Emerging properties of adhesion complexes: what are they and what do they do?

TICB-1128; No. of Pages 10

Review

Emerging properties of adhesion complexes: what are they and what do they do? Jonathan D. Humphries1*, Nikki R. Paul1, Martin J. Humphries1, and Mark R. Morgan2,y 1

Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK 2 Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Nuffield Building, Crown Street, Liverpool, L69 3BX, UK

The regulation of cell adhesion machinery is central to a wide variety of developmental and pathological processes and occurs primarily within integrin-associated adhesion complexes. Here, we review recent advances that have furthered our understanding of the composition, organisation, and dynamics of these complexes, and provide an updated view on their emerging functions. Key findings are that adhesion complexes contain both core and non-canonical components. As a result of the dramatic increase in the range of components observed in adhesion complexes by proteomics, we comment on newly emerging functions for adhesion signalling. We conclude that, from a cellular or tissue systems perspective, adhesion signalling should be viewed as an emergent property of both the core and non-canonical adhesion complex components. Adhesion complexes: integrating extracellular and intracellular signals Cells in multicellular organisms are integrated functionally and structurally with their local microenvironment. Cellular recognition of the extracellular matrix (ECM; see Glossary) permits mechanical, topological, and chemical sensation of the extracellular milieu and leads to localised compartmentalisation of cytoplasmic signalling. Adhesion receptors, including integrins and syndecans, have evolved to control information flow across the plasma membrane and integrate the ECM with the contractile machinery of the cell bi-directionally [1,2]. Ligand binding to the extracellular domain of integrins stabilises activated conformations and leads to recruitment of adaptor and catalytic proteins to the integrin cytoplasmic domains. Conversely, recruitment of specific cytoplasmic proteins to the intracellular face of integrins induces receptor priming and drives ligand engagement Corresponding authors: Humphries, M.J. ([email protected]); Morgan, M.R. ([email protected]). Keywords: integrins; cell adhesion; proteomics; mechanotransduction; receptor trafficking; syndecans. * Twitter: @MJ_Humphries y Twitter: @M_MorganLab 0962-8924/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tcb.2015.02.008

[3–5]. Stable association of ligands and effectors with integrins can therefore be regulated from both sides of the plasma membrane. Proteins recruited to integrin-mediated adhesion complexes perform both membrane-proximal and membrane-distal signalling functions that coordinate processes including migration, proliferation, differentiation, and ECM remodelling [1,6,7]. Consequently, aberrations in cell–ECM interactions or adhesion signalling contribute substantially to a range of diseases and adhesion components are appealing therapeutic targets [8,9]. In this context, a series of recent studies have developed or used reagents targeting adhesion receptor function to treat preclinical models of scleroderma, fibrosis, and thrombosis [10–12] and integrin inhibitors have entered clinical trials for inflammatory and neoplastic diseases [13,14]. Classical experimental approaches do not permit spatially- or temporally-resolved analyses of adhesion receptor function, or the systematic global analysis of adhesion signalling networks. However, recent technological innovations, based on proteomics and advanced imaging techniques, are revealing the complexity, structure, and dynamics of adhesion complexes and leading to a more comprehensive understanding of adhesion function. These analyses have identified a previously unanticipated level of Glossary Adhesome: a set of proteins defined by their localisation to and/or ability to regulate adhesion complexes. ECM: extracellular matrix, a complex mixture of proteins and proteoglycans that cells secrete and organise. FN: fibronectin, a protein that is a constituent of the ECM. FCCS: fluorescence cross-correlation spectroscopy, a microscopy-based technique to study protein interactions in living cells. FRAP: fluorescence recovery after photobleaching, a microscopy-based technique to study the diffusion and exchange of components in living cells. Integrins: a family of cell surface adhesion receptors that exist as heterodimers comprising an a and a b subunit. LIM domain: LIN-11, Isl1, and MEC-3 domain, a protein fold found in many proteins that has been implicated in mechanotransduction through regulation of the actin cytoskeleton. Mass spectrometry: a method to measure accurately the mass of peptides or proteins; it is commonly used to identify the protein components of a system. Mechanotransduction: the transmission of mechanical force into cell signalling events. Proteomics: the study of the complete set of proteins in an organism or system. VCAM-1: vascular cell adhesion molecule-1, a cell surface adhesion receptor.

