Mechanisms of cell membrane repair in corneal cells

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The broad long term goal of this research is to understand the basic mechanisms of cell membrane repair in corneal cells. The major hypothesis to be tested is that cell membrane repair in the cornea is dependent on vesicle trafficking, in which vesicle recruitment, transport, docking and fusion by exocytosis near the site of disruption are essential for corneal cell membrane resealing. This hypothesis will be tested by monitoring membrane resealing, exocytosis and membrane tension after micropuncture wounds. Exocytotic fusion will be disrupted by using neurotoxins to disrupt SNARE complex formation known to be required for vesicle fusion in exocytosis. Specific kinase inhibitors and activators will be used to elucidate the roles of calcium/calmodulin-dependent protein kinase (CaM kinase), protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA). The requirement for conventional kinesin in vesicle transport will be tested with specific function-blocking antibodies and competitive partial constructs. The role of specific myosin subtypes in cell membrane repair in corneal cells will be examined by the use bf by transient knockout of nonmuscle myosin IIA and IIB expression with phosphorothioate-modified antisense oligodeoxynucleotides and by the localization of these proteins by immunofluorescence microscopy to determine their site of action. A second hypothesis to be tested is that long term facilitation of cell membrane repair is dependent on cAMP Responsive Element Binding Protein (CREB) mediated gene transcription. To test this hypothesis, dominant-negative vectors of CREB will be stably expressed in corneal cells to inhibit the CREB mediated signaling pathway and examine the effect on the long term facilitation of membrane resealing. CRE- GFP (CRE-Green Fluorescent Protein) gene transfected cells will be used to detect CREB-mediated gene transcription and study its regulation by kinases. To test for the translocation of PKA to the nucleus after wounding, GFP-tagged PKA subunits will be transfected into corneal cell cultures. DNA microarray analysis will be used to determine significant changes in gene expression after wounding by identifying genes expressed in a CREB-dependent manner. The knowledge gained by these studies will help define optimal conditions for cell membrane repair in cornea under adverse conditions, such as dry eye, long-term contact lens wear, trauma, surgery and the storage of donated corneas in eye banks.