Switching between blebbing and lamellipodia depends on the degree of non-muscle myosin II activity

Abstract

Cells can adopt both mesenchymal and amoeboid modes of migration through membrane protrusive activities, namely formation of lamellipodia and blebbing. How the molecular players control the transition between lamellipodia and blebs is yet to be explored. Here, we show that addition of the ROCK inhibitor Y27632 or low doses of blebbistatin, an inhibitor of non-muscle myosin II (NMII) ATPase activity and filament partitioning, induces blebbing to lamellipodia conversion (BLC), whereas addition of low doses of ML7, an inhibitor of myosin light chain kinase (MLCK), induces lamellipodia to blebbing conversion (LBC) in human MDA-MB-231 cells. Similarly, siRNA-mediated knockdown of ROCK and MLCK induces BLC and LBC, respectively. Interestingly, both blebs and lamellipodia membrane protrusions are able to maintain the ratio of phosphorylated to unphosphorylated regulatory light chain at cortices when MLCK and ROCK, respectively, are inhibited either pharmacologically or genetically, suggesting that MLCK and ROCK activities are interlinked in BLC and LBC. Such BLCs and LBCs are also inducible in other cell lines, including MCF7 and MCF10A. These studies reveal that the relative activity of ROCK and MLCK, which controls both the ATPase activity and filament-forming property of NMII, is a determining factor in whether a cell exhibits blebbing or lamellipodia.

Dynamics of Endothelial Engagement and Filopodia Formation in Complex 3D Microscaffolds

“…On the contrary, ROCK inhibition by Y-27632 (10 µM) inhibited bleb formation ( Figure 7 d, see Movies S6 and S7 ), in agreement with [ 47 ] and as already observed for endothelial cells [ 50 ]. All membrane blebs present before drug addition shifted to an adherent state after Y-27632 treatment (from 5 to 0 blebbing cell islets in three movies).…”

“…Mesenchymal-bleb reversible transitions in function of ROCK/MLCK balance In order to understand the molecular players involved in the transition between blebs and adherent states, we targeted ROCK and MLCK pathways. Indeed, in the literature the transition between lamellipodia and membrane blebs was expected to be governed by the degree of non-muscle myosin II activity, with ROCK favouring a blebbing phenotype and MLCK a mesenchymal adherent phenotype [47]. Different contractility levels (higher with ROCK than with MLCK) may be at play [47], as well as a difference in spatial distribution, ROCK activity being higher in the centre of cells and MLCK in their periphery [48].…”

The tumor suppressor adenomatous polyposis coli regulates T lymphocyte migration

“…Moreover, ERMs, through their Thr phosphorylation, control the interactions between plasma membrane and the cortical actin cytoskeleton, thus regulating cortical rigidity and bleb generation ( 26 ). In addition, ERMs control Rho signaling and may therefore regulate local myosin-mediated contractility and keep the balance between lamellipodial versus bleb-type protrusions ( 27 , 28 ). Hence, we evaluated cell deformability under centrifugal force and ERM phosphorylation ( 29 , 30 ).…”

Is There a Need for a More Precise Description of Biomolecule Interactions to Understand Cell Function?

“…Cell communication with the outer world relies on the adhesion to other cells or surfaces, and recognition of soluble or surface-bound mediators with hundreds of membrane receptors [61]. Generation and propagation of intracellular signaling events relies on the formation and dissociation of multimolecular complexes [62,63]. Molecular transport throughout cells is strongly influenced by interdependent crowding and binding effects.…”