Multiplex Imaging of Signaling Pathways in Cell Motility

Abstract Advancements in cell biological technologies have enabled molecular processes to be interrogated in real time, within living cells, at submicron-level spatial and second-level temporal resolution. Our lab has developed new Förster resonance energy transfer (FRET)-based biosensor technologies utilizing monomeric, single-chain, fluorescent proteins, offering good sensitivity, probe reversibility, and easy quantitation. We are exploring real- world biological applications of our innovative FRET-based multiplex biosensor imaging technology, focusing on the complex coordination of Rho GTPase signaling pathways to better understand their critical roles in regulating cellular morphodynamics. We will continue developing our FRET-based, orthogonal biosensors for direct multiplex imaging to study the coordination of GTPases, their regulators, and their downstream targets during cell motility, adhesion, and migration. In the next 5 years of the program, we will first analyze the coordination of signaling dynamics between Rap GTPases and cytoskeletal Rho GTPases during adhesion, migration, and invasion of polarized cells. We will address newly identified intermediate signaling proteins that connect and coordinate these two GTPase classes during cellular morphodynamic control in both normal and diseased states. We will next target the spatial and temporal coordination of mDia1 and mDia2 formins, immediate downstream effector targets of Rho GTPase that regulate the local availability of activated, non– receptor tyrosine kinases, including Src, during cell motility and invasion. Interestingly, the differential, upstream selectivity of Rho GTPase paralogs and isoforms may play important roles in coordinating the activities of these two formin isoforms during cell migration and invasion, which we will address. We are also exploring the control of cell polarity, macropinocytosis, and phagocytosis through the coordinated activation of RhoG–Rac1 or RhoG–RhoA pathways. We are able to multiplex our new RhoG biosensor with Rac1 or RhoA biosensors to directly address the upstream regulator-initiated pathways of RhoG that coordinate Rac1- and RhoA-mediated cytoskeletal reorganization, driving physiologically important cellular functions. We are also exploring the signal coordination centered around vesicular RhoB GTPase, which interfaces with the cytoskeletal Rac1 and RhoA GTPase pathways and with vesicular trafficking through the Rab GTPase pathway. We will explore next-generation, FRET-based multiplexed approaches using fluorescence lifetime microscopy imaging to analyze FRET-based biosensors in living cells. This approach represents a long-term exploratory goal, and we will initiate our work by extending the utility of multiplexed FRET-based biosensors in living cells, exceeding the current limitation of two simultaneous biosensors in a single living cell. Our proposed works and the research trajectory will dissect the coordination of Rho GTPases and associated pathways governing cell adhesion, motility, and invasion by developing new biosensors, designing new imaging approaches, and extending the biological applications of FRET-based biosensors in direct multiplex imaging to monitor perturbations in signaling networks.