Chapter Seven - Anchors and Signals: The Diverse Roles of Integrins in Development
Introduction
The core problems of developmental biology—cell fate determination, tissue patterning, morphogenesis—remained opaque in terms of underlying mechanism until the revolution in molecular biology allowed a move beyond a description of the event, and uncovered the gene expression patterns and protein interactions that drive development. The discipline became a “science of arrows” (Gilbert, 1998), with a strong focus on how signaling ligands from one cell could elicit fate changes in another, working through conserved signal transduction pathways that culminated in altered gene expression. Dynamic intercellular signaling provided a mechanism for how patterns could emerge from initially uniform tissues, and how one tissue could induce the fate of another (Freeman & Gurdon, 2002).
The “executive” or “decision-making” side of development is only half the story, however; the initiation of a gene expression program marks the intention, not the act. Of equal importance is the question of how cell fate is transformed into cell form (Larsen and Mclaughlin, 1987, Wieschaus, 1996), and thus an understanding of development also has to include how changes in cell mechanics contribute to the building of bodies. A suite of cell behaviors drives development: individual and collective cell migration; proliferation and apoptosis; neighbor exchanges and shape changes. These behaviors commonly involve regulation of the cytoskeleton and the membrane (Lecuit, Lenne, & Munro, 2011), and are co-ordinated to drive higher order morphogenesis as tissues are sculpted into functional forms.
We thus come to a conception of development as orchestrated by chemical signaling pathways, and executed by “realizators” (Garcia-Bellido, 1975) that act to change cell and tissue form. This model, distinguishing signaling from mechanics, can be augmented when we consider how the mechanical environment of the cell feeds back to influence various aspects of cell behavior (Bershadsky et al., 2003, Mammoto et al., 2013). The ability to sense changes in force is clearly crucial for any cell to read its environment. The intracellular response to these changes is mediated by a process termed mechanotransduction, which translates changes in force into chemical signals. Mechanotransduction takes many forms, from stretch activated ion channels in the membrane to changes in protein conformation under tension (DuFort, Paszek, & Weaver, 2011). The mechanical components of a cell's environment can influence cell choices in a similar manner to the chemical components.
Cell adhesion lies at the nexus of signaling and mechanics, as adhesions are both sites of structural connection between the cell and the outside world, and sites of environmental sensing and signaling. The critical importance of cell adhesion to metazoans is self-evident; cells need to stick together to form tissues. Development involves both dynamic changes in adhesion, as seen in transitions between epithelia and mesenchyme (Ciruna & Rossant, 2001), and the formation of stable adhesions that provide tissue integrity, exemplified by Drosophila muscle development (Brown et al., 2002, Zervas et al., 2001). Adhesion takes two primary forms in animals; direct adhesion between cells mediated by cadherins (Halbleib & Nelson, 2006) and adhesion between the cell and the extracellular matrix (ECM), which is mediated by integrins and the focus of this review.
Work in the 1960s to 1980s revealed that cells migrating in dishes interact with the substrate at specific sites of the plasma membrane that are also sites of actin attachment on the cytoplasmic side (Abercrombie, 1980). The first proteins identified at these sites were cytoplasmic, for example, vinculin (Burridge and Feramisco, 1980, Geiger, 1979) and the src kinase (Rohrschneider, 1980). In the following years, the transmembrane receptors that bound the ECM, integrins, were identified in multiple cell types and across many species (Hynes, 1987), and subsequently found to mediate adhesion in diverse developmental and pathological contexts in addition to cell migration. The basic architecture of the cell–matrix adhesion was thus revealed; ECM ligand—transmembrane receptor—cytoplasmic protein complex—actin. This architecture, which is also found in cadherin adhesions (although as homophilic adhesion molecules the extracellular ligand is the same transmembrane receptor on the adjacent cell), provides a continuous physical link between the external and internal worlds of the cell, allowing forces generated within the cell to be transmitted to the outside world, and vice versa (Fig. 1).
