Excitable Networks in Directed Cell Migration

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We are investigating molecular mechanisms of directed cell migration, a critical process in health and disease, using Dictyostelium as a discovery tool to inform our studies of neutrophils, macrophages, and epithelial cells. At the core of our working model are coupled Signal Transduction and Cytoskeletal Excitable Networks, referred to as STEN and CEN which drive motility. The STEN integrates inputs from directional sensing and polarity networks to bring about directed migration. In the last grant period, we found that protrusions are governed by waves of coupled STEN-CEN activities and that manipulation of negatively charged lipids on the inner face of the membrane can alter network excitability and control cell behavior. The STEN-CEN concept is conserved in mammalian cells and the networks are hyperactivated in transformed cells, augmenting motility and macropinocytosis. Since these processes require geranyl geranylation, statins cause starvation of cancer cells. Finally, we found that vesicles internalized from retracting protrusions carry “back” components to the rear of the cell contributing to polarity. How do diverse cellular protrusions depend on the setpoint/threshold of STEN-CEN? We are combining imaging, synthetic biological, and computational studies to prove that pseudopods, lamellipods, forming phagosomes, and so on are closely related on a spectrum and interconvertible. We will show that spatiotemporal patterns of activities and responses to acute molecular perturbations are consistent across these protrusions and parallel those established in propagating STEN-CEN waves. What explains the extraordinary coordination of activities in STEN and CEN? Surmising that charge on the inner leaflet of the membrane is an organizer, we are 1) designing methods to directly monitor charge in local regions; 2) determining how anionic lipids transiently decrease; 3) manipulating charge locally with optogenetic systems; 4) examining how the location of key proteins is regulated by surface charge. Is lowered STEN threshold a general property of transformed cells and can it be exploited? To address this question, we are 1) comparing threshold indicators, such as propagating waves, with macropinocytosis and statin sensitivity in a series of increasingly metastatic cell lines and organoids; 2) identifying the essential geranylgeranylated proteins in STEN; 3) genetically engineering cells to increase threshold to normalize cancer cells or further decrease threshold to induce cell death. How does control of STEN and CEN at the cell poles mediate directional sensing and polarity? First, using a novel suppression assay, we are screening kinase and substrate deficient cells to identify global inhibitors. Second, we are studying membrane flow in a variety conditions to pursue our “reverse fountain” model and it reconcile with alternate models that argue membrane flows from front to back. 1