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.
