We have now examined the different mechanisms used by cells to create movement—from the assembly of actin filaments and the formation of actin filament bundles and networks to the contraction of bundles of actin and myosin and the transport of organelles by myosin molecules along actin filaments. Some of these same mechanisms constitute the major processes whereby cells generate the forces needed to migrate. Cell migration results from the coordination of motions generated in different parts of a cell, integrated with a directed endocytic cycle.
The study of cell migration is important to many fields of biology and medicine. For example, an essential feature of animal development is the migration of specific cells along predetermined paths. Epithelial cells in an adult animal migrate to heal a wound, and white blood cells migrate to sites of infection. Less obvious are the continuous slow migration of intestinal epithelial cells along the villi in the intestine and of endothelial cells that line the blood vessels. The inappropriate migration of cancer cells after breaking away from their normal tissue results in metastasis.
Cell migration is initiated by the formation of a large, broad membrane protrusion at the leading edge of a cell. Video microscopy reveals that a major feature of this movement is the polymerization of actin at the membrane. Actin filaments at the leading edge are rapidly cross-linked into bundles and networks in a protruding region, called a lamellipodium in vertebrate cells. In some cases, slender, fingerlike membrane projections, called filopodia, also extend from the leading edge. These structures form stable contacts with the underlying surface (such as the extracellular matrix) that the cell moves across. In this section, we take a closer look at how cells coordinate various microfilament-based processes with endocytosis to move across a surface. We also consider the role of signaling pathways in coordinating and integrating the actions of the cytoskeleton, a major focus of current research.
Cell Migration Coordinates Force Generation with Cell Adhesion and Membrane Recycling
A moving fibroblast (connective tissue cell) displays a characteristic sequence of events: initial extension of a membrane protrusion, attachment to the substratum, forward flow of cytosol, and retraction of the rear of the cell (Figure 1). These events occur in an ordered pattern in a slowly moving cell such as a fibroblast, but in rapidly moving cells, such as macrophages, all of them are occurring simultaneously in a coordinated manner. We first consider the role of the actin cytoskeleton in cell movement, involving assembly at the leading edge as well as attachment to the substratum via stress fibers (Figure 2a, b), and then discuss the role of the endocytic cycle.
Fig1. Steps in cell locomotion. Movement begins with the extension of one or more lamellipodia from the leading edge of the cell 1; some lamellipodia adhere to the substratum by focal adhesions 2. Then the bulk of the cytoplasm in the cell body flows forward due to contraction at the rear of the cell 3. The trailing edge of the cell remains attached to the substratum until the tail eventually detaches and retracts into the cell body. During this cytoskeleton-based cycle, the endocytic cycle internalizes membrane and integrins at the rear of the cell and transports them to the front of the cell (arrows) for reuse in making new adhesions 4.
Fig2. Actin-based structures involved in cell locomotion. (a) Localization of actin in a fibroblast expressing GFP-actin. (b) Diagram of the classes of microfilaments involved in cell migration. The network of actin filaments in the leading edge advances the cell forward. Contractile fibers in the cell cortex squeeze the cell body forward, and stress fibers terminating in focal adhesions also pull the bulk of the cell body up as the rear adhesions are released. (c) The structure of focal adhesions involves the attachment of the ends of stress fibers through integrins to the underlying extracellular matrix. Focal adhesions also contain many signaling molecules important for cell locomotion. (d) The dynamic actin network in the leading edge is nucleated by the Arp2/3 complex and employs the same set of factors that control assembly and disassembly of actin filaments in the Listeria tail (see Figure 17-17). [Part (a) courtesy of J. Vic Small.]
Membrane Extension The network of actin filaments at the leading edge is a type of cellular engine that pushes the mem brane forward in a manner very similar to the propulsion of Listeria by actin polymerization (Figure 2d; for Listeria). At the membrane of the leading edge, actin is nucleated by the activated Arp2/3 complex, and filaments are elongated by assembly onto (+) ends adjacent to the plasma membrane. Because the actin network is fixed with respect to the substratum, the front membrane is pushed out as the filaments elongate. This process is very similar to the movement of Listeria, which “rides” on the polymerizing actin tail, which is also fixed within the cytoplasm. Actin turnover, and thus treadmilling, is mediated, as it is in the comet tails of Listeria, by the action of profilin and cofilin (see Figure2d).
