Integrin Complex Nucleation of Actin

肌动蛋白整合素复合物成核

• 批准号: 6868395
• 负责人: SCOTT D BLYSTONE
• 金额: $ 7.6万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6868395
• 关键词: actins; bioassay; biochemistry; biological signal transduction; cell adhesion; cell motility; chemical kinetics; cytoskeleton; electron microscopy; extracellular matrix; fluorescence microscopy; inflammation; integrins; microfilaments; molecular assembly /self assembly; posttranslational modifications; protein protein interaction; protein purification; protein structure; protein structure function; thrombosis; tissue /cell culture

DESCRIPTION (provided by applicant): Adhesion of leukocytes and platelets is crucial to the inflammatory and thrombotic response of the body to injury and infection. For leukocyte migration and platelet clot retraction, a connection must be established between the extracellular matrix and the actin cytoskeleton via the integrin family of adhesion receptors. Recent work has defined the assembly of actin into stress fibers and also the signaling underlying successful integrin-mediated adhesion. However, it remains unknown how actin stress fibers become associated with adhesion complexes. It is our hypothesis that the mature adhesion complex sequesters actin assembly :machinery and nucleates the formation of new actin fibers. We have developed an in vitro assay to qualitatively and quantitatively assess actin fiber formation by purified hematopoietic beta-3 integrin complexes. Integrin mediated adhesion in these cell types is non-constitutive, requiring agonist stimulation. For beta-3 integrins, the effects of agonist require coincident phosphorylation of the beta-3 subunit provided by encounter with specific ligand. This requirement for additional activation prevents unwarranted thrombosis and inflammation. Applying this knowledge, integrin complexes are purified in inactive or active states. Following addition of unpolymerized fluorescent actin, the kinetics of actin assembly can be monitored morphologically and quantitatively. In these studies actin assembly will be characterized to determine if active integrin complexes meet the criteria for actin nucleation. Wild type and characterized loss-of-function mutant integrins will be used to determine whether known adhesion defects result from ligand binding inefficiencies or failures to engage actin machinery. We will also characterize the regulated and non-regulated components of beta-3 integrin complexes to determine which molecules are necessary for the production of actin fibers. This approach to understanding integrin-mediated adhesion will merge the adhesion cell biology and actin biochemistry fields uniquely, resulting in a complete depiction of events mediating leukocyte migration and platelet clot retraction and allowing more detailed analysis of adhesion disturbances and testing of future therapeutics targeting inflammation and thrombosis.

描述（由申请方提供）：白细胞和血小板的粘附对于机体对损伤和感染的炎症和血栓形成反应至关重要。对于白细胞迁移和血小板凝块收缩，必须通过粘附受体的整合素家族在细胞外基质和肌动蛋白细胞骨架之间建立连接。最近的工作已经确定了肌动蛋白组装成应力纤维，也成功的整合素介导的粘附信号。然而，它仍然是未知的肌动蛋白应力纤维如何成为与粘附复合物。我们的假设是，成熟的粘附复合物隔离肌动蛋白组装：机械和核形成新的肌动蛋白纤维。我们已经开发了一种体外试验，以定性和定量评估纯化的造血β-3整合素复合物的肌动蛋白纤维的形成。这些细胞类型中的整合素介导的粘附是非组成型的，需要激动剂刺激。对于β-3整联蛋白，激动剂的作用需要与特异性配体相遇所提供的β-3亚基的同时磷酸化。这种额外激活的要求防止了不必要的血栓形成和炎症。应用这一知识，整联蛋白复合物被纯化为非活性或活性状态。加入未聚合的荧光肌动蛋白后，可以从形态学和定量上监测肌动蛋白组装的动力学。在这些研究中，肌动蛋白组装将被表征以确定活性整合素复合物是否满足肌动蛋白成核的标准。将使用野生型和表征的功能丧失突变体整联蛋白来确定已知的粘附缺陷是否是由配体结合效率低下或未能接合肌动蛋白机制引起的。我们还将表征β-3整合素复合物的调节和非调节组分，以确定哪些分子是产生肌动蛋白纤维所必需的。这种理解整合素介导的粘附的方法将独特地合并粘附细胞生物学和肌动蛋白生物化学领域，导致介导白细胞迁移和血小板凝块收缩的事件的完整描述，并允许更详细地分析粘附紊乱和测试未来的治疗靶向炎症和血栓形成。