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Figure 1. The adhesome complex landscape: integrating recent advances from proteomics and imaging studies. The core functional adhesion complex has been defined using complementary information from unbiased global proteomic and targeted microscopy approaches. Analysis by mass spectrometry revealed tension-dependent and integrin heterodimer/extracellular matrix (ECM)-dependent components of adhesions [19–23]. For example, the recruitment of specific components to a5b1 integrin- or aV integrin-mediated adhesion complexes leads to differential regulation of tension sensing via small GTPases such as Rac and RhoA. Proteomic analyses also suggest the existence of a large number of non-canonical adhesome components. By contrast, candidate-based microscopy studies have focused on the architecture, interactions, and dynamics of adhesion complex assembly and disassembly [34,36,37]. For example, recent studies have identified that many adhesome components are pre-assembled as multi-protein building blocks in the cytosol, allowing rapid adhesion complex maturation and turnover [34]. Together these studies have contributed to our understanding of integrin signalling, force generation, and actin regulation and their impact on numerous downstream cellular processes.

complexity and regulation [15–17] (Figure 1). Thus, it is timely to reassess, and redefine, what constitutes the ‘adhesome’ of an adhesion complex and re-appraise the range of its biological functions. In this review, we discuss the adhesion complex architecture and describe its emerging role in mechanotransduction and modulation by receptor trafficking. We also discuss how the more variable, labile, and dynamic components of adhesion complexes fine-tune signalling and cellular function, and may modulate a range of non-canonical adhesion-associated functions. The core adhesion complex: molecular and structural architecture Decades of candidate-based research focusing on the localisation and interactions of individual proteins have provided a wealth of information on adhesion complex composition and regulation [1,18]. One outstanding question, however, is whether a restricted set of components can be defined as the core or canonical adhesion complex? Recently, mass spectrometry-based proteomics and microscopy have shed new light on this issue. Proteomics illuminates the field A variety of proteomic approaches have been used to identify and characterise components of the cellular 2

machinery and signalling events associated with integrin-mediated adhesion (Box 1). Recent advances have resulted from coupling novel adhesion complex isolation strategies with mass spectrometry [19–24]. In essence, proteomic studies of isolated adhesion complexes provide a context-dependent viewpoint of adhesion complex composition that complements the established model of the adhesome (constructed from collation of data derived from studies of specific interactions reported in the literature [17,25]). To date, the main findings resulting from the proteomic analysis of adhesion complexes can be grouped into studies that illustrate integrin heterodimer- or ECMdependent adhesion complex components [19,22,23], reveal novel mechanisms of regulation for the small GTPase Rac [19,20,26], or provide insight into mechanisms that regulate tension-dependent sensing by adhesion complexes [20,21,23] (Figure 1). Integrins, and adhesion complex components, play major roles as mechanotransductive signalling effectors and sensors of environmental stiffness and cell-generated force [7,16]. Cell–matrix adhesion complexes are dynamic structures that form at the periphery of cells as nascent adhesions and undergo myosin II-dependent maturation into focal adhesions anchored to load-bearing bundled actin stress fibres [27]. To determine force-regulated components of adhesion complexes, two groups independently


Review Box 1. Proteomics of integrin-mediated adhesion: an historical perspective Historically, integrins and adhesion complex components have been studied using mass spectrometry-based proteomics in a candidate-focussed manner identifying post-translational modifications or interacting partners of individual proteins [95–99], characterising adhesion-dependent binding partners for a small number of adhesion proteins [79,100,101], or identifying proteins from a restricted compartment of a cell [102]. Many of these efforts have been coordinated by the Cell Migration Consortium (http://www. cellmigration.org/index.shtml) and a substantial amount of their published and unpublished data is freely available (http://www. cellmigration.org/resource/proteomics/). Until recently, the isolation and comprehensive proteomic analysis of adhesion complexes was hindered because the complex exists as a labile junction of cytoskeleton- and membrane-bound assemblies. In 2009, however, a methodology was described that enabled the first global analysis of isolated adhesion complexes by mass spectrometry [19], and since then a number of studies have used similar approaches to probe adhesion complex composition and abundance changes from a variety of cellular perspectives [20–24]. The most immediate finding of note from the proteomic studies was the increased scale and complexity of isolated adhesion complexes, in comparison to the adhesome. Depending on the study, proteomic analysis led to the identification of two- to threefold more proteins than the 232 components reported in the most recent adhesome analysis [13,15]. Interestingly, analysis of the novel proteins contained in the proteomic datasets, that do not appear in the published ‘adhesome’, revealed that many of the proteins had molecular functions linked to those of focal adhesion structural and regulatory proteins, or contained protein domains characteristic of adhesome adaptors [15,16]. From these data, it is apparent that the extent of overlap between proteins identified by proteomics and those within the published adhesome is relatively low, with approximately 10–25% of the adhesome being identified in any one dataset [15]. These findings highlight that the adhesome comprises all known components and interactions of adhesion complex proteins from all cell types and conditions studied, while the proteomic datasets identify a context-specific subset of these components. Furthermore, the datasets confirmed the presence of many existing adhesion components but also identified numerous additional elements, suggesting that the complexity of the adhesome landscape may far exceed current candidate-derived models.

