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

Abstract

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.

Self-organizing glycolytic waves fuel cell migration and cancer progression

“…Emerging studies in many different types of cells have unveiled fascinating phenomena underlying these protrusive events. Waves of signal transduction and cytoskeletal activity propagate across the cell membrane and cortex matching closely the protrusive events [16][17][18][19][20][21][22][23][24] . Perturbation of the wave behavior has a predictable, causal effect on the protrusions and dependent physiological events [25][26][27] .…”

“…A stable HL-60 cell line co-expressing CIBN-CAAX and LifeActmiRFP703 was generated using a lentiviral-based approach described in previous studies 23,28 . In this dual expressing cell line, we stably expressed CRY2PHR-mCherry-AldolaseA via transposon integration 23,28,54 .…”

“…A 40X/1.30 Plan-Neofluar oil objective was used. Pre-treated, differentiated HL-60 cells were prepared for Zeiss LSM780 confocal microscopy on fibronectin-coated chambered coverglass as described earlier 23,28,54 . For global recruitment, the Argon laser was switched on after imaging for 3 min.…”