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Maintaining persistent migration in complex environments is critical for neutrophils to reach infection sites. Neutrophils avoid getting trapped, even when obstacles split their front into multiple leading edges. How they re-establish polarity to move productively while incorporating receptor inputs under such conditions remains unclear. Here, we challenge chemotaxing HL60 neutrophil-like cells with symmetric bifurcating microfluidic channels to probe cell-intrinsic processes during the resolution of competing fronts. Using supervised statistical learning, we demonstrate that cells commit to one leading edge late in the process, rather than amplifying structural asymmetries or early fluctuations. Using optogenetic tools, we show that receptor inputs only bias the decision similarly late, once mechanical stretching begins to weaken each front. Finally, a retracting edge commits to retraction, with ROCK limiting sensitivity to receptor inputs until the retraction completes. Collectively, our results suggest that cell edges locally adopt highly stable protrusion/retraction programs that are modulated by mechanical feedback., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

To control their movement, cells need to coordinate actin assembly with the geometric features of their substrate. Here, we uncover a role for the actin regulator WASP in the 3D migration of neutrophils. We show that WASP responds to substrate topology by enriching to sites of inward, substrate-induced membrane deformation. Superresolution imaging reveals that WASP preferentially enriches to the necks of these substrate-induced invaginations, a distribution that could support substrate pinching. WASP facilitates recruitment of the Arp2/3 complex to these sites, stimulating local actin assembly that couples substrate features with the cytoskeleton. Surprisingly, WASP only enriches to membrane deformations in the front half of the cell, within a permissive zone set by WASP's front-biased regulator Cdc42. While WASP KO cells exhibit relatively normal migration on flat substrates, they are defective at topology-directed migration. Our data suggest that WASP integrates substrate topology with cell polarity by selectively polymerizing actin around substrate-induced membrane deformations in the front half of the cell., (© 2021 Brunetti et al.)

The physicist Edward Purcell wrote in 1977 about mechanisms that cells could use to propel themselves in a low Reynolds number environment. Reporting in Developmental Cell, O'Neill et al. (2018) provide direct evidence for one of these mechanisms by optogenetically driving the migration of cells suspended in liquid through RhoA activation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Neutrophils are key early responders in the innate immune response that use chemotaxis, the directed migration along chemical gradients, to reach sites of infection or inflammation. This process requires integrating inputs from cell surface receptors with the cell's polarity and motility signaling network, in which highly dynamic and interconnected signaling by Rho-family GTPases plays a central role. To understand this fundamentally important behavior, we describe a high-resolution, under-agarose chemotaxis assay for use with neutrophil-like cell lines (HL-60 or PLB-985) or with primary neutrophils. We also describe how to use optical uncaging of chemoattractants to stimulate cells in this assay. These techniques are compatible with epifluorescence, total internal reflection fluorescence (TIRF), and confocal microscopy. Additionally, we cover how to measure the activities of Rho-family GTPases in this context using Förster resonance energy transfer (FRET)-based biosensors. The specific experimental steps outlined in this chapter include how to (1) set up the under-agarose assay, (2) optically pattern chemoattractant gradients, (3) image cells, and (4) conduct basic image analysis for FRET biosensors.