reported proteomic characterisation of adhesion complex components from myosin II-inhibited cells [20,21]. Both studies reported hundreds of proteins recruited to cell– matrix adhesions in a myosin-dependent manner and intriguingly these force-sensitive components were enriched for proteins containing LIN-11, Isl1, and MEC3 (LIM) domains. The functional relevance of LIM domains in effecting the mechanosensitive response of adhesion complexes has not yet been fully characterised. However, there are precedents for the involvement of LIM domaincontaining proteins (e.g., zyxin, Hic5, and paxillin) in force modulation [28]; supporting the hypothesis that LIM domains are key components of mechanosensory modules. Proteomic analyses of isolated adhesion complexes have also determined compositional distinctions between integrin heterodimer- or ECM-dependent adhesion complex components [19,22,23] (Figure 1). Analysis of adhesion complexes formed upon cell adhesion to fibronectin (FN) or vascular cell adhesion molecule-1 (VCAM-1), identifying a5b1 and a4b1 complexes, respectively, revealed both common and receptor-specific components that, despite sharing the integrin b1 subunit, differed both in scale and composition. Further analysis identified regulator of


chromosome condensation 2 (RCC2), a protein isolated specifically in FN-bound a5b1 complexes, as being a negative regulator of both Rac1 and Arf6, with a previously unappreciated role in cell migration, thereby validating this approach as a way of identifying novel signalling components [19]. Subsequent integration of multiple proteomic datasets, for proteins within this local network, enabled identification of IQ motif-containing GTPase activating protein-1 (IQGAP1) and filamin A as Rac1 regulators. Further proteomic analysis identified RacGAP1 as an IQGAP1-binding partner controlling Rac1 activation during cell migration [26,29]. Interestingly, the myosin-sensitive proteomic analysis of Kuo et al. revealed proteins whose recruitment to adhesion complexes was negatively regulated by contractility and clarified the role of the Rac guanine nucleotide exchange factor (GEF), b-Pix, as a regulator of Rac1 that limits focal adhesion maturation to regulate cell migration [20]. More recently, the myosin sensitivity-based proteomic approach was combined with analysis of heterodimer-specific proteomes [23]. Generation of cell lines with restricted expression of FN-binding integrin subunits permitted the definition of complex compositions specific for b1 and aV integrins. These analyses revealed that different integrin heterodimers cooperate to mediate environmental rigidity sensing via integrin heterodimer-specific coupling of active RhoA to different effectors: a5b1 integrins generating force via RhoA, ROCK, and myosin II, whereas aV integrins mediate structural reinforcement of stress fibres via GEFH1, mDia, and RhoA [16,23]. To summarise, proteomic analyses of isolated adhesion complexes have provided important insights into adhesion biology. Distillation of the accumulated data [15,19–23] reveals adhesome proteins that are commonly identified in these analyses [including integrin-linked kinase (ILK), talin, vinculin, a-actinin, LIM and SH3 domain protein (LASP), vasodilator-stimulated phosphoprotein (VASP), zyxin, filamin, IQGAP1, and kindlin], which may represent a putative ‘core adhesome’. Future studies will no doubt help address current limitations of proteomic approaches and answer important outstanding questions (Box 2). Nanoscale architecture and dynamics of the adhesion machinery The complexity of adhesion complex organisation, associations, and dynamics has been highlighted recently through advanced microscopy (Figure 1). Fluorescence recovery after photobleaching (FRAP) studies have shown large variations in the dynamic exchange of adhesome proteins within adhesive structures [1,30–33], including differences in both the proportion of molecules that are available for exchange and the exchange rate. Developing this concept further, FRAP and fluorescence cross-correlation spectroscopy (FCCS) experiments have revealed that an array of adhesion complex components are pre-assembled in the cytosol and act as building blocks for the rapid formation of adhesion structures [34]. These findings are interesting as they suggest that mechanistic checkpoints exist to enable accurate and efficient regulation of complex assembly at the plasma membrane, while preventing formation in the 3


