Integrin Complex Nucleation of Actin

描述(申请人提供)：白细胞和血小板的粘附性对机体对损伤和感染的炎症和血栓反应至关重要。对于白细胞迁移和血小板凝块回缩，必须通过黏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。最近的工作定义了肌动蛋白在应力纤维中的组装，以及整合素介导粘着成功的信号基础。然而，目前尚不清楚肌动蛋白应激纤维如何与黏附复合体相关联。我们的假设是，成熟的黏附复合体隔离肌动蛋白组装：机械和核形成新的肌动蛋白纤维。我们已经建立了一种体外实验来定性和定量地评估纯化的造血β-3整合素复合体的肌动蛋白纤维形成。整合素介导的这些细胞类型的黏附是非结构性的，需要激动剂的刺激。对于β-3整合素，激动剂的作用需要与特定的配体相遇而使β-3亚基发生一致的磷酸化。这种额外激活的要求可以防止不必要的血栓形成和炎症。应用这一知识，整合素复合体在非活性或活性状态下被提纯。在加入未聚合的荧光肌动蛋白后，肌动蛋白组装的动力学可以从形态和定量上进行监测。在这些研究中，肌动蛋白组装的特征将被用来确定活性整合素复合体是否符合肌动蛋白成核的标准。野生型和特征性功能丧失的突变体整合素将用于确定已知的粘连缺陷是由于配体结合效率低下还是未能连接肌动蛋白机械造成的。我们还将确定β-3整合素复合体的调节和非调节成分，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黏附的方法将独特地融合黏附细胞生物学和肌动蛋白生化领域，导致对介导白细胞迁移和血小板凝块回缩的事件的完整描述，并允许更详细地分析黏附障碍和测试未来针对炎症和血栓的治疗方法。

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.