Fibronectin (FN) is an indispensable extracellular matrix (ECM) protein that is required for tissue repair and remodeling. In the wound, cells assemble newly synthesized FN into a three-dimensional (3D) fibrillar matrix that regulates a variety of cell behaviors including contractility. The receptor responsible for FN matrix assembly is the integrin a5b1. Although the structural features of FN required for matrix assembly have been examined, the specific receptor requirements have not been well defined. During the previous funding period, we demonstrated the novel finding that the a5 integrin cytoplasmic domain regulates FN matrix assembly and as a consequence, confers strong intercellular cohesion to 3D cellular aggregates. In this new proposal, we show for the first time that FN matrix assembly is regulated by endocytosis, leading us to hypothesize that regulated internalization of a5b1 and its recycling to the plasma membrane are required for the initiation and stabilization of the FN matrix. To test this hypothesis, we will accomplish the following Specific Aims: 1) The mechanisms of a5b1 receptor trafficking, in the presence or absence of endogenous FN matrices, will be determined using pharmacologic blockade, immunofluorescent imaging and molecular biological techniques. Stimulation or inhibition of receptor trafficking will be used to correlate FN matrix formation with receptor endocytosis; 2) Recombinant FN proteins will be used to analyze how FN structure regulates a5b1 receptor trafficking. The role of the actin and tubulin cytoskeletons in a5b1 receptor trafficking will be determined using pharmacologic agents that alter actin organization and microtubule stabilization, respectively; 3) Sites on the a5 integrin cytoplasmic domain that regulate receptor trafficking will be mapped by generating point mutations in wild type or chimeric a5 cDNAs that block interactions with chaperone proteins involved in endocytosis; 4) The effect of de novo a5b1 expression on the trafficking of the av|33 integrin will be analyzed and correlated with changes in avb3 receptor function. Excessive FN matrix deposition is associated in vivo with pathologic fibrosis and scarring. Therefore, an understanding of the molecular mechanisms that regulate FN matrix assembly will be important to the design of new therapies to prevent abnormal wound repair and to improve functional recovery following injury.
Showing the most recent 10 out of 14 publications
