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

肌动蛋白重塑在膜融合微域 R01 GM076038-01A1 中的作用

• 批准号: 7138764
• 负责人: WILLIAM Tobey WICKNER
• 金额: $ 12.79万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7138764
• 关键词: actins; fluorescence microscopy; fungal genetics; genetic regulation; genetic screening; guanosinetriphosphatases; intermolecular interaction; liposomes; membrane activity; membrane fusion; membrane lipids; membrane model; membrane proteins; membrane reconstitution /synthesis; microorganism culture; molecular assembly /self assembly; molecular site; mutant; polymerization; protein isoforms; protein structure; protein structure function; transcription factor; vesicle /vacuole; yeasts

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.

描述（由申请人提供）：调节的膜融合对于细胞分隔和生长、神经传递和激素释放至关重要。融合通过 SNARE、Ypt/Rab、其效应子和伴侣（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。除了调节细胞器运动之外，肌动蛋白重塑对于对接和融合膜微域的组装和功能也至关重要，如酵母液泡所示。液泡融合是由蛋白质和脂质（包括肌动蛋白）组成的“顶点”环催化的，它围绕着紧密贴合的膜表面。 Jasplakinolide 敏感的肌动蛋白重塑是 SNARE 和 Ypt/Rab 蛋白以及 3-磷酸肌醇组装成该环微结构域所必需的，并且它们反过来控制肌动蛋白在环处的富集。对接后，融合需要进一步的 latrunculin 敏感肌动蛋白重塑。为了测试我们的工作模型，即从 F 到 G 的肌动蛋白重塑调节脂质和蛋白质组装到顶环中，然后通过从 G 到 F 的进一步重塑来促进融合，我们将： 1. 在液泡融合的每个步骤中建立 F 和 G 肌动蛋白的丰度和微域定位，利用它们独特的物理特性和配体结合，2. 进行遗传筛选以识别具有特定效果的肌动蛋白突变体 使用广泛且可用的突变体集合，研究重塑液泡肌动蛋白的液泡融合和肌动蛋白调节蛋白级联， 3. 使用肌动蛋白亲和分离方法鉴定肌动蛋白的液泡受体和调节其重塑的蛋白质， 4. 建立肌动蛋白和融合调节脂质（甾醇、磷酸肌醇和二酰甘油）之间的相互关系 使用顶点富集和肌动蛋白和脂质配体以及修饰酶的比例荧光显微镜测定，它们调节彼此组装成环结构域，5.使用可逆抑制配体和我们的比色测定，建立反式SNARE配对、肌动蛋白聚合和脂质重排以进行液泡融合的顺序或相互依赖性。 体外液泡融合，和 6. 通过系统纯化肌动蛋白和肌动蛋白调节因子并重新构建融合子反应，首先在液泡上，然后在脂质体模型系统中，重建液泡融合每个步骤对纯化肌动蛋白和肌动蛋白调节蛋白的功能依赖性。