Cell-Substratum Adhesions When the membrane has been extended and the actin network has been assembled, the plasma membrane becomes firmly attached to the substratum. Time-lapse microscopy shows that actin bundles in the leading edge become anchored to structures known as focal adhesions (Figure 2c). The attachment serves two purposes: it prevents the lamellipodium from retracting, and it attaches the cell to the substratum, allowing the cell to move forward. Given the importance of focal adhesions and their regulation during cell locomotion, it is not surprising that they have been found to be very rich in molecules involved in signal transduction pathways. Focal adhesions are discussed in more detail in Chapter 20, where we discuss cell-matrix interactions.
The cell-adhesion molecules that mediate most cell matrix interactions are membrane proteins called integrins. These proteins have an external domain that binds to specific components of the extracellular matrix, such as fibronectin and collagen, and a cytoplasmic domain that links them to the actin cytoskeleton (see Figure 2c). The cell makes adhesions at the leading edge, and as the cell migrates forward, the adhesions eventually assume positions toward the rear.
Cell-Body Translocation After the forward adhesions have been made, the bulk contents of the cell body are translocated forward (see Figure 1, step 3). It is believed that the nucleus and the other organelles embedded in the cytoskeleton are moved forward by myosin II–dependent cortical contraction in the rear part of the cell, like toothpaste when the lower half of the tube is squeezed. Consistent with this model, myosin II is localized to the rear cell cortex.
Breaking Cell Attachments Finally, in the last step of movement (de-adhesion), the focal adhesions at the rear of the cell are broken, the integrins recycled, and the freed tail brought forward. In the light microscope, the tail is often seen to “snap” loose from its connections—perhaps by the contraction of stress fibers in the tail or by elastic tension—and it sometimes leaves a little bit of its membrane behind, still firmly attached to the substratum.
Cells cannot move if they are either too strongly attached or not attached to a surface. 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. This relationship can be demonstrated by measuring the rate of movement in cells that express varying levels of integrins. 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.
Recycling of Membrane and Integrins by Endocytosis The dynamic changes in the actin cytoskeleton alone are not sufficient to drive cell migration; it is also dependent on endocytic recycling of membranes. The membrane needed during lamellipodium extension is provided by internal endosomes following their exocytosis. Adhesion molecules in focal adhesions at the rear of the cell are internalized as those adhesions are disassembled and transported by an endocytic cycle to the front to make new substratum attachments (Figure 1, step 4). This cycling of adhesion molecules in a migrating cell resembles the way a tank uses its treads to move forward. The movement of membrane internally through the cell also generates a rearward membrane flow across the surface of the cell. Indeed, this type of flow may contribute to the mechanics of cell locomotion, as it has recently been found that white blood cells can move in a liquid (“swim”) in the absence of attachment to a substratum, presumably as surface structures operating like paddles move backward across the cell surface.
The Small GTP-Binding Proteins Cdc42, Rac, and Rho Control Actin Organization
A striking feature of a moving cell is its polarity: it has a front and a back. When a cell makes a turn, a new leading edge forms in the new direction of movement. If these extensions formed in all directions at once, the cell would be unable to pick a new direction of movement. To sustain movement in a particular direction, a cell requires signals to coordinate events at the front of the cell with events at the back and, indeed, signals to tell the cell where its front is. Our understanding of how this coordination occurs emerged from studies with growth factors.
Growth factors, such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), bind to specific cell-surface receptors (see Chapter 16) and stimulate cells to move and then to divide. For example, in a wound, blood platelets become activated by being exposed to collagen in the extracellular matrix at the wound edge, which helps the blood to clot. Activated platelets also secrete PDGF to attract fibroblasts and epithelial cells to enter the wound and repair it. It is possible to watch part of this process in vitro. If you grow cells in a culture dish and, after starving them of growth factors, you add some fresh growth factor, within a minute or two the cells respond by forming membrane ruffles. Membrane ruffles are very similar to the lamellipodia of migrating cells: they are a result of activation of the machinery that controls exocytosis of endosomes coupled with actin assembly.