Review

Box 2. Adhesion complex proteomics: current limitations and future perspectives The application of proteomic approaches to current biological methodologies is subject to limitations including those imposed by sample heterogeneity and the isolation of co-purifying proteins. The use of control isolations, or stable isotopes, is a widely used strategy to reduce irrelevant identifications for comparative subtractive proteomic approaches. Sample heterogeneity, however, does present some specific challenges to adhesion complex proteomics. For example, cells spread on ECM ligands exhibit numerous types of adhesion structures such as focal complexes, focal adhesions, and fibrillar adhesions that have been classified according to their size, subcellular distribution, and maturation state [27]. Therefore, proteomic characterisation of adhesion complexes from cells in culture results in the identification of proteins from heterogeneous adhesion complex structures. In some respects this issue has begun to be addressed through the use of the myosin inhibitor blebbistatin, as proteomic analysis of adhesions isolated under these conditions essentially identified components of less mature adhesion complexes such as focal complexes [20,21,23]. However this approach only partially resolves the issue and more elaborately designed studies will be required to resolve these questions. Another important question is how does adhesion complex composition in vivo differ from that of cultured cells? Adhesion complex proteomics could be used to address this question. While the enrichment of complexes containing adhesion components from smooth muscle has been described [103], the specific isolation or enrichment of adhesion complex structures from tissues has not yet been fully optimised, and even when these technological hurdles have been overcome, sample heterogeneity could still be an issue as it would be difficult to discriminate from which cell types or anatomical regions of the tissue the identified proteins originally resided. Despite these limitations, some immediate important adhesion biology questions that can be addressed still remain, such as: (i) are core components conserved in adhesion complexes from all ECM ligands and cell types?; (ii) what complement of post-translational modifications is located at adhesion sites? Future studies will no doubt help to address these issues, and to this end studies have begun to catalogue phosphorylation signalling downstream of integrin engagement [23,104]. Looking to the future, the use of imaging mass spectrometry [105,106] with increased spatial resolution may be usefully employed to overcome issues surrounding sample heterogeneity and at the same time may help to bridge proteomics with spatial findings at the nanoscale afforded by advanced imaging technologies.

cytosolic compartment due to restrictions upon protein interactions. Super-resolution microscopy techniques are now being applied to visualise the molecular architecture and dynamics of integrin-based adhesion complexes. Initial studies demonstrated the close proximity of integrins in adhesion structures to those within the endoplasmic reticulum [35]. Subsequent super-resolution investigations identified unappreciated dynamic fluctuations within adhesion complexes – revealing that mobility of individual b1 integrins differs from that of b3, and that integrin activation leads to immobilisation [36]. Generally, our view of adhesion complex organisation has been restricted spatially to two dimensions in conjunction with the temporal regulation of assembly, maturation, and disassembly [1]. However, analysis of the 3D spatial organisation of adhesion complexes using super-resolution microscopy demonstrated a highly organised stratification of adhesion complex architecture in the vertical axis [37]. These analyses illustrated that the integrin–actin connection was vertically separated by 40 nm and comprised a membrane-proximal signalling 4


stratum [focal adhesion kinase (FAK) and paxillin], a middle stratum of force-transduction elements (talin and vinculin), and a layer proximal to actin but distal to integrin comprising of actin regulators (zyxin, VASP, a-actinin). Interestingly, many of these components are commonly observed in proteomic datasets (see above) and this 3D view may represent an initial illustration of how core components of the adhesion complex connect to actin. Whether a core complex of adhesion components and molecular stratification is conserved to organise and regulate integrin-mediated adhesion across multiple cell types and tissues is still unknown and will be revealed by future studies. However, what does seem clear is that the observations from these sophisticated approaches closely match those that one might predict from the conceptual models developed over the past 20 years (based on classical biochemical and cell biological approaches). Primary functions of adhesion complexes The basic control mechanisms that modulate and are controlled by adhesion complexes can be broadly defined as either cytoskeletal- or trafficking-associated. These processes feed into nearly every function that is regulated by cell–matrix adhesion. Here we will not attempt to review all facets of integrin signalling, as they have been reviewed elsewhere [38–41]; rather we focus on emerging developments relating to cytoskeleton-mediated mechanosensation and receptor trafficking. Mechanotransduction and mechanosensation Mechanical forces, generated externally via the ECM or internally by actomyosin-based contractility, play important physiological roles acting at both the cellular and tissue level to direct a multitude of biological processes [42], ranging from control of cell cycle progression[43,44] to coupling of the cell migration machinery [41]. Extracellular and intracellular forces are transmitted, and sensed, bidirectionally via transmembrane molecules linking the external microenvironment to the cytoskeleton. Thus, integrins, and their associated core adhesion complexes, serve as mechanical conduits, across which forces can be applied directly [7,45]. Matrix stiffness and mechanosensation, through its downstream regulation of gene expression programs [46], co-ordinate normal developmental processes [42], including stem cell propagation and lineage specification [42,47,48] and contribute to cancer, fibrosis and diseases involving tissues exposed to mechanical force, such as the cardiovascular system [42,49,50]. For example, differentiation of mesenchymal stem cells is directed towards bone lineages by stiff environments whereas soft substrates promote differentiation to adipose phenotypes [48]. Matrix stiffness is thought to regulate cell fate through the shuttling of the transcriptional co-activators YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) between the cytoplasm and the nucleus [51,52], whereby stiff matrix promotes the nuclear localisation of YAP/TAZ, resulting in the transcriptional regulation of a number of cellular programmes [53]. In addition to YAP/TAZ, altered cell and tissue stiffness results in transformed transcriptional programming of a wide variety of other factors including


