Cell locomotion;  Cell motility

Cell migration can be defined as the movement of cells from one site to another and is a central process in the development and homeostasis of multicellular organisms. The orchestrated movement of cells in a particular direction to a specific location is essential for tissue formation during embryonic development, wound healing, and immune responses. Deregulation of cell migration during any of these processes has serious consequences and can contribute to mental retardation, vascular disease, rheumatoid arthritis, tumor formation, and metastasis.

Cell migration (the movement of cells from one site to another) is an essential process for normal development and homeostasis that can also contribute to important pathologies such as neoplasia. For example, one of the major mechanisms involved in tumor cell  invasion is cell migration, a process in which cells demonstrating higher invasive capacity typically show higher migratory ability. Cell migration is a complex and highly coordinated processes which involves changes in the expression of several genes. Cell migration requires an intricate balance between extracellular cues and responsive intracellular signals that lead to dynamic regulation of the interactions between actin microfilaments, microtubules, intermediate filaments, and associated  cell adhesion molecules. The driving force for cell movement, however, is normally provided by dynamic reorganization of the actin  cytoskeleton, directing protrusion at the front of the cell and retraction at the rear. In general, cell migration can be conceptualized as a cyclic process (Fig. 1). For a cell to move, it must establish morphological polarity resulting in leading and trailing edges with directionalized forces. To accomplish this, the initial step requires extension of protrusions in the direction of movement. These protrusions can be large, broad lamellipodia or spike-like filopodia that are typically formed by active actin polymerization. Frequently, lamellipodia evolve into ruffles either de novo or when the membrane protrusions fail to adhere and are swept backward on the dorsal surface.

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Protrusions are then stabilized by adhering to the extracellular matrix (ECM) or to adjacent cells via transmembrane receptors which are in turn linked to the actin cytoskeleton. Different forms of focal contacts exist that serve as adhesive structures to connect the cell with the ECM. They consist of two main elements: (i) a transmembrane component comprised of clusters of integrin molecules linked to the ECM and (ii) a submembranous component comprised of a complex of proteins such as vinculin, talin,  FAK, and other proteins. Although focal contacts react to actin-myosin tension, they also transmit their own tension upon the ECM to which they are linked. Since migrating cells must be able to detach, yet exert traction on the substratum, the velocity of migration is a biphasic function dependent on the strength of cell attachment. Finally, a contractile force, derived by the interaction of actin with myosin II to form a functional actin-myosin motor unit, propels the cell forward and contributes to the release of adhesive contacts in the rear of the cell and cell body translocation.

Considerable research with in vitro model systems has led to an increased understanding of cancer cell motility and invasion. The difficulty with many of these systems, however, is their relevance to the in vivo situation. Cell movement on a glass surface in a two-dimensional plane is clearly different than invasion in a three-dimensional environment. To improve our understanding of tumor cell migration, scientists have used in vivo imaging by intravital multiphoton technology, three-dimensional in vitro models, and histological analyses as techniques to further characterize migration in a more physiological and representative context. The best understood system at present relates to mesenchymal cell motility which is characterized by an elongated morphology with established cell polarity. This polarity is dependent upon proteolysis to degrade the ECM. It is estimated that cancer cells which undergo  epithelial to mesenchymal transition (EMT), a phenomenon which takes place in ~10% of all solid tumors, use this form of motility. Intravital imaging has demonstrated that some cancer cells move at very high speeds with amoeboid morphology, very similar to the rounded form of migration of leukocytes and Dictyostelium. Another form of cell motility is called collective cell motility which involves the movement of whole clusters of sheets of tumor cells. Collective cell motility has been observed in infiltrating breast carcinoma and some  ovarian carcinomas. In several other cancers, including gliomas, the type of motility employed has not been clearly defined and will require intravital imaging techniques to help in this assessment in the future.

Mechanisms of Cell Migration: Rho-GTPases Are Key Players

In the past decade, the analysis of a model cell line, Swiss 3 T3 fibroblasts, has led to the identification of three  signal transduction pathways responsible for controlling actin remodeling leading to directional cell migration. Each of these pathways is controlled by a member of the  Rho family of small GTPases. Rho-GTPases are members of the  Ras superfamily of proteins. They are essentially molecular switches that become activated when they are bound to GTP and inactivated by hydrolysis of GTP to GDP. Rac, Cdc42, and Rho are the best characterized members of this family (Fig. 2).

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Rac

Rac is required to regulate actin polymerization and formation of lamellipodia at the leading edge of cells and is thought to be the driving force for cell movement. Formation of small  adhesions known as focal complexes which help stabilize the lamellipodium to the ECM also depends on Rac. To accomplish this, Rac recruits high-affinity integrins to the area where new membrane protrusions are occurring. Members of the WASP/SCAR/WAVE family of scaffold proteins are key regulators of actin polymerization. In their stimulated state, each of these proteins is able to stimulate Arp2/3 complex which can initiate actin polymerization. Rac stimulates lamellipodia extension by activating SCAR/WAVE proteins indirectly and involves the Nck-adaptor complex.