Integrins function as heterodimers of α and β subunits (for a more detailed account of the structure and biochemistry of integrins, see Campbell & Humphries, 2011). The β subunits can bind to more than one α subunit, and some α subunits can bind to more than one β subunit (Fig. 2). Different heterodimers provide functional diversity by interacting with distinct ligands, and this can be developmentally regulated by differential expression of the subunits (Meighan & Schwarzbauer, 2008). For example, the Drosophila αPS1βPS heterodimer binds to laminin, while the αPS2βPS heterodimer binds to molecules containing the RGD peptide, such as thrombospondin and tiggrin (Fogerty et al., 1994, Gotwals et al., 1994, Subramanian et al., 2007). Transcriptional switching between αPS1 and αPS2 is observed at a specific point in egg chamber development, and this switch leads to the recruitment of the adapter protein tensin (Delon & Brown, 2009). The number of subunits per species increases with organismal complexity; worms have two α and one β subunit, flies have five α and two β subunits, while mammals have 18 α and 8 β subunits, which can assemble into 24 heterodimer pairs (Fig. 2; Bouvard et al., 2001, Brown, 2000, Humphries et al., 2006).
Integrins bind a variety of proteins with both their extracellular and intracellular domains, and can also bind “in cis” to other transmembrane proteins such as growth factor receptors. The extracellular ligands are predominantly ECM proteins such as laminin, fibronectin, and collagen. Originally considered mere intercellular filler, the ECM, a highly crosslinked meshwork of insoluble proteins, is now known to contribute to multiple aspects of cell and tissue biology during development (Brown, 2011, Rozario and DeSimone, 2010). Integrins not only bind ECM proteins but also help assemble them into functional, organized matrices. For example, the assembly of a fibronectin matrix from component dimers depends on integrin binding both in cell culture (Akiyama, Yamada, Chen, & Yamada, 1989) and in embryos (Koshida et al., 2005, Marsden and DeSimone, 2001). An ordered ECM can act as a tract for cell migration, define tissue boundaries, and provide physical instructive cues to the cell (Brown, 2011, Rozario and DeSimone, 2010). The demonstration that the ECM can move in concert with moving cells during avian primitive streak formation (Zamir, Rongish, & Little, 2008) and in Xenopus extracts (Davidson, Dzamba, Keller, & Desimone, 2008), and that the basement membrane can slide to facilitate tissue interactions in worms (Ihara et al., 2011), emphasizes the dynamic nature of the ECM; we cannot always think of it as a stationary reference point for cell behaviors. As well as functions mediated through integrins, the ECM can interact with numerous other membrane receptors, provide a general, supportive function for tissues, and provide a reservoir of signaling molecules to influence cell fate.
On the intracellular side, integrins do not bind actin directly, but indirectly via some of the proteins they recruit with their short cytoplasmic tails. The dynamic complex of proteins at cell–ECM adhesions has been termed the integrin adhesome (Zaidel-Bar, Itzkovitz, Ma'ayan, Iyengar, & Geiger, 2007). A crucial adhesome component is talin, which can simultaneously bind the cytoplasmic tail of integrin β subunits and actin, as well as mediating recruitment of a number of other proteins to the adhesion (Critchley, 2009). Depleting talin in fibroblasts leads to the failure of normal adhesion formation in vitro (Zhang et al., 2008), and talin loss causes phenotypes that are identical to loss of βPS-containing integrins in Drosophila (Brown et al., 2002) and β1-containing integrins in mouse skeletal muscle (Conti, Monkley, Wood, Critchley, & Muller, 2009), suggesting that all integrin adhesive functions require talin.
Aside from proteins with actin binding or regulating activity, the intracellular adhesome also contains proteins that function as scaffolds, promoting interactions between other proteins (for example, paxillin (Deakin & Turner, 2008)), and proteins that enzymatically modify proteins to change their activity, such as kinases (for example, focal adhesion kinase (Mitra, Hanson, & Schlaepfer, 2005)). The precise attribution of function to any single protein in the adhesome can be confounded by the degree of interconnectivity (Zaidel-Bar et al., 2007). In general, integrins only provide a functional output when they are bound to proteins on both sides of the plasma membrane (though there may be exceptions to this rule; for example, unliganded integrins can play a role in promoting cell death (Stupack, Puente, Boutsaboualoy, Storgard, & Cheresh, 2001)). We can thus think of the “functional unit” as an integrin bound to other proteins in both intracellular and extracellular environments.