Review myocardin-related transcription factor (MTRF)/megakaryocyte acute leukemia protein (MAL), transforming growth factor-beta (TGFb)/SMAD, and nuclear factor-kappa-B (NFkB) [53,54]. Moreover, tissue mechanics modulate the expression of micro RNAs that impact upon malignant progression [55]. Upon ligand binding, integrins such as a5b1 form catch bonds whose conversion to a high-affinity state is stabilised by force [56] and whose bond strength ‘remembers’ force from previous applications [57]. This characteristic facilitates efficient and sustained mechanotransmission. Integrin-dependent catch bonds and the ability of adhesion complex proteins such as vinculin, talin, and p130Cas to transmit forces to actin, or other intracellular signalling moieties, via conformational modulation [58–61], place integrins and adhesion complexes in a central mechanosensing role. As the core adhesion complex machinery highlighted by recent proteomic and microscopy investigations (see above) comprises many actin-binding or -regulating proteins, it seems reasonable to propose that this core module performs key mechanoregulatory roles in the cell, providing a spatially and temporally controlled structural platform for signal propagation. Which of these proteins perform key mechanosensing roles, or whether specific adhesion complex components initiate specific pathways, is yet to be resolved. In this respect, LIM domain-containing proteins are promising candidates as they have been implicated in cytoskeletal mechanotransduction [28]. However, in order for bi-directional application of tension across the membrane to co-ordinate cellular function, mechanical signals need to be refined and/or propagated distally. It is likely that components of the adhesion complex environment (i.e., the broader adhesion-associated signalling machinery) may enable integration of membrane-proximal and membranedistal signals. It is also apparent that regulation of these processes via the cytoskeleton can occur in both directions with the cytoskeleton feeding back to regulate plasma membrane organisation and signalling [62]. In this respect, it is intriguing that membrane tension can drive ligand-independent integrin signalling via urokinase receptor (uPAR) [63] and that mechanotransduction can be augmented by the glycosaminoglycan matrix component hyaluronic acid, which is frequently upregulated in cancer [64]. Furthermore, the cancer glycocalyx, a layer of glycans and glyoproteins surrounding cells, can bypass the actomyosin machinery, applying tension to matrix-bound integrins and facilitating signalling to support cancer growth and survival [65]. Unravelling the specific role of adhesion complex components in these processes therefore has direct implications for disease pathogenesis. Receptor trafficking Intracellular trafficking is a dynamic process controlling adhesion receptor engagement and signalling spatially and temporally (Figure 2). While initially under-appreciated, a growing body of evidence suggests that receptor trafficking mechanisms play a critical role in adhesiondependent processes including GTPase signalling, migration, cell division, matrix remodelling, and invasion [66,67]. Proteomic analyses have demonstrated that the adhesome contains many proteins with functions associated


with receptor trafficking mechanisms [15]. For example, AP2 components, clathrin, disabled homologue 2 (DAB2), flotillin, Arf6, and Rab35 are identified in multiple adhesion complex proteomic studies. Other Rabs, Arfs and sorting nexins, are also represented in some of these datasets [19– 21]. Thus, receptor trafficking should now be considered a principal regulatory mechanism co-ordinating adhesion complex function. By constraining where and when adhesion receptors are engaged, vesicular trafficking mechanisms fine-tune the response of cells to their extracellular microenvironment. Precise regulation of receptor internalisation, sorting, recycling, and degradation enables spatiotemporal control of integrin–ECM engagement (Figure 2 and Box 3). As different integrin heterodimers exhibit distinct biomechanical and signalling properties, it is likely that these mechanisms spatially and temporally control adhesion dynamics and mechanotransduction. Importantly, these processes can be co-ordinated by other receptors, such as syndecans and growth factor receptors [66,68,69], suggesting that these molecules function as environmental sensors modulating integrin engagement to control cell behaviour in response to extracellular stimuli. Interestingly, matrix elasticity/rigidity has been shown to influence b1 integrin endocytosis [70], suggesting that the capacity of adhesion complexes to relay mechanical signals may directly impact adhesion receptor trafficking mechanisms. Given the range of intracellular signals and cellular functions regulated by adhesion complexes, it is likely that precisely co-ordinated trafficking mechanisms have evolved to constrain and fine-tune adhesion receptor engagement and signalling. Consistent with this, perturbation of integrin trafficking is a key driver of cancer progression [66]. We are slowly developing an understanding of the mechanisms, and relative contributions of intracellular and extracellular signals, that control receptor trafficking and adhesome function (Figure 2). However, to fully comprehend this complexity, it is likely that systems-level analyses, integrating advanced proteomic and imaging data with mathematical modelling, will be required to attain a global understanding of adhesion complex regulation. The ‘non-canonical adhesome’ and unexpected roles for adhesion complexes A striking feature of the proteomic characterisation of adhesion complex components was the notable increase in complexity and number of components identified [15]. Despite the variety of methodologies used, the use of stringent controls and the incorporation of rigorous acceptance criteria for identified proteins, these studies uniformly identified a conservative two- to three-fold more proteins than expected from surveying the literature [13,15]. Using objective informatic analyses, these putative adhesion complex components were linked to an extensive variety of biological functions and contained a wide array of protein domains, many of which are also found in wellcharacterised adhesion proteins, suggesting that they may have adhesion-related roles [15,16] (Figure 1). The relationship of these new non-canonical adhesome components to adhesion function requires further examination. However, as Geiger and Zaidel-Bar note, adhesion 5


