The main objective of this Project is to employ a combination of molecular cell biology and bioengineering approaches to test the following central hypothesis: that integrin/ligand binding properties (e.g., integrin expression level, ligand concentration) governing the number of integrin/ligand bonds affect integrin-mediated signaling responses to mechanical stimuli in a manner quantitatively depending on identifiable metrics characterizing signal activation curves.
Specific Aim 1 Measure MAPK/SAPK activation dynamics in CHO cells responding to a range of membrane deformation, as a integrin/ligand bond number is manipulated by varying receptor number and ligand density, using CHO cells expressing different levels of alpha5beta1 integrin on substrate presenting different fibronectin concentrations.
Specific Aim 2 Compare signaling and gene expression responses in smooth muscle cells in response to a range of membrane deformation degrees, as integrin/ligand bond number is manipulated by varying ligand density, determining: (a) MAPK/SAPK activation dynamics, and consequently certain metrics quantitatively characterizing the activation curves; (b) expression dynamics of the Egr-1 and PAI-1 genes.
Specific Aim 3 Determine the relative numbers of integrin/ligand bonds as integrin and ligand properties are varied, using fluorescence lifetime microscopy in multi-photon mode, incorporating fluorescently-labeled (Alex 488) Fab antibody fragments to the alpha5beta1 integrin in CHO cells and fluorescently-labeled (Cy3) fibronectin as the substratum ligand.
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