One of the best characterized effectors that transmits the Rac signal is the Ser/Thr kinase, PAK. PAK plays an important role in regulating actin remodeling and cell adhesion during cell migration. The effects of PAK are partially controlled by its subcellular localization and interaction with binding proteins. In quiescent cells, PAK is distributed throughout the cytoplasm; however, when cells are stimulated, PAK is targeted to focal adhesions and membrane ruffles through binding of PIX. PAK has also been shown to phosphorylate and activate LIM kinase (LIMK), which in turn phosphorylates cofilin. Cofilin facilitates subunit dissociation from the pointed end of actin filaments and induces filament severing. It is also essential for promoting filament treadmilling (assembly/disassembly of free filament ends) at the front of migrating cells. PAK can also alter the phosphorylation state of myosin II. However, there are conflicting data on the role of PAK in myosin phosphorylation. Expression of constitutively activated PAK resulted in decreased phosphorylation of the regulatory light chain of myosin in Hela cells which is in contrast to activated PAK in NIH3T3 cells, where activated PAK promoted MLC phosphorylation.

Cdc42

Cdc42 is the major regulator of cell polarity and plays a crucial role in controlling the direction of migration in eukaryotic organisms ranging from yeast to humans. One way in which Cdc42 influences polarity is by restricting where lamellipodia form. Cdc42 can also affect polarity by localizing the microtubule-organizing center (MTOC) and Golgi apparatus in front of the nucleus, oriented toward the leading edge. The effects on MTOC position appear to be exerted mainly through a pathway involving the Cdc42 effector protein PAR6 which exists in a complex with PAR3 and an atypical protein kinase C (aPKC). However, the exact molecular mechanism by which PAR6/PAR3/aPKC complex orients the MTOC is still not well defined. PAK, also a downstream target of Cdc42, can itself mediate Cdc42 activation downstream of heterotrimeric G-protein-coupled receptors, which are activated by many chemoattractants. These signaling events mediate a positive feedback loop between Cdc42 and PAK.

In response to bradykinin and interleukin 1, Cdc42 also induces parallel actin filament assembly to form filopodia. Filopodia are particularly well designed to serve as sensors and to explore the local environment, although they are not essential for chemotaxis. Cdc42 binds to WASP proteins and in vitro stimulates the Arp2/3 complex to induce dendritic actin polymerization and subsequent filopodia formation. WAVE/WASP proteins may themselves regulate the activity of Rac and Cdc42 by binding to GAPs and GEFs and could therefore generate positive or negative feedback loops to regulate the extent of actin polymerization.

Rho

Rho activity in migrating cells is associated with focal adhesion assembly and stress fiber formation and is responsible for cell body contraction and rear end retraction in response to a variety of extracellular stimuli such as lysophosphatidic acid administration or integrin engagement. The inhibition of Rho in cells like fibroblasts decreases adhesion causing a retraction of lamellae and rounding of the cell body. Rho can stimulate the clustering of integrins and thereby increases the strength of adhesion. The clustering is very pronounced and results from tension aggregating dispersed integrins, such that they align through their attachment with the ends of stress fibers in focal adhesions. Contractile actin-myosin filaments are found in all mammalian cells. In cultured fibroblasts, these are readily seen as well-organized bundles known as stress fibers that are tethered to the plasma membrane at focal adhesions. The ability of Rho to induce the assembly of actin-myosin filaments and therefore contractility appears to depend on the activation of two distinct targets, Ser/Thr kinase p160ROCK (also known as ROCK, Rho-kinase) and the formin family protein, mDia.

ROCK has many targets but a key one is the myosin binding subunit of myosin light chain phosphatase. Phosphorylation leads to inactivation of the phosphatase which leads to an increase in the levels of myosin light chain (MLC) phosphorylation and cross-linking of myosin II into actin filaments and generation of cell tension. ROCK can also take the place of myosin light chain kinase (MLCK) and directly phosphorylate MLC. In its active state, ROCK, like PAK, can also phosphorylate and activate LIMK, which in turn phosphorylates and inactivates cofilin leading to stabilization of actin filaments within actin-myosin filament bundles. Ezrin/radixin/moesin (ERM) family proteins, adducin, and intermediate filaments (IFs) are also substrates of ROCK, and the phosphorylation states of these targets are implicated in specific cell functions. mDia is another major player for promoting actin polymerization in eukaryotic cells. Upon binding Rho-GTP, mDia adopts an open conformation and binds to the barbed ends of actin filaments. It has the property of allowing addition of actin monomers to filaments while preventing the binding of capping proteins which would otherwise block elongation. mDia also cooperates with ROCK to assemble properly aligned stress fibers.