Nanotopography modulates intracellular excitable systems through cytoskeleton actuation.

Cellular sensing of most environmental cues involves receptors that affect a signal-transduction excitable network (STEN), which is coupled to a cytoskeletal excitable network (CEN). We show that the mechanism of sensing of nanoridges is fundamentally different. CEN activity occurs preferentially on nanoridges, whereas STEN activity is constrained between nanoridges. In the absence of STEN, waves disappear, but long-lasting F-actin puncta persist along the ridges. When CEN is suppressed, wave propagation is no longer constrained by nanoridges. A computational model reproduces these experimental observations. Our findings indicate that nanotopography is sensed directly by CEN, whereas STEN is only indirectly affected due to a CEN-STEN feedback loop. These results explain why texture sensing is robust and acts cooperatively with multiple other guidance cues in complex, in vivo microenvironments.

Complementary Cytoskeletal Feedback Loops Control Signal Transduction Excitability and Cell Polarity.

To move through complex environments, cells must constantly integrate chemical and mechanical cues. Signaling networks, such as those comprising Ras and PI3K, transmit chemical cues to the cytoskeleton, but the cytoskeleton must also relay mechanical information back to those signaling systems. Using novel synthetic tools to acutely control specific elements of the cytoskeleton in Dictyostelium and neutrophils, we delineate feedback mechanisms that alter the signaling network and promote front- or back-states of the cell membrane and cortex. First, increasing branched actin assembly increases Ras/PI3K activation while reducing polymeric actin levels overall decreases activation. Second, reducing myosin II assembly immediately increases Ras/PI3K activation and sensitivity to chemotactic stimuli. Third, inhibiting branched actin alone increases cortical actin assembly and strongly blocks Ras/PI3K activation. This effect is mitigated by reducing filamentous actin levels and in cells lacking myosin II. Finally, increasing actin crosslinking with a controllable activator of cytoskeletal regulator RacE leads to a large decrease in Ras activation both globally and locally. Curiously, RacE activation can trigger cell spreading and protrusion with no detectable activation of branched actin nucleators. Taken together with legacy data that Ras/PI3K promotes branched actin assembly and myosin II disassembly, our results define front- and back-promoting positive feedback loops. We propose that these loops play a crucial role in establishing cell polarity and mediating signal integration by controlling the excitable state of the signal transduction networks in respective regions of the membrane and cortex. This interplay enables cells to navigate intricate topologies like tissues containing other cells, the extracellular matrix, and fluids.

Ras suppression potentiates rear actomyosin contractility-driven cell polarization and migration.

Ras has been extensively studied as a promoter of cell proliferation, whereas few studies have explored its role in migration. To investigate the direct and immediate effects of Ras activity on cell motility or polarity, we focused on RasGAPs, C2GAPB in Dictyostelium amoebae and RASAL3 in HL-60 neutrophils and macrophages. In both cellular systems, optically recruiting the respective RasGAP to the cell front extinguished pre-existing protrusions and changed migration direction. However, when these respective RasGAPs were recruited uniformly to the membrane, cells polarized and moved more rapidly, whereas targeting to the back exaggerated these effects. These unexpected outcomes of attenuating Ras activity naturally had strong, context-dependent consequences for chemotaxis. The RasGAP-mediated polarization depended critically on myosin II activity and commenced with contraction at the cell rear, followed by sustained mTORC2-dependent actin polymerization at the front. These experimental results were captured by computational simulations in which Ras levels control front- and back-promoting feedback loops. The discovery that inhibiting Ras activity can produce counterintuitive effects on cell migration has important implications for future drug-design strategies targeting oncogenic Ras.

Ras-mediated homeostatic control of front-back signaling dictates cell polarity.

Studies in the model systems, Dictyostelium amoebae and HL-60 neutrophils, have shown that local Ras activity directly regulates cell motility or polarity. Localized Ras activation on the membrane is spatiotemporally regulated by its activators, RasGEFs, and inhibitors, RasGAPs, which might be expected to create a stable 'front' and 'back', respectively, in migrating cells. Focusing on C2GAPB in amoebae and RASAL3 in neutrophils, we investigated how Ras activity along the cortex controls polarity. Since existing gene knockout and overexpression studies can be circumvented, we chose optogenetic approaches to assess the immediate, local effects of these Ras regulators on the cell cortex. In both cellular systems, optically targeting the respective RasGAPs to the cell front extinguished existing protrusions and changed the direction of migration, as might be expected. However, when the expression of C2GAPB was induced globally, amoebae polarized within hours. Furthermore, within minutes of globally recruiting either C2GAPB in amoebae or RASAL3 in neutrophils, each cell type polarized and moved more rapidly. Targeting the RasGAPs to the cell backs exaggerated these effects on migration and polarity. Overall, in both cell types, RasGAP-mediated polarization was brought about by increased actomyosin contractility at the back and sustained, localized F-actin polymerization at the front. These experimental results were accurately captured by computational simulations in which Ras levels control front and back feedback loops. The discovery that context-dependent Ras activity on the cell cortex has counterintuitive, unanticipated effects on cell polarity can have important implications for future drug-design strategies targeting oncogenic Ras.