Scientists knew that growth factors bind to very specific receptors on the cell surface and induce a signal transduction pathway on the inner surface of the plasma membrane, but how that process linked up to the actin machinery was mysterious. Research then revealed that the signal transduction pathway activates Rac, a member of the small GTPase superfamily of Ras-related proteins. Rac is one member of a family of proteins that regulate microfilament organization; two others are Cdc42 and Rho. Unfortunately, due to the history of their discovery, the family of proteins of which Cdc42, Rac, and Rho are members has also been collectively named “Rho proteins.” To understand how these proteins work, we first have to recall the way small GTP-binding proteins function.
Like all small GTPases of the Ras superfamily, Cdc42, Rac, and Rho act as molecular switches, inactive in the GDP-bound state and active in the GTP-bound state (Figure 3). In their GDP-bound state, they exist free in the cytoplasm in an inactive form bound to a protein known as guanine nucleotide dissociation inhibitor (GDI). Growth factors can bind and activate their receptors to turn on specific membrane-bound regulatory proteins, called guanine nucleotide exchange factors (GEFs), which activate Rho proteins at the membrane by releasing them from GDI and catalyzing the exchange of GDP for GTP. The GTP-bound active Rho protein associates with the plasma membrane, where it binds effector proteins to transmit the biological response. The small GTPase remains active until the GTP is hydrolyzed to GDP, a process that is stimulated by specific GTPase-activating proteins (GAPs).
Fig3. Regulation of the Rho family of small GTPases. The small GTPases of the Rho family are molecular switches regulated by accessory proteins. Rho proteins exist in the Rho-GDP bound form complexed with a protein known as GDI (guanine nucleotide dissociation inhibitor), which keeps them in an inactive state in the cytosol. Membrane-bound signaling pathways bring Rho proteins to the membrane and, through the action of a GEF (guanine nucleotide exchange factor), exchange the GDP for GTP, thus activating them. Membrane-bound activated Rho-GTP can then bind effector proteins that cause changes in the actin cytoskeleton. The Rho protein remains in the active Rho-GTP state until acted on by a GAP (GTPase-activating protein), which allows it to interact with the GDI and be returned to the cytoplasm. See S. Etienne-Manneville and A. Hall, 2002, Nature 420:629.
An important approach to unraveling the functions of the Rho proteins has been to introduce into cells mutant proteins that are locked either in the active—Rho-GTP—state or in the inactive—Rho-GDP—state. A mutant small GTPase that is locked in the active state, called a dominant-active protein, binds the effector molecules constitutively, and one can then assess the biological outcomes. Alternatively, one can intro duce a different mutant that is dominant negative, which binds and inhibits the relevant GEF protein. Thus introduction of a dominant-negative protein, even in the presence of the endogenous wild-type protein, interferes with the signal transduction pathway, so one can now assess what processes are blocked.
Cdc42, Rac, and Rho were implicated in the regulation of microfilament organization because introduction of dominant-active mutant proteins had dramatic effects on the actin cytoskeleton, even in the absence of growth factors. It was discovered that dominant-active Cdc42 results in the appearance of filopodia, dominant-active Rac results in the appearance of membrane ruffles, and dominant-active Rho results in the formation of stress fibers, which then contract (Figure 4). How can one tell if dominant-active Rac and growth factor stimulation, both of which stimulate membrane ruffle formation, operate in the same signal trans duction pathway? If growth factor stimulation leads to Rac activation, then introduction of a dominant-negative Rac protein into a cell should block the ability of a growth fac tor to induce membrane ruffling. This is precisely what was found. Using this and many other biochemical strategies, scientists have identified the signaling pathways involving Cdc42, Rac, and Rho (Figure 5).