Review


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Figure 2. Receptor trafficking and cell adhesion. Many adhesion complex components are involved in adhesion receptor trafficking. For example, the clathrin adaptor disabled homologue 2 (DAB2) interacts with the cytoplasmic tail of b-integrins and regulates adhesion disassembly during cell migration. In addition, protein kinase C alpha (PKCa) and Rab21 can bind to b-integrins and control endocytosis. Moreover, ontological interrogation of proteomic datasets reveals that many ‘non-canonical’ adhesome components are known regulators of receptor trafficking, although their role co-ordinating integrin engagement remains unclear. Following internalisation, integrins follow multiple trafficking routes (blue arrows) whereby they are recycled back to the cell surface or targeted for degradation in lysosomes. Upon fibronectin binding, integrins can be ubiquitinated and undergo ESCRT (endosomal sorting complexes required for transport)-dependent trafficking to the multivesicular body/late endosome prior to degradation [107,108]. Conversely, sorting nexin 17 (SNX17) can be recruited to the kindlin binding site of b-integrins and promotes integrin recycling back to the cell surface [109,110]. Co-receptors of integrins can also coordinate trafficking and therefore cell migration [68,69]. Src-mediated phosphorylation of syndecan-4 suppresses the activity of the adhesome component Arf6, leading to increased recycling of aVb3 integrin to the cell surface, supressed a5b1 integrin recycling, and stabilisation of adhesion complexes [68]. Dynamic regulation of integrin availability at the cell surface through receptor trafficking pathways is essential for microenvironmental sensing during cell migration and invasion, differentiation, and progression through the cell cycle. Adhesion receptors can also be co-trafficked with growth factor receptors, impacting signalling from both classes of receptor (for clarity, these pathways are not depicted here).

complexes are not membrane-bound organelles and their localisation and function in the cell is closely linked to other cellular processes and structures. It follows that proteomic analyses may have captured the true complexity of the ECM-adhesion nexus [15]. Consistent with the emerging complexity of adhesion complexes, and the identification of unexpected classes of molecules at sites of cell–matrix interaction, there is a growing body of evidence suggesting that adhesion complexes regulate a diverse range of previously unanticipated cellular functions. Interestingly, these processes are not necessarily regulated at the site of cell–matrix engagement. So, the capacity of non-canonical adhesome components to propagate long-range signals may play a critical role in the regulation of these processes (Figure 1). The non-canonical roles of adhesion complexes can broadly be categorised as: (i) new roles for classical adhesome components; (ii) adhesiondependent roles for novel non-canonical adhesome components; (iii) hypothetical roles of adhesion signalling based on known functions of adhesion regulatory molecules. Here we 6

identify three novel functions that fall within each of these categories and that are likely to be adhesion-dependent. Actin repair mechanisms Application of extracellular and intracellular mechanical strain leads to thinning of actin stress fibres at defined sites of extension. If strain sites are not repaired, stress fibres will rupture and retract. Zyxin is a mechanosensitive core adhesome component that is recruited from adhesion complexes to actin stress fibres upon force application [71]. Importantly, zyxin translocates specifically to stochastic or force-induced strain sites, in a LIM domaindependent manner, where it recruits a-actinin and VASP to repair and stabilise damaged fibres [72,73]. Thus, zyxin controls cellular repair mechanisms in a manner that is independent of its role in adhesion complexes. Paxillin, another LIM-domain-containing core adhesome component, can also be recruited to strain sites to initiate actin repair [72,74]. However, unlike zyxin, recruitment of paxillin to strain sites is not driven by cyclical stretch


