mTORC2 coordinates the leading and trailing edge cytoskeletal programs during neutrophil migration

Abstract

By acting both upstream and downstream of 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 mechanoresponsive mTORC2 program 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 mTORC2 kinase arm is essential for regulation of Rho activity and Myosin II-based contraction at the trailing edge. Cells required mTORC2 for spatial and temporal coordination between the front and back polarity programs and persistent migration under confinement. mTORC2 is in a mechanosensory cascade, but membrane stretch did not suffice to stimulate mTORC2 unless the co-input PIP3 was also present. Our work suggests that different signalling arms of mTORC2 regulate spatially and molecularly divergent cytoskeletal programs allowing efficient coordination of neutrophil shape and movement.

Do cell membranes support or resist tension propagation?

“…To investigate membrane tension propagation upon endogenous force generation, we employed optogenetic regulation (Opto-PI3K) (22) in neutrophil-like HL-60 cells to activate localized actin-driven membrane protrusions (Fig. 1A, Fig.…”

iLID-antiGFP-nanobody is a flexible targeting strategy for recruitment to GFP-tagged proteins

“…These experiments require overexpression of the signaling molecules fused to SspB but also the target protein, which by itself might already have a signaling effect. iLID is widely used and often in combination with a plasma membrane tag (Dessauges et al, 2022; Gonzalez-Martinez et al, 2022; Saha et al, 2022; Vaidžiulytė et al, 2022). The approach can be improved by targeting proteins to a more specific location, for example recruiting to focal adhesions or adherens junctions where local GEF and GAP activity have been detected (Müller et al, 2020).…”