An ezrin-rich, rigid uropod-like structure directs movement of amoeboid blebbing cells

Abstract

SummaryMelanoma cells can switch between an elongated mesenchymal-type and a rounded amoeboid-type migration mode. The rounded 'amoeboid' form of cell movement is driven by actomyosin contractility resulting in membrane blebbing. Unlike elongated A375 melanoma cells, rounded A375 cells do not display any obvious morphological front-back polarisation, although polarisation is thought to be a prerequisite for cell movement. We show that blebbing A375 cells are polarised, with ezrin (a linker between the plasma membrane and actin cytoskeleton), F-actin, myosin light chain, plasma membrane, phosphatidylinositol (4,5)-bisphosphate and b1-integrin accumulating at the cell rear in a uropod-like structure. This structure does not have the typical protruding shape of classical leukocyte uropods, but, as for those structures, it is regulated by protein kinase C. We show that the ezrin-rich uropod-like structure (ERULS) is an inherent feature of polarised A375 cells and not a consequence of cell migration, and is necessary for cell invasion. Furthermore, we demonstrate that membrane blebbing is reduced at this site, leading to a model in which the rigid ezrin-containing structure determines the direction of a moving cell through localised inhibition of membrane blebbing. Journal of Cell ScienceIn confined spaces, blebbing cells can move forward without adhesion by a process termed 'chimneying' (Hawkins et al., 2009;Malawista and de Boisfleury Chevance, 1997). In fibrillar threedimensional matrices, such as collagen gels, blebs can expand into pores in the matrix, causing the squeezing phenotype observed in amoeboid lymphocytes and cancer cells in collagen gels (Haston and Shields, 1984;Wolf et al., 2003a). Cells that form small blebs can use them to 'elbow' their way through the meshwork of the ECM (Friedl and Wolf, 2009). The site of bleb formation determines the direction of cell movement (Keller and Bebie, 1996). However, some cells, such as A375 melanoma cells, form multiple blebs over the membrane and would therefore not be able to move directionally. However, A375 melanoma cells are able to invade into a collagen matrix and move in vitro and in vivo (Sahai and Marshall, 2003;Sanz-Moreno et al., 2008). As asymmetry is a prerequisite of cell movement (Petrie et al., 2009), this suggests some form of cellular polarisation. To understand cell movement driven by multiple blebs, we investigated polarisation in blebbing 'amoeboid' A375 melanoma cells. We show that A375 cells form an ezrin-rich uropod-like structure (which we call the ERULS) at their cell rear, and that this is required for invasion into a three-dimensional matrix. We present a model whereby accumulation of ezrin, membrane and actin in this structure reflects strong connections between the plasma membrane and the submembrane cortex, leading to the formation of a rigid structure that inhibits blebbing at the back of the cell. This leads to net movement of the cell in the direction opposite to the uropod-like structure. Our model explains how cells can move ...

Apoptotic membrane dynamics in health and disease

“…66 Alteration of ezrin expression in A375 melanoma cells by knockdown or overexpression to change the strength of plasma membrane coupling to the submembrane cortex resulted in corresponding increased or decreased blebbing, respectively. 67 This supports the conclusion that bleb protrusion results from changes in the strength of membrane-cortex coupling.…”

How blebs and pseudopods cooperate during chemotaxis

“…Such factors could include the density of membrane-cortical linkers and the local concentration of PIP2 in the membrane, as this can modulate the attachment of these linkers (18,27). Consistent with this, in melanoma cells, it has been argued that a high level of the membranecytoskeleton linker ezrin inhibits blebbing from the uropod (28).…”

At the leading edge of three-dimensional cell migration

“…The term amoeboid might in fact encompass at least three distinct modes of motility in rounded cancer cells (Fig. 2C) (Lorentzen et al, 2011). The adenocarcinoma cell line MTLn3 is morphologically round in 3D collagen, yet it has a leading edge that is enriched in F-actin (Wyckoff et al, 2006), similar to border cells migrating in Drosophila melanogaster egg chambers (Wang et al, 2010).…”

“…Finally, several cancer cell lines move as round cells through 3D Matrigel and collagen by rapidly protruding and retracting multiple small blebs. These blebs can occur anywhere on the plasma membrane except at the rear of the cell, where ezrin links the plasma membrane to the actin cytoskeleton to inhibit blebbing by mediating the formation of a rigid uropod, thereby facilitating directional migration (Lorentzen et al, 2011;Poincloux et al, 2011). It will be important to distinguish between these different sub-types of amoeboid cancer cell migration in the future.…”

“…These cells generally require integrin-mediated adhesion to migrate and can deform the surrounding matrix as they move (Lorentzen et al, 2011;Wyckoff et al, 2006). However, their diffuse pattern of integrin localization in the plasma membrane might reflect weaker cell-matrix interactions Box 1.…”