EXPERIMENTAL Fig4. Dominant-active Rac, Rho, and Cdc42 induce different actin-containing structures. To look at the effects of constitutively active Rac, Rho, and Cdc42, growth-factor-starved fibroblasts were microinjected with plasmids to express dominant-active versions of the three proteins. The cells were then treated with fluorescent phalloidin, which stains filamentous actin. Dominant-active Rac induces the formation of peripheral membrane ruffles, whereas dominant-active Rho induces abundant contractile stress fibers, and dominant-active Cdc42 induces filopodia. [Republished with permission of AAAS, from Hall, A., “Rho GTPases and the actin cytoskeleton,” Science, 1998, 279 (5350):509-14; per mission conveyed through Copyright Clearance Center, Inc.]
Fig5. Summary of signal-induced changes in the actin cytoskeleton. Specific signals, such as growth factors and lysophos phatidic acid (LPA), are detected by cell-surface receptors. Detection leads to the activation of the small GTP-binding proteins, which then interact with effectors to bring about cytoskeletal changes as indicated.
Some of the pathways that these proteins regulate contain proteins we are familiar with. Activation of Cdc42 stimulates actin assembly by Arp2/3 through activation of WASp, a nucleation promoting factor (NPF), resulting in the formation of filopodia. Activation of Rac also induces Arp2/3, mediated by the WAVE com plex, leading to the assembly of branched actin filaments in the leading edge. Activation of Rho has at least two effects. First, it can activate a formin for unbranched actin filament assembly. Second, through activation of Rho kinase, it can phosphorylate the myosin light chain to activate nonmuscle myosin II and can also inhibit light-chain dephosphorylation by phosphorylating myosin light-chain phosphatase to inhibit its activity. Both actions of Rho kinase lead to a higher level of myosin light-chain phosphorylation and therefore higher myosin activity and contraction. The three Rho proteins, Cdc42, Rac, and Rho, are also linked by activation and inhibition pathways, as shown in Figure5.
Cell Migration Involves the Coordinate Regulation of Cdc42, Rac, and Rho
How does each of these small GTP-binding proteins con tribute to the regulation of cell migration? To answer this question, researchers developed an in vitro wound-healing assay (Figure 6a). Cells in culture are grown in a petri dish with growth factors until they are confluent and form a tight monolayer, at which point they stop dividing. The cell monolayer is then scratched with a needle to remove a swath of cells, generating a “wound” containing a free edge of cells. The cells on the edge sense the loss of their neighbors and, in response to components of the extracellular matrix now exposed on the dish surface, move to fill up the empty wound area (Figure 6b). To do this, they orient themselves toward the empty area, first extending a lamelli podium and then moving in that direction. In this way, one can study the induction of directed cell migration in vitro.
EXPERIMENTAL FIGURE 6. The wounded-cell monolayer assay can be used to dissect signaling path ways in directed cell movement. (a) A confluent layer of cells is scratched to remove a swath about three cells wide to generate a free cell border. The remaining cells detect the free space and newly exposed extracellular matrix and, over a period of hours, fill in the area. (b) Localization of actin in a wounded monolayer 5 minutes and 3 hours after scratching; after 3 hours, cells have migrated into the wounded area. (c) Effect of introducing dominant-negative Cdc42, Rac, and Rho into cells at the wound edge; all affect wound closure. [Part (b) ©1999 Nobes & Hall et al., J. Cell Biol. 144:1235–1244. doi:10.1083/jcb.144.6.1235. Part (c) data from C. D. Nobes and A. Hall, 1999, J. Cell Biol. 144:1235.]