Review Box 3. Adhesion receptor trafficking Integrins are constitutively internalised via clathrin-dependent and clathrin-independent endocytic mechanisms (including caveolindependent endocytosis and macropinocytosis) [86,111,112] (see Figure 2 in main text). Numerous trafficking regulators, adaptors, and scaffolding proteins regulate endocytosis of integrins, and many of these are detected at adhesion contacts [19–21,66,113– 117]. While mechanisms co-ordinating selection of different endocytic routes are not fully understood, they are likely to be dependent on cellular context, integrin heterodimer specificity, integrin activation, and microenvironmental cues. Following internalisation to early endosomes, integrins are sorted for recycling back to the membrane or for degradation; controlling localised ligand engagement and overall receptor availability (see Figure 2 in main text). The precise mechanisms targeting integrins differentially along these alternative pathways are not yet clear; however, recent studies suggest a critical role for sorting nexins. SNX17 interacts with b-subunit cytoplasmic domains to drive integrin heterodimers through recycling pathways, whereas inhibition of SNX17 binding redirects integrins towards lysosomal degradation routes [109,110]. Following endosomal sorting, integrins are classically thought to be recycled to the membrane directly from early endosomes (short-loop) or via the perinuclear recycling compartment (long-loop). However, it is now clear that this model of a binary selection of recycling routes, from two distinct subcellular compartments, is too simplistic. Recent studies suggest that many different GTPases and subcellular compartments contribute to adhesion receptor trafficking. For example, spatially constrained endocytosis and recycling of a5b1, via Rab25-positive endosomes, serves to restrict integrin recycling and engagement within invasive pseudopods to drive cellular invasion [118]. As with integrin endocytosis, selection of recycling routes is likely to be controlled by integrin heterodimer composition, activation status, extracellular stimuli (e.g., growth factors/cytokines) and accessory molecules (e.g., syndecans) [119–121]. Relative to integrin recycling, little is known about the mechanisms controlling integrin degradation. However, FN binding has been shown to induce a5b1 ubiquitination and drives ESCRT-dependent delivery of FN-a5b1 to multi-vesicular bodies for lysosomal degradation [107,108] (see Figure 2 in main text). It is not yet known whether similar mechanisms regulate degradation of all integrin heterodimers; however, potential ubiquitination sites have been identified in other a-subunits [122]. If it transpires that all integrin heterodimers are subject to ubiquitin-dependent degradation, it will be important to determine how different ubiquitin ligases and deubiquitinating enzymes are spatially and temporally co-ordinated to regulate heterodimer-specific degradation. Intriguingly, while lysosomal delivery might be considered the end of the road for integrins, this is not necessarily the case – expression of chloride intracellular channel protein 3 (CLIC3) can drive recycling of a5b1 integrin from late endosomes and lysosomes back to the cell–matrix interface [123].

[73,75], suggesting that specific regulatory mechanisms exist to direct recruitment of individual adhesome components to repair actin stress sites. Given the key role that adhesion complexes play in mechanotransduction, it is tempting to speculate that adhesion signalling might coordinate such regulatory mechanisms. LIM domains play a critical role in the regulation of zyxin- and paxillin-dependent actin repair. Since LIM domain-containing proteins are highly represented in adhesion complex proteomic data sets, it will be important to determine whether other mechanoresponsive adhesome components also co-ordinate such cellular repair mechanisms [16,28]. Localised translation Proteomic analyses of adhesion complexes have identified a range of unexpected proteins involved in the global


control of cellular functions, including translation [15,19–21]. Protein translation was classically thought to involve production of proteins at the endoplasmic reticulum or in the cytosol and subsequent trafficking of those proteins to their functional sites. However, recent studies suggest that translational machinery can localise to a range of other subcellular structures. Spatial compartmentalisation of translation ensures that proteins are generated near their sites of activity and binding partners and is therefore energetically favourable [76,77]. Ribosomes, mRNA translation initiation factors, and heterogeneous nuclear ribonucleoproteins (hnRNPs) have all been detected at sites of cell–matrix engagement, and ribosomes and mRNA are dynamically recruited in response to mechanical force [77–79]. Moreover, localisation of b-actin mRNA at adhesion sites increases adhesion complex stability to control cell migration [80]. So localised translation of cytoskeletal proteins at adhesion sites has a direct impact on adhesive function and cell behaviour. Consistent with this, paxillin binds to the mRNA binding protein, PABP1 (poly(A)-binding protein-1). Paxillin promotes nucleocytoplasmic PABP1 shuttling and redistribution to sites of cellular protrusion to regulate adhesion dynamics and cell migration [81,82]. The energetically favourable nature of localised translation is a particular advantage in neurons, where the length of axons precludes the efficient generation of proteins in the cell body and trafficking to the growth cone in response to dynamic extracellular stimuli. Indeed, in neurons, the non-receptor tyrosine kinase and core adhesome component Src phosphorylates the mRNA binding protein ZBP1 (Z-DNA-binding protein 1) to localise b-actin translation spatially and control neurite outgrowth [83]. In addition, it has recently been shown that b-actin mRNA and ribosomes adopt a masked and inactive conformation in neurons, which can be activated by extracellular chemical stimulation [84]. However, the extent to which adhesion signalling contributes to the unmasking of adhesionrelated mRNA is currently unknown. Thus, there is growing evidence that adhesion signalling modulates local translation, and that adhesion complex-associated translational machinery regulates cell adhesion and migration. While there is a long way to go before we fully understand the nature of these relationships, and there is a danger that many of the identified RNA-binding proteins are contaminants [85], these data provide a compelling indication that adhesion and translation are fundamentally linked. Consequently, it will be fascinating to determine the extent to which adhesion signalling influences, and is controlled by, the other regulators of global control of cellular function identified by adhesion complex proteomics. Membrane stress responses and lipid order Membrane mobilisation and homeostasis play key roles in regulating normal cellular function and the ability of cells to respond rapidly to extracellular stresses. While adhesion signalling drives receptor trafficking, until now, little consideration has been given to the role that it plays in directing trafficking, or the lipid order, of the plasma membrane itself. Despite this, accumulating evidence 7


