In Vitro Reconstitution of Calcium-Mediated Membrane Reorganization by Annexin A2

膜联蛋白 A2 钙介导的膜重组的体外重建

• 批准号: 8317234
• 负责人: Michael D Vahey
• 金额: $ 4.92万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8317234
• 关键词: ANXA2 gene; Address; Annexins; Behavior; Binding; Biological Process; Biology; Calcium; Calcium Signaling; Caveolae; Cell membrane; Cell physiology; Cell-Cell Adhesion; Cells; Cholesterol; Clathrin; Complex; Cues; Development; Disease; Elements; Encapsulated; Endocytosis; Environment; Equilibrium; Exocytosis; Fluorescence Microscopy; Fluorescence Recovery After Photobleaching; In Vitro; Intercellular Junctions; Ionophores; Journals; Knowledge; Ligands; Link; Lipids; Measures; Mediating; Membrane; Membrane Lipids; Methodology; Microfluidics; Modeling; Monitor; Pathology; Phospholipids; Play; Process; Protein Family; Proteins; Research; Role; Science; Second Messenger Systems; Side; Signal Transduction; Stimulus; System; Techniques; Technology; Testing; Vesicle; aqueous; cell motility; extracellular; insight; link protein; lipid transport; new technology; novel; porin; protein distribution; receptor; receptor internalization; reconstitution; response; scaffold; second messenger; trafficking; trend; two-dimensional; unilamellar vesicle

DESCRIPTION (provided by applicant): The internalization of receptors and ligands via endocytosis requires dramatic reorganization of the cell membrane, coordinated by extracellular and intracellular signals. Although membrane reorganization is a feature of both clathrin- and caveolae-mediated endocytosis, the mechanism by which it is achieved - and how it is coordinated with specific cues - remains unclear. One set of candidates involved in membrane reorganization prior to caveolae-mediated endocytosis are the annexins, a family of proteins which translocate to the membrane in a calcium-dependent manner, where they associate with lipid microdomains and may form two-dimensional scaffolds [Gerke, Nat. Rev. Mol. Cell Biol., 2005]. This behavior suggests a possible role for annexins in organizing and maintaining caveolae, regions of the membrane where lipid microdomains and GPI-anchored proteins in the outer leaflet are believed to cluster prior to internalization [Pelkmans, Traffic, 2002]. While certain annexins bind cholesterol in a calcium-independent manner and localize to the cytoplasmic side of caveolae [Mayran, EMBO Journal, 2003; Lisanti, J. Cell Biol., 1994], it remains an open question as to how the calcium-independent interactions of annexins with cholesterol are balanced with their calcium-dependent interactions with other membrane components, and if annexins themselves play an active role in coordinating the clustering of lipid microdomains and GPI-anchored proteins into caveolae from the cytoplasmic side of the membrane. This research proposes to address this question by reconstructing elements of membrane- annexin interactions in vitro, where the stimulating calcium signal can be spatially and temporally controlled, and the annexin response determined under biochemically well-defined conditions. This will be accomplished by encapsulating the annexin AnxA2 in lipid vesicles with controlled membrane composition (Aim 1), studying the effects of cholesterol on AnxA2 translocation (Aim 2), and investigating the effects of annexin A2 translocation and distribution on the distributions of proteins linked to the outer leaflet of the membrane (Aim 3). Accomplishing these aims will help to elucidate the mechanism through which annexins participate in caveolae-mediated endocytosis. A better mechanistic understanding of this fundamental cellular process could help in identifying new targets for treating pathologies associated with caveolae, including many lipid storage diseases [Marks, Trends Cell Biol., 2002]. PUBLIC HEALTH RELEVANCE: The completion of this research will advance current knowledge of how cells coordinate the complex traffic of lipids in their interior, and the role of external signals in this coordination. Understanding how cells regulate their internal composition in response to external cues will provide insight into how this process can malfunction during disease, and may suggest treatments that could be developed to correct these malfunctions.

描述（由申请人提供）：通过内吞作用的受体和配体的内在化需要对细胞膜进行戏剧性的重组，并由细胞外和细胞内信号协调。尽管膜的重组是网格蛋白和小窝介导的内吞作用的特征，但它实现的机制以及如何与特定提示协调的机制尚不清楚。在小窝介导的内吞作用之前，一组参与膜重组的候选者是膜联蛋白，它是一个蛋白质家族，它们以钙依赖性的方式转移到膜上，它们与脂质微域相关，并可能形成二维cackolds [Gerke [Gerke] [Gerke，Nat。摩尔牧师。 Cell Biol。，2005]。这种行为表明，膜联蛋白在组织和维持小窝，膜的区域，膜的区域中，外部小叶中的脂质微区和GPI锚定的蛋白质在内在化之前会聚集[Pelkmans，交通，2002年]。而某些膜毒素以钙独立的方式结合胆固醇，并定位于小窝的细胞质侧[Mayran，Embo Journal，2003年； Lisanti, J. Cell Biol., 1994], it remains an open question as to how the calcium-independent interactions of annexins with cholesterol are balanced with their calcium-dependent interactions with other membrane components, and if annexins themselves play an active role in coordinating the clustering of lipid microdomains and GPI-anchored proteins into caveolae from the cytoplasmic side of the membrane.这项研究建议通过在体外重建膜 - 膜联蛋白相互作用的元素来解决这个问题，其中刺激的钙信号可以在空间和时间控制上进行，并且在生化定义良好的条件下确定的膜联蛋白反应。这将通过封装具有控制膜组成的脂质囊泡中的膜联蛋白Axcha2（AIM 1），研究胆固醇对AnxA2易位的影响（AIM 2），并研究膜联蛋白A2易位和分布对蛋白质分布的影响与蛋白质的分布链接到膜外叶的外部传叶（AIM 3）。实现这些目标将有助于阐明膜联蛋白参与小窝介导的内吞作用的机制。对这种基本细胞过程的更好的机械理解可以帮助识别治疗与小窝相关的病理学的新靶标，包括许多脂质储存疾病[Marks，Trends Cell Biol。，2002]。 公共卫生相关性：这项研究的完成将促进当前有关细胞如何协调脂质在其内部的复杂流量以及外部信号在此协调中的作用的知识。了解细胞如何根据外部提示调节其内部组成将提供有关该过程在疾病期间如何故障的洞察力，并可能提出可以开发出来纠正这些故障的治疗方法。