HL-60 neutrophils heat-treated for 10 minutes at 45°C form a tethered morphology, which reduces diffusion between the leading edge and the cell body. Image courtesy of Dr Andrew Houk, University of California San Francisco, California, USA.

In a wide range of migrating cells, including neutrophils, Rac activity at the leading edge drives actin polymerization and protrusion. It is widely assumed that the leading edge prevents further fronts from forming by producing long-range diffusible inhibitory molecules or by sequestering limiting polarity components, but a report in Cell outlines an alternative method of long-range inhibition — mechanical tension.

For hypothetical diffusion-based systems to generate cell polarity, the components must diffuse or be sequestered more rapidly than the products of the positive feedback loops that generate the initial leading edge. Theoretically, then, altering cell morphology might perturb diffusion and thereby affect polarity. But when Houk et al. induced neutrophils to adopt a stretched morphology, they still observed a single zone of actin assembly tethered to the cell body.

When the researchers simulated the response of three published diffusion-based inhibition models to stretching, the cell body was predicted to remain inactive. It was similarly predicted to remain inactive after being severed from the pseudopod, provided that any inhibitory molecules were long lasting or that limiting components were not resynthesized. By contrast, according to a tension-based inhibition mechanism, in which mechanical tension generated at the leading edge by protrusion functions as a long-range inhibitor, severing would be expected to reanimate the cell body by reducing tension. Indeed, the cell body underwent rapid reanimation after laser-based severing, indicating that the pseudopod sequesters rapidly generated limiting components, generates short-lived diffusible inhibitory molecules or generates mechanical tension to inhibit the cell body.

Next, Houk et al. showed that the tethered morphology significantly reduced diffusion-based exchange between the pseudopod and the cell body, and calculated that the diffusion rate required to prevent cell body activity would be much higher than is attainable by cytoplasmic proteins, even if the diffusible component was resynthesized. A more rapid mode of communication is therefore required.

Focusing on mechanical tension as a hypothetical mode of long-range inhibition, Houk et al. confirmed that plasma membrane tension did increase in conjunction with cell protrusion in response to chemoattractant. They then tested the ability of an artificial increase in cell tension to prevent protrusion throughout the cell by applying micropipette aspiration. Deformation of the cell as a result of suction not only prevented protrusion but also inhibited recruitment of the SCAR/WAVE complex and Rac activation throughout the cell. So, increased tension can confer long-range inhibition of protrusion and leading-edge signals in migrating neutrophils, but is it required to restrict signals to the leading edge? And where does it emanate from — the plasma membrane or the cytoskeleton, or both?

To address these questions, the authors reduced membrane tension using hypertonic buffers or cytoskeletal tension using the myosin inhibitor blebbistatin, and analyzed leading-edge signalling. Although blebbistatin caused cells to elongate, leading-edge signals did not spread. By contrast, treatment with hypertonic buffer induced the spatially uniform distribution of SCAR/WAVE as well as causing the neutrophils to spread uniformly and display several pseudopodia.

These results indicate that pseudopod protrusion increases plasma membrane tension, which functions as a long-range inhibitor of leading-edge formation. This seems to constitute the dominant inhibitory mechanism for cell polarization in neutrophils, and might be physiologically important during leukocyte migration when cells have to crawl through narrow openings which would impede diffusion-based inhibition. In other cells, or in different circumstances, however, it is possible that tension might collaborate with diffusion-based inhibitors for polarity, or might be transmitted by the cytoskeleton. Elucidating how cells sense and respond to tension will be the next challenge.

Author: Katrin Legg - Copyright © 2012 Cell Migration Consortium