The Role of Actin Remodeling in Membrane Fusion Microdomains R01 GM076038-01A1

描述（由申请人提供）：受调节的膜融合对于细胞区室化和生长、神经传递和激素释放至关重要。融合通过SNARE、Ypt/Rab、它们的效应物和伴侣（NSF/SNAP和SM蛋白）、磷酸肌醇和肌动蛋白的高度复杂且保守的途径发生。除了调节细胞器的运动，肌动蛋白重塑是必不可少的组装和功能的对接和融合膜微区，如所示的酵母液泡。蛋白质和脂质的“顶点”环（包括肌动蛋白）催化蛋白质融合，其围绕紧密贴壁的膜表面。SNARE和Ypt/Rab蛋白和3-磷酸肌醇组装到这个环微结构域中需要对Jasplakin敏感的肌动蛋白重塑，并且它们反过来控制环上的肌动蛋白富集。对接后，融合需要进一步的latrunculin敏感的肌动蛋白重塑。为了测试我们的工作模型，肌动蛋白从F到G的重塑调节脂质和蛋白质组装到顶点环，然后通过从G到F的进一步重塑促进融合，我们将：1。建立F和G肌动蛋白在液泡融合的每个步骤中的丰度和微区定位，利用其独特的物理性质和配体结合，2。进行遗传筛选，以确定这两个肌动蛋白突变体与液泡融合和肌动蛋白调节蛋白级联重塑液泡肌动蛋白的具体影响，使用广泛和可用的突变体，3。利用肌动蛋白亲和分离方法鉴定肌动蛋白的空泡受体和调控其重构的蛋白质。建立肌动蛋白和融合调节脂质（固醇，磷酸肌醇，和甘油二酯）之间的相互关系，通过它们调节彼此的组装成环域，使用比率荧光显微镜测定顶点富集和肌动蛋白和脂质配体和修饰酶，5。建立顺序，或相互依存，反式SNARE配对，肌动蛋白聚合，和脂质重排的液泡融合，使用可逆的抑制配体和我们的比色测定在体外液泡融合，和6。通过系统地纯化肌动蛋白和肌动蛋白调节因子并重建融合子反应，首先在液泡上，然后在脂质体模型系统中重建液泡融合的每个步骤对纯化的肌动蛋白和肌动蛋白调节蛋白的功能依赖性。

DESCRIPTION (provided by applicant): Regulated membrane fusion is essential for cell compartmentation and growth, neurotransmission, and hormone release. Fusion occurs by a highly complex, and conserved, pathway of SNAREs, Ypt/Rabs, their effectors and chaperones (NSF/SNAP and SM proteins), phosphoinositides, and actin. In addition to regulating organelle movement, actin remodeling is essential for the assembly and function of docking and fusion membrane microdomains, as shown with the yeast vacuole. Vacuole fusion is catalyzed by a "vertex" ring of proteins and lipids, including actin, which surrounds the closely apposed membrane surfaces. Jasplakinolide sensitive actin remodeling is required for SNARE and Ypt/Rab proteins and 3- phosphoinositides to assemble into this ring microdomain, and they in turn govern actin enrichment at the ring. After docking, fusion requires further latrunculin-sensitive actin remodeling. To test our working model, that actin remodeling from F to G regulates lipid and protein assembly into the vertex ring and then contributes to fusion by being further remodeled from G to F, we will: 1. Establish the abundance and microdomain localization of F and G actin at each step of vacuole fusion, exploiting their distinct physical properties and ligand binding, 2. Perform genetic screens to identify both actin mutants with specific effects on vacuole fusion and actin regulatory protein cascades which remodel vacuolar actin, using the extensive and available collections of mutants, 3. Identify actin's vacuolar receptor and the proteins which regulate its remodeling, using actin-affinity isolation methods, 4. Establish the mutual relationships between actin and the fusion regulatory lipids (sterol, phosphoinositides, and diacylglycerol) by which they regulate each other's assembly into the ring domain, using ratiometric fluorescence microscopy assays of vertex enrichment and actin and lipid ligands and modifying enzymes, 5. Establish the order, or interdependence, of trans-SNARE pairing, actin polymerization, and lipid rearrangements for vacuole fusion, using reversible inhibitory ligands and our colorimetric assay of in vitro vacuole fusion, and 6. Reconstitute the functional dependence of each step of vacuole fusion on purified actin and actin regulatory proteins through systematic purification of actin and actin regulatory factors and reconstitution into fusion subreactions, on the vacuole at first and then in liposome model systems.