Cell Movement Coordinates Force Generation with Cell Adhesion

A moving keratinocyte (skin cell) and a moving fibroblast (connective tissue cell) display the same sequence of changes in cell morphology—initial extension of a membrane protru

► FIGURE 19-27 Forces produced by assembly of the actin network. (a) As shown in this diagram, actin filaments are assembled into a branched network in which the ends of filaments approach the plasma membrane at an acute angle. ATP-G-actin (red) adds to the filament end and pushes the membrane forward ( 1 ). The Arp2/3 complex (blue) binds to sides of filaments ( 2) and forms a branch at a 70° angle from the filament. With time, filaments ends are capped by capping protein (yellow) ( 3); the ATP-G-actin subunits convert into ADP-G-actin subunits (white) ( 4|) and dissociate from the filament through the action of the severing proteins cofilin and gelsolin (gray) ( 5). The released ADP-G-actin subunits form complexes with profilin (green) ( 6) to regenerate ATP-G-actin subunits. (b) The network of actin filaments supports the elongation of filaments and the generation of pushing forces. An actin filament is stiff but can bend from thermal fluctuations. In the elastic Brownian ratchet model, bending of filaments at the leading edge ( 1 ), where the (+) ends contact the membrane, creates space at the membrane for subunits to bind to the ends of filaments ( 2). The elastic recoil force of the filaments then pushes the membrane forward. [Part (a) adapted from T M. Svitkina and G. G. Borisy, 1999, J. Cell Biol. 145:1009.]

sion, attachment to the substratum, forward flow of cytosol, and retraction of the rear of the cell (Figure 19-26).

Membrane Extension The network of actin filaments at the leading edge (see Figure 19-12) is a type of a cellular engine that pushes the membrane forward by an actin polymerization-based mechanism (Figure 19-27a). The key

Plasma membrane

ATP-G-actin

Profilii

ADP-G-actin ilfji

Filamin membrane

Filamin

ATP-G-actin

Profilii

ADP-G-actin

Capping protein

Cofilin, gelsolin

Capping protein

Cofilin, gelsolin

membrane

f step in generating the force is (step 1) the addition of actin subunits at the ends of filaments close to the membrane. New filament ends are created (step 2) by branches formed by the Arp 2/3 complex. The branched network of filaments are stabilized by cross-linking proteins such as filamin. As the filaments grow, the ATP-actin subunits are converted into ADP-actin subunts. Consequently, (step 3) capping protein caps the (+) ends of filaments, and (step 4) cofilin and gel-solin fragment actin filaments and (step 5) cause actin sub-units to dissociate. Profilin converts the ADP-actin monomers into a polymerization-competent ATP-actin monomer ready to participate in the next cycle.

A mechanism to explain what propels the membrane forward, called the elastic Brownian ratchet model, is based on the elastic mechanical property of an actin filament (Figure 19-27b). Electron micrographs show that the ends of actin filaments abut against the membrane, leaving no space for an actin subunit to bind. However, thermal energy causes a filament to bend, creating room for subunit addition. Because actin filaments have the same stiffness as that of a plastic rod, the energy stored in bending straightens the filament. The concerted action of numerous filaments undergoing similar movements and their cross-linkage into a mechanically strong network generate sufficient force (several piconew-tons) to push the membrane forward.

Cell-Substrate Adhesions When the membrane has been extended and the cytoskeleton has been assembled, the membrane becomes firmly attached to the substratum. Time-lapse microscopy shows that actin bundles in the leading edge be-

come anchored to the attachment site, which quickly develops into a focal adhesion. The attachment serves two purposes: it prevents the leading lamella from retracting and it attaches the cell to the substratum, allowing the cell to push forward.

Cell Body Translocation After the forward attachments have been made, the bulk contents of the cell body are translocated forward (see Figure 19-26). How this translocation is accomplished is unknown; one speculation is that the nucleus and the other organelles are embedded in the cy-toskeleton and that myosin-dependent cortical contraction moves the cytoplasm forward. The involvement of myosin-dependent cortical contraction in cell migration is supported by the localization of myosin II. Associated with the movement is a transverse band of myosin II and actin filaments at the boundary between the lamellipodia and the cell body (Figure 19-28).

Breaking Cell Attachments Finally, in the last step of movement (de-adhesion), the focal adhesions at the rear of the cell are broken and the freed tail is brought forward. In the light microscope, the tail is seen to "snap" loose from its connections—perhaps by the contraction of stress fibers in the tail or by elastic tension—but it leaves a little bit of its membrane behind, still firmly attached to the substratum.

The ability of a cell to move corresponds to a balance between the mechanical forces generated by the cytoskeleton and the resisting forces generated by cell adhesions. Cells cannot move if they are either too strongly attached or not

exerts traction forces on the substratum. A keratinocyte plated on a thin silicon membrane exerts lateral forces from contraction of the cell body and causes the membrane to buckle. [Part (a) from T M. Svitkina and G. G. Borisy, 1999, J. Cell Biol. 145:1009; courtesy of T M. Svitkina. Part (b) from K. Burton et al., 1999, Mol. Biol. Cell 10:3745; courtesy of D. L. Taylor.]

▲ EXPERIMENTAL FIGURE 19-28 Contractile forces are generated by a moving cell. (a) A fluorescence micrograph of a keratlnocyte shows that the network of actin filaments (blue) is located at the front of the cell, whereas myosin II (red) is at the rear of the cell. However, both are located in a band (white) that traverses the cell just anterior to the nucleus. Contraction of this band is postulated to pull the cell body forward. (b) A moving cell exerts traction forces on the substratum. A keratinocyte plated on a thin silicon membrane exerts lateral forces from contraction of the cell body and causes the membrane to buckle. [Part (a) from T M. Svitkina and G. G. Borisy, 1999, J. Cell Biol. 145:1009; courtesy of T M. Svitkina. Part (b) from K. Burton et al., 1999, Mol. Biol. Cell 10:3745; courtesy of D. L. Taylor.]

attached to a surface. This relation can be demonstrated by measuring the rate of movement in cells that express varying levels of integrins, the cell-adhesion molecules that mediate most cell-matrix interactions (Chapter 6). Such measurements show that the fastest migration occurs at an intermediate level of adhesion, with the rate of movement falling off at high and low levels of adhesion. Cell locomotion thus results from traction forces exerted by the cell on the underlying substratum. The traction forces can be detected by the effects of cells on extremely thin sheets of silicon (Figure 19-28b). As a cell moves forward, contractile forces exerted at the front and the back of the cell cause the membrane to buckle. On a stiffer membrane that resists deformation, the buckling forces will be transformed into the forward movement of the cell.

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