Curated Optogenetic Publication Database

Abstract: To migrate efficiently, neutrophils must polarize their cytoskeletal regulators along a single axis of motion. This polarization process is thought to be mediated through local positive feedback that amplifies leading edge signals and global negative feedback that enables sites of positive feedback to compete for dominance. Though this two-component model efficiently establishes cell polarity, it has potential limitations, including a tendency to "lock" onto a particular direction, limiting the ability of cells to reorient. We use spatially defined optogenetic control of a leading edge organizer (PI3K) to probe how neutrophil-like HL-60 cells balance "decisiveness" needed to polarize in a single direction with the flexibility needed to respond to new cues. Underlying this balancing act is a local Rac inhibition process that destabilizes the leading edge to promote exploration. We show that this local inhibition enables cells to process input signal dynamics, linking front stability and orientation to local temporal increases in input signals.

Abstract: Membrane tension is thought to be a long-range integrator of cell physiology. Membrane tension has been proposed to enable cell polarity during migration through front-back coordination and long-range protrusion competition. These roles necessitate effective tension transmission across the cell. However, conflicting observations have left the field divided as to whether cell membranes support or resist tension propagation. This discrepancy likely originates from the use of exogenous forces that may not accurately mimic endogenous forces. We overcome this complication by leveraging optogenetics to directly control localized actin-based protrusions or actomyosin contractions while simultaneously monitoring the propagation of membrane tension using dual-trap optical tweezers. Surprisingly, actin-driven protrusions and actomyosin contractions both elicit rapid global membrane tension propagation, whereas forces applied to cell membranes alone do not. We present a simple unifying mechanical model in which mechanical forces that engage the actin cortex drive rapid, robust membrane tension propagation through long-range membrane flows.

Abstract: By acting both upstream of and downstream from biochemical organizers of the cytoskeleton, physical forces function as central integrators of cell shape and movement. Here we use a combination of genetic, pharmacological, and optogenetic perturbations to probe the role of the conserved mechanosensitive mTOR complex 2 (mTORC2) programs in neutrophil polarity and motility. We find that the tension-based inhibition of leading-edge signals (Rac, F-actin) that underlies protrusion competition is gated by the kinase-independent role of the complex, whereas the regulation of RhoA and myosin II-based contractility at the trailing edge depend on mTORC2 kinase activity. mTORC2 is essential for spatial and temporal coordination of the front and back polarity programs for persistent migration under confinement. This mechanosensory pathway integrates multiple upstream signals, and we find that membrane stretch synergizes with biochemical co-input phosphatidylinositol (3,4,5)-trisphosphate to robustly amplify mTORC2 activation. Our results suggest that different signaling arms of mTORC2 regulate spatially and molecularly divergent cytoskeletal programs for efficient coordination of neutrophil shape and movement.

Abstract: T cells use kinetic proofreading to discriminate antigens by converting small changes in antigen binding lifetime into large differences in cell activation, but where in the signaling cascade this computation is performed is unknown. Previously, we developed a light-gated immune receptor to probe the role of ligand kinetics in T cell antigen signaling. We found significant kinetic proofreading at the level of the signaling lipid diacylglycerol (DAG) but lacked the ability to determine where the multiple signaling steps required for kinetic discrimination originate in the upstream signaling cascade (Tischer and Weiner, 2019). Here we uncover where kinetic proofreading is executed by adapting our optogenetic system for robust activation of early signaling events. We find the strength of kinetic proofreading progressively increases from Zap70 recruitment to LAT clustering to downstream DAG generation. Leveraging the ability of our system to rapidly disengage ligand binding, we also measure slower reset rates for downstream signaling events. These data suggest a distributed kinetic proofreading mechanism, with proofreading steps both at the receptor and at slower resetting downstream signaling complexes that could help balance antigen sensitivity and discrimination.