Lecture 9 Cellular protrusions and cortactin, FAT cadherins and laminin and epithelial morphogenesis Feb 9, 2011.doc

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University of Toronto St. George
Cell and Systems Biology
Maurice Ringuette

Lecture 9 February 9, 2011 Cortactin: Coordinating Adhesion and the Actin Cytoskeleton at Cellular Protrusions Cell Motility and the Cytoskeleton 66: 865873 (2009) Neither cadherins nor integrins act alone in supporting morphogenesis and tissue homeostasis. Instead, they cooperate with intracellular signaling and the cytoskeleton to achieve specific biological outcomes. Cytoskeletal cooperation impinges upon actin filaments, intermediate filaments and microtubules and is regulated by cell signaling. Beyond these broad brush statements, however, we are confronted by the challenge that a bewildering number of cytoskeletal effectors and regulatory signals have been implicated in adhesive interactions. Even without having a comprehensive catalog of these players, the challenge is to identify some guiding principles that can help us parse this diversity. In this article, we will use the actin regulator, cortactin, to illustrate one approach: to identify modules of signals and effectors that couple distinct adhesive and cytoskeletal states together for specific functional purposes and characterize the integrator proteins that help coordinate the elements of such modules. Actin-driven surface protrusions: a functional module common to cell-cell and cell-matrix adhesions: The embarrassment of molecular richness is well exemplified by the functional relationship between adhesion receptors and the actin cytoskeleton. Both classical cadherins and integrins functionally interact with actin. Cell-matrix and cell-cell interactions are disrupted when actin filament integrity is perturbed with drugs (e.g., cytochalasins, latrunculin) or in Drosophila embryos mutant for fundamental actin regulators such as profilin. Conversely, actin organization is altered when cells make either integrin or cadherin-based adhesions and a host of actin regulatory proteins are implicated at adhesive interactions. The latter include actin nucleators (Arp2/3, formins), regulators of filament dynamics (e.g., Ena/VASP proteins), cross-linkers (e.g., a-actinin) and myosin motors. Despite this molecular diversity, it is becoming possible to identify distinct cytoskeleton- driven morphogenetic events that are common both to integrin- and cadherin-based adhesive interactions. This is exemplified by the process of cell protrusion, which is well-described in cell culture models of cell locomotion. In this latter case, actin-driven protrusion of leading edges is commonly thought to constitute the earliest stage in the sequence of events that lead to cell translocation. Moreover, while integrin-based adhesion is not essential for protrusion to occur, the two processes are functionally coupled. Protrusion of leading edges allows new adhesive interactions to be made, thereby stabilizing extension of the anterior margin of cells. Integrin complexes can also induce actin assembly, a process closely implicated in generating protrusive force. Moreover, stable adhesive interactions might also be predicted to facilitate the productive generation of the forward directed forces necessary for ongoing protrusion. Something analogous also occurs as cells make contacts with one another, where characteristically cells spread upon one another. This is observed in cell culture and also in Xenopus embryos, where oriented cell protrusion distinguishes morphogenetic cell-upon-cell locomotion during gastrulation (We will examine cell movements associated with Xenopus gastrulation later in the course). Indeed, when cells are allowed to make cadherin-based adhesive contacts with substrata coated with cadherin ligands, they spread in two dimensions, as they do on matrix proteins, with cell protrusion at leading margins being the initiating event in this process. As well, cell-cell contacts are sites for actin assembly, which is also evident at the protrusive leading margins of cells spreading on cadherin ligands. Overall, then, functional coupling of surface protrusion and adhesion plays an important role in extending and modeling adhesive contacts both at cell-matrix and cell-cell interactions. Importantly, a common molecular apparatusthe Arp2/3 actin nucleator complexparticipates in surface protrusion in both these forms of adhesive interactions. Arp2/3 is 1 commonly thought to nucleate branched actin filament networks that are capable of generating force in vitro. This has made it an attractive candidate to participate in surface protrusion in locomoting cells, by supporting the assembly of filaments that are orientated with their growing plus-ends directed towards the plasma membrane. Indeed, Arp2/3 is found at the leading margins of cells as they spread or locomote on matrix proteins and the branched filament