Review suggests that several molecules that regulate membrane functions are modulated by adhesion signalling. The plasma membrane is a highly compartmentalised subcellular structure ordered by lipid–lipid, lipid–protein, and protein–protein interactions. Lipid rafts and caveolae are highly ordered cholesterol-enriched microdomains that spatially constrain signalling and trafficking events [86]. When cells are subjected to mechanical or hypoosmotic stress, caveolae rapidly disassemble to buffer membrane tension via a mechanism involving disassociation of caveolin from cavin-1 [87]. In addition, the small GTPase and adhesome component, Arf6, regulates recycling of internalised lipid rafts to the cell–ECM interface to control adhesion-dependent Rac1 activity [88]. Given these roles, it is interesting that integrin engagement constrains caveolin phosphorylation and restricts caveolin-dependent internalisation of membrane microdomains [89,90] and syndecan-4 engagement independently regulates both Arf6 activity and caveolin-dependent adhesion disassembly [68,69] (Figure 2). Thus it is likely that mechanoresponsive adhesion receptor signalling might directly regulate membrane stress responses and exert exquisite control over the lipid order of the plasma membrane and compartmentalisation of signal initiation. Consistent with these proposed roles, it has been reported that plasma membrane glycosphingolipids directly bind integrins and modulate integrin-ligand binding capacity [91–94]. By controlling plasma membrane trafficking and composition, it is conceivable that adhesion signalling can regulate membrane integrity, co-ordinate cellular responses to extracellular mechanical insults, and control the distribution and function of signal modulators. Concluding remarks Adhesion complexes functionally integrate the extracellular microenvironment with the inside of the cell. The signalling machinery associated with adhesion complexes regulates a diverse range of cellular functions that influence nearly all biological processes. Recent technological advances have provided insight into adhesion signalling and function. These studies highlight the fundamental complexity, structure, and dynamics of adhesion complexes, and consequently raise more questions than they answer (Box 4).


By re-appraising these data, certain key properties of adhesion complexes emerge. It would appear that adhesive structures comprise a conserved core adhesion complex that links integrins to actin, and a more variable and labile non-canonical adhesome, that is recruited to sites of adhesion in a context-dependent manner. The core adhesion complex has a well-organised stratified structure. However, components within the complex (including integrins themselves) undergo dynamic recruitment, translocation, and exchange even within established complexes. Thus, the core adhesion module can be viewed as meta-stable, undergoing continuous turnover to allow it to be simultaneously robust and dynamic. It is likely that these qualities are essential for the mechanoresponsive properties of adhesion complexes and that adhesion receptor trafficking mechanisms contribute directly to the dynamics and reinforcement of the core adhesion complex. Given the bidirectional interplay between mechanotransduction and receptor trafficking, it could be argued that mechanosensitive adhesion complexes represent a platform to drive adhesion receptor trafficking. Proteomic studies of adhesion signalling differ from the literature-curated adhesome as they offer cellular and environmental context. Despite this, the degree of variation between the datasets is surprising. Given this variability, these studies illuminate what we term the noncanonical adhesome. This ill-defined and labile protein network associated with adhesion complexes is contextdependent (e.g., modulated by cell type, environment, or synergy with other receptors) and introduces an additional level of complexity to adhesive function. Thus, one would speculate that some changes in the adhesion-associated environment might fine-tune adhesion signalling, while others might elicit highly divergent functions. Therefore we hypothesise that adhesion signalling is an emergent property of the adhesion complex as a whole. When we consider the potential functions of the non-canonical adhesome, there are numerous unexpected, sometimes controversial, roles for adhesion complexes and their regulators, and it is clear that their sphere of influence extends far beyond adhesion sites. So the major outstanding question for the field now is: to what extent, and precisely how, do adhesion complexes regulate global cellular functions?

References Box 4. Outstanding questions Are core adhesion complex components and the observed spatial molecular stratification conserved across multiple cell types and tissue systems? What complement of post-translational modifications are observed at adhesion sites, and how does adhesion complex composition in vivo or in 3D environments differ from those observed for cultured cells on 2D and stiff substrates? What roles, if any, do the new ‘non-canonical’ adhesome components play in adhesion function? To this end, can a continually-evolving meta-analysis of proteomic datasets assess at a global level the conserved and context-specific roles of adhesion complex components? From a systems perspective, how do signals relay across the adhesion complex? How robust are these signalling relay mechanisms to change? And how do adhesion complexes regulate ‘global cellular functions’? 8

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