Temporary plasma membrane disruptions in corneal epithelium and keratocytes.

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Transient plasma membrane disruptions (TPMD) are minor, quickly repairable plasma membrane tears that result from normal stresses on cells within tissues. We hypothesize that keratocyte and cornea epithelial (CE) TPMDs represent a novel cornea mechanosensation pathway, with TPMDs routinely occurring in keratocytes and CE following mechanical stressors such as eye rubbing and application of contact lenses. We further hypothesize that the resulting TPMD-induced Ca++ waves (TPMD-Ca++ Wvs) initiate signaling cascades that coordinate specific activities of keratocytes and CE, including corneal matrix maintenance and cell actin dynamics, respectively, which become disrupted in pathologies such as diabetes. The first Aim of this proposal is to identify the specific Ca++ channels responsible for keratocyte and CE TPMD-Ca++ Wvs. The only Ca++ transporters identified to date in keratocytes are TRPV1 and TRPM8. We determined that neither are significantly involved in TPMD-Ca++ Wvs, and our preliminary data indicate that L- type Ca++ channels are likely involved. Identifying the specific Ca++ channels involved will allow us to determine the signaling pathways triggering these Ca++ waves. Channel identification will be carried out by creating TPMDs in individual mouse and human keratocytes and CE using a multiphoton microscope, either in culture or within ex-vivo corneas, and pharmacological blockers and genetic knockdown of the identified channels will be utilized to identify channel subtypes contributing to the TPMD-Ca++ Wvs. The second Aim is to identify the cell signaling pathways responsible for initiating and propagating TPMD- Ca++ Wvs in keratocytes and CE in normal and diabetic corneas. We previously examined the general contribution of several signaling pathways leading to keratocyte TPMD-Ca++Wvs, including ATP, release of intracellular Ca++ stores, and gap junctions. This aim is designed to identify the specific signaling molecules responsible for keratocyte and CE TPMD-Ca++Wvs. Pharmacological blockers and genetic knockdown of the signaling molecules we have identified to date and their different receptor subtypes will be employed to determine which specific receptors and signaling proteins are responsible for initiating and propagating keratocyte and CE TPMD-Ca++ Wvs. Aim 3 will test the hypothesis that TPMD-Ca++ Wvs initiate Ca++-activated activities associated with cornea matrix remodeling and CE actin dynamics, and that these are altered in diabetic corneas. Our preliminary data indicate that keratocyte and CE TPMD-Ca++ Wvs influence cornea matrix and actin remodeling, respectively, along with other physiological activities. We also find that diabetes alters TPMD responses. TPMDs will be created by gentle manipulation of healthy and diabetic cells in culture and in diabetic mice. TPMD-Ca++ Wvs will be blocked pharmacologically or with genetic knockdown, and matrix protein synthesis and CE actin remodeling will be examined, along with additional activities including ATP release, apoptosis, migration, and proliferation.