The ease with which large integrin adhesions can be observed and perturbed in cell culture led to such studies dominating integrin research. The role of integrins and their many associated proteins in coordinating the movement of migrating cells—commonly fibroblasts migrating on two-dimensional substrates such as gels or artificial matrices—has become one of the most intensively studied processes in cell biology (Huttenlocher & Horwitz, 2011). Other modes of cell migration have also been revealed in different cell types, utilizing distinct cytoskeletal, membrane and adhesion mechanisms, and indeed requirements for integrins; the 2D fibroblast model is, clearly, not universally applicable to cell migration (Friedl et al., 2012, Huttenlocher and Horwitz, 2011). Furthermore, many migration events in vivo are collective, and through complex three-dimensional environments, such that the physical requirements and constraints might differ considerably (Friedl & Gilmour, 2009). Finally, the way that integrins work in cell migration may differ to its multitude of other roles in tissue construction, where the adhesion turnover will be a lot slower than that seen in quickly migrating cells (Bokel & Brown, 2002). For example, muscle development in Drosophila involves the progressive stabilization of adhesions, as seen by reductions in protein mobility, in response to increases in force from muscle contraction (Pines et al., 2012, Yuan et al., 2010). Thus, although cells migrating in culture have taught us a lot about how integrins work, the lessons need not all be universal.
Alongside work on cells in culture, studies that addressed the role of integrins during development also mushroomed, with analysis of genetic knockouts or knockdowns in a number of model organisms (Brower, 2003, Wickstrom et al., 2011), and, in certain models, tissue-specific knockouts. As we will discover, integrin adhesion regulates a whole host of developmental processes, with both similarities and distinctions between the ways integrins work in different developmental contexts within and between model organisms, and indeed between studies using cells in culture versus those in intact animals (Bokel & Brown, 2002). Developmental defects arising from integrin loss include defects in morphogenesis (tissues are not formed properly), structural defects (tissue integrity is lost), and in some cases, alterations to basic cellular properties (cells do not divide, survive, or differentiate properly).
The discovery that integrin adhesions contained both actin-binding proteins and proteins like kinases led to the current model that integrins perform two key functions; first, provide a mechanical link between the ECM and cytoskeleton, anchoring the cell and transducing force; and second, influence cell behavior by regulating cell signaling (Fig. 1). The signaling side of integrin function has many component parts, depending on how restrictive one wishes to be with the definition of a signal. A strict definition may confine this to effects on signal transduction pathways that terminate in the nucleus. This can happen by a number of means, from “cis” interactions with membrane receptors enhancing their activation, to the recruitment and activation of adhesome components that themselves influence a given pathway (Streuli & Akhtar, 2009). A broader definition of signaling might involve all of the protein interactions that do not play a direct role in linking to the cytoskeleton, for example, recruitment cascades. The complexity is that many of these interactions may feed back onto the mechanical link. For example, adhesion signaling crucially impinges on RhoGTPases, multifunctional proteins that play a role multiple cell behaviors (Huveneers & Danen, 2009). Among their primary targets are regulators of the actin cytoskeleton, and hence they are important in regulating the mechanical link provided by the adhesion; signaling feeds into mechanics, and vice versa. Signaling can also control local features of adhesion within the cell, such as adhesion polarity and localized disassembly. In a sense, any signaling aspect of integrin function is necessarily downstream of the anchorage; you cannot have a signaling platform without the ECM–actin link.
As determined in vitro, both integrin and cadherin adhesions are sites of mechanotransduction (le Duc et al., 2010, Ross et al., 2013); the structures that bear force in the cell play a key role in sensing that force (Schwartz, 2010). One of the key responses of adhesions to increases in tension is reinforcement, which can be driven by changes in protein recruitment and cytoskeleton dynamics, and allows the adhesion to withstand force across it. While integrins themselves may behave as mechanosensors—force influences the strength of interaction between α5β1 and fibronectin, for example (Friedland, Lee, & Boettiger, 2009)—certain adhesome proteins certainly do. Force across talin leads to the exposure of cryptic vinculin-binding sites and subsequent binding of vinculin (del Rio et al., 2009), and vinculin itself regulates protein composition in response to changes in tension across the adhesion via a conformational change (Carisey et al., 2013).
