The body's first line of defense against infection is the circulating white blood cells that detect and respond to tissue damage. These cells move rapidly towards the site of injury using an amoeboid type of motility. This movement is characterized by the formation of broadly distributed, low affinity contacts between the cell and surrounding substrate. The amoeboid adhesions generated by leukocytes are composed of many of the same components as the tightly organized, high affinity adhesions formed by slowly moving fibroblastic cells (focal contacts), but the organization and regulation of these molecules is distinct in amoeboid cells. Identifying the distinct mechanisms by which shared adhesion molecules are employed in amoeboid cells versus fibroblastic cells is key to understanding how rapid cell migration is achieved. The cell adhesion machinery that provides a dynamic link between extracellular matrix receptors and the actin cytoskeleton has been conserved throughout evolution. An unexpected role for a highly conserved unconventional myosin, myosin VII (M7), in amoeboid adhesion has been discovered in the amoeba Dictyostelium. The goal of this project is to define the role of M7 amoeboid adhesion. The first question that will be addressed is how M7 is targeted to adhesion sites, a critical step towards understanding the relationship between this motor protein and the cell adhesion machinery. The contribution of the conserved M7 tail domains to M7 localization, membrane dynamics and cellular adhesion will be systematically investigated. Essential domains will then be used as probes to identify binding partners (most likely other adhesion molecules or regulators). The next question is whether M7 motor activity is required for its role in amoeboid adhesion. M7 could participate in adhesion by passively linking adhesion components to the cytoskeleton or it could be required to generate tension at sites of adhesion. Cells expressing M7 motor mutants (either strong or weak actin binding) will be characterized to determine if cycles of M7 interaction with actin are necessary for its role in amoeboid adhesion. An in vitro analysis of the motor properties of M7 will also be performed to determine if it has the potential to function as an anchor or a transport motor. The results of this study will provide basic new insights into the molecular basis of amoeboid adhesion and how a myosin contributes to this critical type of adhesion to promote rapid cell migration.
The body's ability to fight infection depends on the ability to white blood cells to move rapidly to the site of bacterial invasion. The proposed work will provide new insight into the basic mechanism of how cells of the immune system optimize their interactions with their surrounding substrates to ensure their timely arrival at sites of injury to ward off infection.
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