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

Amalia Hadjitheodorou,Sean R Collins,Shashank Shastry,Julie A Theriot,Daniel Irimia,George R R Bell,Felix Ellett

doi:10.1038/s41467-021-26622-z

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.

Highlights

To migrate efficiently to target locations, cells must integrate receptor inputs while maintaining polarity: a distinct front that leads and a rear that follows
We found that persistent optogenetic receptor activation at the rear of neutrophils migrating in 1D microfluidic channels is sufficient to reorient weakly polarized and slowly migrating cells
We show that by applying pharmacological treatments known to alter the phosphorylation of myosin regulatory light chain (MRLC), we can modulate the activity and localization of myosin II and the amenability of the cell rear and the ability of cells to reverse their direction of motion

Summary

Introduction

To migrate efficiently to target locations, cells must integrate receptor inputs while maintaining polarity: a distinct front that leads and a rear that follows. To examine the role of polarity in chemoattractant sensing, researchers have historically challenged neutrophils migrating on two-dimensional (2D) planar substrates using point sources of chemoattractant at different angles with respect to the original direction of migration[3,9,10,11] In these experiments, chemoattractant is typically delivered using a micropipette positioned near the cell, resulting in diffusion of the attractant over the entire cellular surface. In more strongly polarized cells, myosin II and RhoA activity limit the ability of the rear to respond, even at the level of signal transmission to Cdc[42], creating a cell rear that is refractory to new receptor inputs. We show that by applying pharmacological treatments known to alter the phosphorylation of myosin regulatory light chain (MRLC), we can modulate the activity and localization of myosin II and the amenability of the cell rear and the ability of cells to reverse their direction of motion

Methods

Results

Conclusion