Acquisition of an iLas2 TIRF/FRAP imaging platform

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Project Abstract. Signaling pathways that control cell movement and membrane morphodynamics are tightly regulated at the subcellular level and occur with rapid kinetics. Therefore, the best way to investigate the molecules involved in these processes is to observe and control them in real-time. To achieve this, we need to develop molecular probes that report the activity of target molecules and microscopy systems to visualize and control these probes in living cells. In the parent award, we propose to develop optogenetically controlled inhibitors that modulate the activity of Guanine Exchange Factors of the Dbl family. These molecules are the main activators of Rho family GTPases, the major regulators of cell membrane dynamics. In this supplement, we request funds to acquire an iLas2 TIRF/FRAP imaging platform to characterize GEF-GTPases signaling in real-time in living cells through the application of our engineered inhibitors. The iLas2 TIRF/FRAP system will greatly enhance and expand the scientific premise of the parent awards by providing the ability to: 1) observe biological events where they primarily occur, at the contact sites between the cell and the coverslip, due to the TIRF capability; 2) rapidly uncage engineered inhibitors with accurate subcellular precision; 3) simultaneously observe GTPase activity using fluorescent biosensors.