Cell locomotion is fundamental to development, wound healing, immune system function, and metastatic cancer. It involves a complex, dynamic interplay between proterusion, retraction, and adhesion. This proposal is focused on several major issues that we need to understand more fully in order to explain cell locomotion. The temporal and spatial distributions of traction that a cell applies to the substratum are prime determinants of cell shape and speed. Experiments are proposed to image the distribution of tractions exerted by locomoting cells and to address how traction is generated and regulated. In order to examine the combined role of contractility and adhesion in traction force generation, we will perturb both lamellar contractility and cell substratum contracts and observe the resultant effects on cell behavior. To address how adhesion and traction are regulated in functional contexts, we will apply this assay to cells that are turning and cells that are placed under """"""""load"""""""", and visualize how the traction distribution is altered in these situations. To address the molecular origin of traction force generation, we will apply this assay to cells such as Dictyostelium and its mutants where more is known about molecular motors that may generate traction. Lastly, we need to define, in rapidly locomoting cells, the molecular composition of the cell-substratum contract zones where traction is applied to the substratum. This work will employ a range of cell biological tools including immunofluorescence, transfection, and traditional and novel forms of light microscopy. The latter include release of caged physiological mediators to perturb lamellar contractility and chromophore assisted laser inactivation to locally al=ter cell adhesions. It is anticipated that the information generated in this project will ultimately lead to a deepened understanding of the role of the cell movement in normal and pathological states.
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