Using this wounded-cell monolayer assay, researchers have introduced dominant-negative Rac into cells on the wound edge to see how it affects the ability of the cells to mi grate and fill the wound. Since Rac is needed for activation of the Arp2/3 complex to form the lamellipodium, it is not surprising that the cells fail to form this structure and do not migrate, and so the wound does not close (Figure 6c). A very interesting result is obtained when dominant-negative Cdc42 is introduced into the cells at the wound edge: they can form a lamellipodium, but they do not orient in the correct direction—in fact, they try to migrate in random directions. This observation suggests that Cdc42 is critical for regulating the overall polarity of the cell. Studies from yeast (in which Cdc42 was first described), wounded-cell mono layers, epithelial cells, and neurons reveal that Cdc42 is a master regulator of polarity in many different systems. Part of this regulation in animals involves the binding of Cdc42 to its effector, Par6, a polarity protein that functions in nematodes (in which it was first discovered), neurons, and epithelial cells.
Studies such as these suggest a general model of how cell migration is controlled (Figure 7). Signals from the environment are transmitted to Cdc42, which orients the cell. The oriented cell has high Rac activity at the front, which induces the formation of the leading edge; Rho activity is high in the rear, inducing the assembly of contractile structures and activating the myosin-II-based contractile machinery. It is important to notice that different regions of the cell can have different levels of active Cdc42, Rac, or Rho, so these regulators are controlled locally within the cell. Part of this spatial regulation occurs because some small G proteins can work antagonistically. For example, active Rho can stimulate pathways that lead to the inactivation of Rac. This process might help ensure that no leading-edge structures form at the rear of the cell.
Fig7. Contributions of Cdc42, Rac, and Rho to cell movement. The overall polarity of a migrating cell is controlled by Cdc42, which is activated at the front of a cell. Cdc42 activation leads to active Rac in the front of the cell, which generates the leading edge, and active Rho at the back of the cell, which causes myosin II activation and contraction. Active Rho inhibits Rac activation, ensuring the asymmetry of the two active G-proteins.
Migrating Cells Are Steered by Chemotactic Molecules
Under certain conditions, extracellular chemical cues guide the locomotion of a cell in a particular direction. In some cases, the movement is guided by insoluble molecules in the underlying substratum, as in the wounded-cell monolayer assay described above. In other cases, the cell senses soluble molecules and follows them, along a concentration gradient, to their source—a process known as chemotaxis. For example, leukocytes are guided toward an infection by a tripeptide that is secreted by many bacterial cells (Figure 8). In another example, during the development of skeletal muscle, a secreted protein signal called scatter factor guides the migration of myoblasts to the proper locations in limb buds. One of the best-studied examples of chemotaxis is the migration of Dictyostelium amoebae during their starvation response. When these soil amoebae are stressed, they begin to secrete cAMP, which is an extracellular chemotactic agent in this organism. Other Dictyostelium cells move up the cAMP concentration gradient toward its source (see Figure 8). Thus the amoebae move toward one another, aggregate into a migratory slug, and then differentiate into a fruiting body in which starvation-resistant spores are formed.
Fig8. Chemotactic molecules guide migrating cells by signaling the actin cytoskeleton. Dictyostelium cells migrate toward a pipette of cAMP (left), and human neutrophils (a type of leukocyte) migrate toward a pipette of fMLP (formylated Met-Leu-Phe), a chemotactic peptide produced by bacteria (right). In the lower two panels are individual chemotaxing Dictyostelium and neutrophil cells, which look remarkably similar, despite about 800 million years of evolution separating them. [Republished with permission of Elsevier, from Parent, C. A., “Making all the right moves: chemotaxis in neutrophils and Dictyostelium,” Current Opinion in Cell Biology, 2004,16(1):4-13, 2004; permission conveyed through Copyright Clearance Center, Inc.]
Despite the variety of different chemotactic molecules— sugars, peptides, cell metabolites, cell-wall or membrane lipids—they all work through a common and familiar mechanism: binding to cell-surface receptors, activation of intracellular signaling pathways, and remodeling of the cytoskeleton through the activation or inhibition of various actin-binding proteins. What is quite amazing is that just a 2 percent difference in the concentration of chemotactic molecules between the front and back of the cell is sufficient to induce directed cell migration. Equally amazing is the finding that the internal signal trans duction pathways used in chemotaxis have been conserved between Dictyostelium amoebae and human leukocytes despite almost a billion years of evolution.
