Comparative Maps of Motion and Assembly of Filamentous Actin and Myosin II in Migrating Cells

Abstract

To understand the mechanism of cell migration, one needs to know how the parts of the motile machinery of the cell are assembled and how they move with respect to each other. Actin and myosin II are thought to be the major structural and force-generating components of this machinery (Mitchison and Cramer, 1996; Parent, 2004). The movement of myosin II along actin filaments is thought to generate contractile force contributing to cell translocation, but the relative motion of the two proteins has not been investigated. We use fluorescence speckle and conventional fluorescence microscopy, image analysis, and computer tracking techniques to generate comparative velocity and assembly maps of actin and myosin II over the entire cell in a simple model system of persistently migrating fish epidermal keratocytes. The results demonstrate contrasting polarized assembly patterns of the two components, indicate force generation at the lamellipodium-cell body transition zone, and suggest a mechanism of anisotropic network contraction via sliding of myosin II assemblies along divergent actin filaments. INTRODUCTIONCrawling cell motion involves a cycle of several distinct processes: protrusion at the front of the cell, attachment to the substratum, and forward translocation of the cell body followed or accompanied by detachment and withdrawal of the rear of the cell. Crawling motion is thought to be dependent on the actin-myosin II cytoskeletal system (Mitchison and Cramer, 1996): protrusion is thought to be driven by the assembly of actin network, which is anchored to the substratum through integrin-containing adhesions; forward translocation of the cell body and contraction of the rear are thought to depend on the interaction of the actin network with the motor protein myosin II. Actin assembly during the protrusion at the leading edge of motile cells has recently received the most attention both in experimental (Pantaloni et al., 2001;Pollard and Borisy, 2003;Ridley et al., 2003) and theoretical studies (for review, see Mogilner, 2006). In contrast, the mechanisms involved in the forward translocation of the cell body remain largely unclear: the exact layout and the mode of action of the contractile actin-myosin II machinery are controversial. Qualitative models proposed in the literature include shortening of small contractile units similar to muscle sarcomeres, myosin II-dependent transport along uniformly polarized actin arrays, and a dynamic network contraction mechanism where contraction results from alignment of actin filaments by myosin II assemblies (Cramer, 1999;Verkhovsky et al., 1999a). More sophisticated biophysical models aim to understand the contractile cytoskeletal machinery in quantitative physical terms. In the past, considering the cytoskeleton as a gel made of cross-linked semiflexible polymers has helped understanding its passive visco-elastic properties. More recently, attempts were made to develop biophysical models taking into account intrinsic activity of the cytoskeleton: polarized assembly of acti...

Frustrated Phagocytic Spreading of J774A-1 Macrophages Ends in Myosin II-Dependent Contraction

“…Interestingly, actin bundles similar to those that form during FP can be found spanning the midline of migrating keratocytes behind the Arp2/3-rich lamellipodium (38). In this context, the formation of the midline bundle has been largely attributed to myosin activity (38,(45)(46)(47). According to the dynamic network contraction model, the forces that are generated by myosin II in the lamellipodium are unable to displace and rearrange filaments due to the dense packing and strong filament-filament interconnections (45).…”

“…However, at the rear, where the network is sparse, force exerted across intersecting filaments results in a torque that rotates the filaments into an antiparallel alignment (see Fig. 7 and Schaub et al (46) for illustrations). With the filaments aligned, continued myosin activity causes the network to contract laterally.…”

On a poroviscoelastic model for cell crawling

“…This band of actin is comparable in density to that at the leading edge and exceeds that in the lamellum and the cell body (Svitkina et al, 1997). A forwards flow of the cytosol, the fluid that bathes the actin network, has also been detected in crawling cells (Schaub et al, 2007). Whilst this picture of a steady, translating configuration is broadly true, it neglects some interesting dynamical features of actin networks.…”

“…Fluorescent speckle microscopy has provided evidence for strong polymerization of the network at the leading edge and transport of the actin network backwards away from the leading edge, termed retrograde flow (Schaub et al, 2007;Vallotton et al, 2004Vallotton et al, , 2005. Arc shaped bundles of actin align predominantly parallel to the cell's leading edge where the protrusion joins the main body of the cell (Svitkina et al, 1997).…”

Band-like Stress Fiber Propagation in a Continuum and Implications for Myosin Contractile Stresses

“…For instance, images of cytoplasmic flow in keratocytes show cytoskeletal actin being compressed into the stress fiber. 69,73 Hirata et al 31 showed that quantum-dot labeled actin filaments in cell lamella follow a definite centripetal trajectory into stress fibers. The directed nature of the trajectory cannot be explained by random diffusion, but only by some coarse-grained material continuity.…”