networks that it generates in vitro resembles the actin networks observed at these sites in cells. Arp2/3 is also found at cell-cell contacts it also localizes to the leading protrusive margins of cells as they adhere to cadherin-coated substrata. Moreover, Arp2/3 can interact indirectly with integrins via the accessory protein, vinculin, and is found in a protein complex with the classical cadherin, E-cadherin. Indeed, in the latter case E- cadherin ligation induces the association of Arp2/3 with the E-cadherin/catenin molecular complex. Overall, then, the Arp2/3 complex is found at sites of protrusion for both integrin and cadherin adhesion. This implies that both these adhesion systems can co-opt Arp2/3 depending on the cells functional context. Moreover, the subcellular localization of Arp2/3 at both these forms of adhesive contacts is strikingly dynamic and stringently controlled, being apparently confined to the leading margins, despite the fact that adhesive ligand-receptor interactions occur at many other places, even within the same adhesive contacts. Does such a shared module have a definable conserved function at both integrin and cadherin adhesions? Indeed, actin filament assembly occurs at both integrin and cadherin adhesions precisely at the sites where they recruit Arp2/3. Furthermore, blocking Arp2/3 activity perturbs surface protrusion both at integrin- and cadherin-based adhesive contacts. In the latter case, disruption of Arp2/3 compromises the efficiency with which cells interact with one another, presumably by affecting the ability of cells to extend their contacts upon one another. Overall, these observations suggest that a conserved molecular apparatus serves a common cellular function at both integrin and cadherin-based adhesions. Arp2/3 has only a limited intrinsic capacity to nucleate filament assembly. Instead, its catalytic activity is stimulated by a number of nucleation-promoting factors that allosterically promote nucleation in response to cell signaling. WASP/WAVE family members are the best-characterized of these Arp2/3 activators and, notably, are found at integrin- and cadherin adhesions. N-WASP and WAVE are found at leading margins in locomoting cells as well as in specialized integrin-based structures, such as podosomes (Podosomes and invadopodia are cellular structures which establish close contact with the ECM. Therefore, they are thought to be key structures of cell invasion). Both N-WASP and WAVE are also found at cadherin-based cell-cell junctions. These nucleation-promoting factors, in turn, respond to a variety of intracellular signaling molecules that include Cdc42 and Rac, for WASP/N-WASP and WAVE, respectively, and protein tyrosine kinases, such as those of the Src kinase family (SKF). Of note, all these signals are found at integrin and cadherin adhesive contacts and can be activated in response to ligation of these adhesion receptors themselves. For example, E-cadherin ligation acutely stimulates both Rac and Cdc42, and Rac inhibition significantly reduced the ability of cadherin ligation to induce actin assembly. Expression of Arp2/3 activity at adhesive contacts therefore appears to be embedded in a matrix of cell signaling pathways, as it is in other contexts. Thus, Arp2/3 regulation by adhesion receptors corresponds to a more general paradigm where the subcellular localization and catalytic activity of this core actin nucleator is stringently controlled by extracellular cues and cellular context. Here signaling by integrin and cadherin adhesion receptors mediates their instructive influence. Overall, these observations suggest that molecules must exist to coordinate the matrix of regulators that impinge on Arp2/3 at adhesive contacts. Emerging evidence indicates that cortactin is one candidate to serve just such a role. 2Cortactin as a regulator of Arp2/3 at cell adhesions: Coordinating actin assembly Cortactin is an actin-binding scaffolding protein that regulates dynamic actin networks in many cellular processes, including endocytosis, cell migration, and cell adhesion (the focus of the lecture). Cortactin can directly bind to Arp2/3 through its N-terminal acidic (NTA) domain, directly to actin filaments via the central portion of its repeats region and also to a variety of other proteins through its SH3-domain (see lecture slide). Cortactin is characteristically seen at adhesive sites that are undergoing surface protrusion. These include at the leading margins of integrin-based adhesions in cells that are spreading or locomoting at cadherin-based adhesions as cells assemble contacts with one another; and at the leading edges of cadherin-based protrusions as cells spread on cadherin-coated substrata. In all these locations, cortactin localizes with Arp2/3. Moreover, inhibiting cortactin perturbs Arp2/3-dependent cell protrusion and its coordination with cell adhesion. Depleti
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