Lens formation requires a chronologically and spatially executed program of cell division and proliferation, as well as exit from the cell cycle and differentiation into lens fibers. These processes are controlled in most cell types by the timely degradation of cell cycle regulators by Ubiquitin Proteasome Pathways (UPPs). Aberrations, in these processes frequently result in microphthalmia or cataract. Yet there are few published papers that address either the lens cell cycle or its control by- UPPs. During the ongoing grant, we demonstrated that Ub and Ubc3 (a Ub ligase) are required for proliferation and differentiation. However, regulation of the cell cycle by-these moieties occurs at the G2/M transition and not, as predicted, at G1/S. We now seek to determine if the same controls are observed in vivo by directing expression of mutant Ub to the epithelial and differentiating lens cells in transgenic animals. Our recent data beget two new overall hypotheses: 1) proteolysis involving Ub and Ubc3 is required for proliferation, differentiation, and lens formation in vivo; 2) a UPP which involves an undescribed Ubc3-E3 interaction is involved in control of G2/M events in lens. These overall hypotheses are separated into 4 specific aims, to test the hypotheses that: an active UPP is required for proliferation, differentiation and lens formation in vivo; ubiquitination is required for the lens cell cycle, particularly in G2/M; lens Ubc3 cooperates with an E3, which we will identify, to control the G2/M transition; and control of the cell: cycle at G2/M requires ubiquitination of the APC regulators in a Ubc3-dependent process. These studies will address a major objective of the NEI lens and cataract program: to characterize controls of lens cell division and differentiation, and their roles in formation of secondary cataract. The long-term objective is to prolong function of 1) the natural lens by gaining a better understanding of processes involved in control of lens cell proliferation and differentiation, as well as in lens formation, and 2) implanted lenses by limiting secondary cataract due to overgrowth. Our recent papers show that these results will also lead to a better understanding of corneal wound healing and retina responses to stress. This information, and our novel """"""""reagents"""""""", will also find use in new ways to limit secondary cataract, prolong function of glaucoma medication, limit cancer and in fighting other proliferative maladies. We are joined in this effort by excellent collaborators, each of whom is a leader in his field. ? ? ?
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