Filopodia in Leukocyte and Endothelial Cell Function

白细胞和内皮细胞功能中的丝状伪足

• 批准号: 7217764
• 负责人: RICHARD E CHENEY
• 金额: $ 25.01万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7217764
• 关键词: SDS polyacrylamide gel electrophoresis; biological signal transduction; cell adhesion; cell component structure /function; cell components; chemotaxis; immune response; immunofluorescence technique; inflammation; integrins; leukocytes; matrix assisted laser desorption ionization; myosins; phagocytosis; protein binding; protein localization; protein protein interaction; protein structure function; receptor expression; tissue /cell culture; vascular endothelium

Growing evidence indicates that the slender actin-based extensions known as filopodia play central roles in the cell biology underlying inflammation and angiogenesis. The initial contacts between leukocytes and endothelial cells during leukocyte rolling occur at the tips of filopodia-like structures and endothelial "pathfinder" cells appear to be guided by filopodia during angiogenesis. Despite these critical roles of filopodia in leukocytes and endothelial cells, the molecular mechanisms underlying filopodial extension, adhesion, and signaling are largely unknown. Our discovery that myosin-X (Myo10) is a core component of a putative filopodial tip complex, is involved in filopodia formation, and undergoes a novel form of motility in filopodia, gives us a powerful tool to investigate these questions. We therefore propose to: I. Determine the functional consequences of MyolO's interaction with integrins. Since Myo10 is an unconventional myosin whose tail includes a PERM domain that can binds to the cytoplasmic domains of pintegrins, we will test whether binding to this motor protein activates integrins. We will also test if Myo10 binds to the leukocyte specific beta2-integrins and if Myo10 is required for complement-mediated phagocytosis. II. Analyze the functions of Myo10 in leukocytes and endothelial cells in processes such as filopodial extension, chemotaxis, and transendothelial migration using siRNA and a dominant negative construct. Although Myo10 is the MyTH-FERM myosin expressed in most vertebrate cells and a related myosin in lower organisms is required for filopodia formation, adhesion, and phagocytosis, almost nothing is known about the functions of this family of myosins in leukocytes and endothelial cells. III. Define the basic biochemical properties and components of the filopodial tip complex. Like the focal adhesion, the filopodial tip complex appears to function as a specialized site of cell adhesion, signaling, and actin polymerization, but currently very little is known about the molecular components and properties of the filopodial tip complex. This work is of great relevance to public health because it will investigate the fundamental cell biological mechanisms of inflammation and cell signaling that underlie cardiovascular disease and other serious human health problems.

越来越多的证据表明，被称为丝状伪足的细长肌动蛋白为基础的延伸在 炎症和血管生成的细胞生物学基础。白细胞和 白细胞滚动期间的内皮细胞出现在丝状伪足样结构和内皮细胞的尖端 “探路者”细胞似乎在血管生成期间由丝状伪足引导。尽管这些关键作用， 白细胞和内皮细胞中的丝状伪足，丝状伪足延伸的分子机制， 粘附和信号传导在很大程度上是未知的。我们发现肌球蛋白-X（Myo 10）是 一种假定的丝状伪足尖端复合体，参与丝状伪足的形成，并在 丝状伪足，给了我们一个强大的工具来调查这些问题。因此，我们建议： I.确定MyolO与整联蛋白相互作用的功能后果。由于Myo 10是一个 非常规肌球蛋白，其尾部包括可结合β整联蛋白的胞质结构域的PERM结构域， 我们将测试是否与这种马达蛋白结合激活整联蛋白。我们还将测试Myo 10是否 与白细胞特异性β 2-整联蛋白结合，并且是否需要Myo 10进行补体介导的吞噬作用。 二.分析Myo 10在白细胞和内皮细胞中的功能， 使用siRNA和显性阴性的丝状伪足延伸、趋化性和跨内皮迁移 构建体虽然Myo 10是在大多数脊椎动物细胞中表达的MyTH-FERM肌球蛋白， 在低等生物中，肌球蛋白是丝状伪足形成、粘附和吞噬作用所必需的， 已知该肌球蛋白家族在白细胞和内皮细胞中的功能。 三.定义丝状伪足尖端复合体的基本生化特性和组成。像 粘着斑，丝状伪足尖端复合体似乎作为细胞粘着，信号传导， 和肌动蛋白聚合，但目前对肌动蛋白的分子组成和性质知之甚少。 丝状伪足尖端复合体。 这项工作与公共卫生密切相关，因为它将研究基本的细胞生物学过程。 炎症和细胞信号传导机制是心血管疾病和其他严重疾病的基础。 人类健康问题。