞吐作用中，有额外的蛋白质被添加到这一“核心”机制中，这些蛋白质似乎在中间阶段冻结了这一过程，从而允许从这些中间体快速动员(例如，在钙进入时)，从而允许迅速分泌(例如，胰岛素)。这些蛋白质是如何做到这一点的，或者可能直接促成双层融合事件本身，一直很难准确地辨别出来。该项目的长期目标是弥合这一差距，利用功能研究来验证调控蛋白是否以及如何通过它们与SNARE蛋白的相互作用来影响胞吐作用。

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.