Directional reorientation of migrating neutrophils is limited by suppression of receptor input signaling at the cell rear through myosin II activity

Abstract

To migrate efficiently to target locations, cells must integrate receptor inputs while maintaining polarity: a distinct front that leads and a rear that follows. Here we investigate what is necessary to overwrite pre-existing front-rear polarity in neutrophil-like HL60 cells migrating inside straight microfluidic channels. Using subcellular optogenetic receptor activation, we show that receptor inputs can reorient weakly polarized cells, but the rear of strongly polarized cells is refractory to new inputs. Transient stimulation reveals a multi-step repolarization process, confirming that cell rear sensitivity to receptor input is the primary determinant of large-scale directional reversal. We demonstrate that the RhoA/ROCK/myosin II pathway limits the ability of receptor inputs to signal to Cdc42 and reorient migrating neutrophils. We discover that by tuning the phosphorylation of myosin regulatory light chain we can modulate the activity and localization of myosin II and thus the amenability of the cell rear to ‘listen’ to receptor inputs and respond to directional reprogramming.

Mechanical competition promotes selective listening to receptor inputs to resolve directional dilemmas in neutrophil migration

“…Notably, and consistent with our previous observations in straight channels (Hadjitheodorou et al, 2021), the direction of migration reverses considerably before the two cell edges reach equal levels of Cdc42 activity and myosin II localization (cross-point) (Supplementary Figs. 2A-2D).…”

“…5G). Similarly to what we previously found for optogenetic receptor-directed cell reversals in 1-D straight channels (Hadjitheodorou et al, 2021), this suggests that the refractory nature of the retracting edge is at least in part mediated by local myosin II/RhoA activity.…”

“…Prior work has shown that myosin II contractile activity at the cell’s rear contributes to the maintenance of polarity, while also inhibiting cell front polarity programs (Hadjitheodorou et al, 2021; Smith et al, 2007; Uchida et al, 2003; Xu et al, 2003). We therefore sought to test the effects of altering myosin contractility in our optogenetic reversal assay.…”

“…The parental HL60 cell line was a generous gift from Orion Weiner’s lab. In this work, we used the previously published HL60 cell line expressing actin-YFP and MRLC-mApple (Tsai et al, 2019), an HL60 cell line expressing the tdTomato/tdKatushka2 FRET biosensor for Cdc42 and the light-controllable parapinopsin GPCR (Hadjitheodorou et al, 2021), and an HL60 cell line expressing mScarlet-I-tagged Myl9 and parapinospin (Hadjitheodorou et al, 2021). Cells were cultured and differentiated into a neutrophil-like state as previously described (Millius and Weiner, 2009).…”

Cell size and actin architecture determine force generation in optogenetically activated adherent cells

“…In particular, non-neuronal optogenetics for the Rho-system has been used to control single cell contractility, using either the CRY2/CIBN-construct (Valon, Etoc, Remorino, Di Pietro, et al 2015; Valon et al 2017) or the LOV2-construct (Wagner and Glotzer 2016; Oakes et al 2017). Optogenetic activation of Rho has also been used to reveal mechanical adaptation responses in epithelial cell junctions (Staddon et al 2019; Cavanaugh et al 2020), the feedback loops that structure the Rho-responses in cells (Kamps et al 2020) and even cell migration (Hadjitheodorou et al 2021).…”