Aside from shoring up the adhesion in response to stress, integrin-mediated mechanotransduction may influence cell fate choices. Integrins allow stem cells to sense the stiffness of the matrix and make decisions on when and how to differentiate accordingly (Engler et al., 2006, Trappmann et al., 2012); the mechanical properties of the cell's environment can thus play a similarly inductive role as chemical signals. In the context of development, mechanical forces have been shown to both augment cell signaling to reinforce cell fate decisions, and even in some cases play a primary instructive role (Miller & Davidson, 2013). Here, integrins could be crucial in mediating mechanical induction, but examples from development are limited.
Thus, during development, phenotypes following integrin removal could result from putative mechanical or signaling functions of integrins, or both. Do we see integrins primarily as anchors, or as “interactive information interfaces” (Geiger & Yamada, 2011) that allow the cell to sense and influence its environment? In this review, we take examples of integrin function from the development of various model organisms—mice, worms, flies, zebrafish, chickens, frogs—and consider how they give us a better understanding of how integrins work. Given the depth of the literature we do not hope to be comprehensive, rather to identify threads that link functions in different organisms, and give a picture of the diversity of processes in which integrins are implicated. We start by asking whether integrins regulate basic cellular properties of viability and fate, and then move on to some of the morphogenetic processes influenced by integrin function.
Section snippets
Integrins in Choices of Life and Death: Animal Lethal, not Always Cell Lethal
Metazoan development requires integrins; mutations in some or all of the α or β subunits are lethal in flies (reviewed in Brown, 2000), worms (Williams & Waterston, 1994), mice (reviewed in Bouvard et al., 2001), and zebrafish (Julich, Geisler, Holley, & Tübingen 2000 Screen Consortium, 2005), while nongenetic reductions in integrin function perturb development in frogs (Marsden & DeSimone, 2001), chickens (Rallis et al., 2010, Tucker, 2004), and sea urchins (Marsden & Burke, 1998). Mutations
Mechanotransduction of matrix properties by stem cells
Metazoan development involves the segregation of cells into distinct cell fates, and the subsequent morphological differentiation of the cells to construct different tissues. Although, the role of integrins in the latter process is clear, for example, by adhering different cell types together via an intervening matrix, how integrins influence cell fate choices is less certain. Instructive induction is canonically mediated by extracellular ligands from a variety of peptide families, for example,
Integrins in Single and Collective Cell Migration: Beyond the Fibroblast Model
Migration, whether by single cells, small groups of cells, or tissues en masse, is a crucial component of development, as cells are often not born where they need to end up and organs require the coming together of tissues of different origins. The critical importance of integrins to cell migration on 2D substrates has been firmly established (Huttenlocher & Horwitz, 2011), but the particular features of this migration event are not universally shared. Even before the molecular delineation of
Building Tissues and Organs: Snags and Anchors
Binding between the extracellular domains of integrins and ECM ligands can have profound consequences for both the cell and its environment. During development, integrins can either act to snag dispersing ECM components and enrich them in one region of the tissue, or act as anchors to halt migration in moving cells (Fig. 4).
Loss of integrins can lead to a loss of normal accumulation and patterning of the ECM. In frog gastrulation, expression of dominant negative β1 integrin suppressed
Integrins in Epithelia: Cell Polarity and Division Orientation
Our final examples from development come from the role of integrins in epithelial cell biology. On their basal surface, epithelial cells contact the ECM via integrins, while the apical surface can either contact other cells, in the case of a multilayered epithelium, contact an apical ECM, as is the case in the arthropod epidermis, or face the lumen, in the case of tube-forming epithelia. The basal contact mediated by integrins can play a fundamental role in telling the cell which way is down,
Outlook
Animal development provides a seemingly inexhaustible supply of events in which integrins play an important role. As we have seen, integrins establish the ECM–actin link to stick cells together, provide the means of migration, and sculpt tissues. In this sense, the role of integrins in many contexts can be explained without the need to invoke much signaling; the anchorage itself is the primary functional output of the protein, and the most obvious integrin phenotypes tend to be adhesion
Acknowledgments
The authors wish to apologize to colleagues whose work has been omitted due to space constraints. The Brown lab acknowledges the funding it has received from the Wellcome Trust, the BBSRC, and the Human Frontier Science Program.
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