Cross-linking of actin filaments by myosin II is a major contributor to cortical integrity and cell motility in restrictive environments

Abstract

Cells are frequently required to move in a local environment that physically restricts locomotion, such as during extravasation or metastatic invasion. In order to model these events, we have developed an assay in which vegetative Dictyostelium amoebae undergo chemotaxis under a layer of agarose toward a source of folic acid [Laevsky, G. and Knecht, D. A. (2001). Biotechniques 31, 1140-1149]. As the concentration of agarose is increased from 0.5% to 3% the cells are increasingly inhibited in their ability to move under the agarose. The contribution of myosin II and actin cross-linking proteins to the movement of cells in this restrictive environment has now been examined. Cells lacking myosin II heavy chain (mhcA -) are unable to migrate under agarose overlays of greater than 0.5%, and even at this concentration they move only a short distance from the trough. While attempting to move, the cells become stretched and fragmented due to their inability to retract their uropods. At higher agarose concentrations, the mhcA -cells protrude pseudopods under the agarose, but are unable to pull the cell body underneath. Consistent with a role for myosin II in general cortical stability, GFP-myosin dynamically localizes to the lateral and posterior cortex of cells moving under agarose. Cells lacking the essential light chain of myosin II (mlcE -), have no measurable myosin II motor activity, yet were able to move normally under all agarose concentrations. Mutants lacking either ABP-120 or α-actinin were also able to move under agarose at rates similar to wild-type cells. We hypothesize that myosin stabilizes the actin cortex through its cross-linking activity rather than its motor function and this activity is necessary and sufficient for the maintenance of cortical integrity of cells undergoing movement in a restrictive environment. The actin cross-linkers α-actinin and ABP-120 do not appear to play as major a role as myosin II in providing this cortical integrity. Research Article 3762 measurable actin activated ATPase activity (Chen et al., 1995;Xu et al., 1996). While not generally thought of as an actin cross-linking protein, myosin II minifilaments presumably also have this capability (Wachsstock et al., 1994;Humphrey et al., 2002). Mutants lacking myosin II (mhcA -) are able to accomplish both random and chemotactic motility; however, they move slowly and have defects in pseudopod extension (Peters et al., 1988;Wessels et al., 1988). Although mhcA -cells are able to aggregate, they are unable to complete the developmental program .The developmental defect of mhcA -cells appears to be due to their inability to move in a restrictive environment . During early development, cells acquire surface adhesion proteins and so movement occurs while cells are continually making and breaking adhesive contacts with their neighbors as well as the substratum. Unlike movement on a planar substratum, this form of motility is analogous to the movement of metastatic cancer cells away from a primary tumor, or the extravasation of immune...

Spatio-temporal analysis of eukaryotic cell motility by improved force cytometry

“…This result suggests that an important contribution to the traction forces is made by actin polymerization (2) with possible contributions from myosin I (29). The importance of MyoII for motility on a flat surface may stem from its function in cytoskeletal organization and cortical integrity (9), rather than from the traction forces produced by its motor activity. A notable fraction of the myoII Ϫ cells in our experiments never become organized, remaining round and static.…”

“…It has been suggested that MyoII contractility facilitates rear retraction. Supporting this idea are the inability of myoII Ϫ cells to move on highly adhesive substrates (7), and their failure to move in the constrained space under a layer of agarose or in a 3D aggregate of cells (8,9). Despite significant progress in the understanding of the biochemistry of the cytoskeleton, the analysis of the spatiotemporal events that enable cell movement is in its infancy.…”

Revealing Early Steps of α2β1Integrin-mediated Adhesion to Collagen Type I by Using Single-Cell Force Spectroscopy

“…Although unbound integrins are freely diffusive in the membrane plane, ligand binding promotes rapid attachment of integrins to actin filaments (Felsenfeld et al, 1996). Myosin II is an effective F-actin cross-linker (Laevsky and Knecht, 2003), and myosin-driven bundling and alignment of actin filaments carrying ligand-bound integrin complexes could then lead to the clustering of integrin-cytoskeleton complexes (Chrzanowska-Wodnicka and Burridge, 1996). Alternatively, myosin-dependent forces acting on integrin complexes could induce conformational changes in associated mechanosensitive proteins.…”

Talin couples the actomyosin cortex to the plasma membrane during rear retraction and cytokinesis

“…5B). Laevsky and Knecht (20) have also reported that myosin II-null cells drag a long tail containing GFP-ABD, which is especially prominent under an agarose sheet but also present in liquid media. In certain mammalian cell lines, depletion of myosin II results in the formation of similar F-actin-containing long tails (37).…”

“…By analogy to the cytokinesis of myosin II-null cells, in which the division process is driven by opposite movement of the daughter cells (2), the above results suggest that the active polar elongation by the pseudopodia at both polar regions substantially contributes to the separation of the daughter cells in talin Anull cells. When the polar elongation was inhibited by pressing the cells under a sheet of 3% (wt/vol) agarose (20), wild-type cells still completed furrow ingression (Fig. 3B, Upper), but talin A-null cells failed to constrict the furrow.…”