Online

Senatore, Amanda J.

Pharmacology

2021

M.S.

Cellular adhesion to the extracellular environment is a critical component of physiological processes and its dysregulation drives pathological processes such as cancer metastasis. The input cells derive from their interaction with the extracellular matrix (ECM) is largely mediated through specialized organelles known as focal adhesions (FAs). FAs tether extracellular matrix-bound integrins to the contractile actin cytoskeleton and serve as attachment points which allow the cell to exert force on the extracellular environment via acto-myosin contractility. In addition to this structural role, FAs function as dynamic signaling hubs that respond to changes in force. Reversible phosphorylation plays a major role in relaying these mechanical signals. While tyrosine phosphorylation within this context is well studied, serine and threonine phosphorylation, despite being more abundant within FAs, are comparatively less well characterized and understood. Our lab has evidence indicating that protein kinase A (PKA), a patriarchal serine/threonine kinase which is involved in mechanically regulated signaling in other subcellular compartments, may also be active within FAs. To investigate this possibility, we used immunofluorescence and live cell imaging approaches to characterize changes in FA morphology and cellular traction force upon treatment of cells with a specific inhibitor of PKA. PKA inhibition increased FA size and aspect ratio and increased traction force in spreading cells. Subsequent studies suggested these changes might be a consequence of reduced phosphorylation of RhoA by PKA. We utilized microtubule depolymerization and regrowth as a tool to discretely study changes in adhesion dynamics and found that PKA inhibition prevented FA disassembly during microtubule regrowth. However, it is unknown whether any of these changes were due to the activity of PKA within adhesions themselves or elsewhere in the cell. Since global inhibition of PKA makes it impossible to target PKA activity only within adhesions, we provide preliminary evidence of the utility of the optogenetic tool OLAF (Optogenetic Localization of A kinase to Focal Adhesions) in this endeavor. OLAF allows for discrete and reversible recruitment of active PKA to FAs and will be an invaluable tool in understanding how PKA activity contributes to FA dynamics. The work presented here shows a role for PKA in controlling FA dynamics and traction force and provides a preliminary investigation of an optogenetic method to specifically study the role of PKA activity within FAs.