ReviewImaging of cell adhesion events in 3D matrix environments
Introduction
Cell adhesion to extracellular matrix (ECM) is an essential process in development and homeostasis, and plays a key role in physiological and pathological states. Cell adhesions work both as a molecular scaffold to physically connect the cell cytoskeleton and surrounding environment and as a signalling hub to translate extracellular signals to intracellular signalling cascades. Cell adhesions are essential in adherent cells to regulate efficient cell motility. Most of the information and studies accumulated to date on the biology of adhesion have been from experiments performed in two-dimensional (2D) surfaces. The study of adhesion structures in cells that usually interact with a three-dimensional (3D) environment in vivo is still in its infancy. Formation and regulation of these structures in environments where the cells are surrounded by complex matrix, as well as other cell types, is still a key question that remains to be elucidated. In following sections we focus on the most widely used cell-based experimental models for 3D matrix studies and recent imaging techniques to analyse them.
Efficient cell migration on 2D surfaces involves the initial formation of a protrusion at the cell front followed by formation of new adhesion sites to stabilise the protrusion. These adhesions can mature, and eventually support the tension generated by cell contractile machinery to push the cell body forward. At the same time older adhesions at the rear of the cell must be disassembled, so a tight and controlled regulation of these processes is needed in order to drive effective forward movement (Ridley et al., 2003). Adherent cells plated on an ECM-coated rigid surface initially form small, dot-like adhesive contacts called nascent adhesions formed by small clusters (<0.25 mm) of integrins, integrin activators, such as talin, and adaptor proteins, like paxillin (Choi et al., 2008). Integrins are a family of 24 αβ heterodimeric receptors in mammals with a different affinity for ECM components. They can be activated by intracellular factors, such as talin and kindlins, but they are also primary docking sites for matrix-dependent outside-in signalling that acts to recruit a myriad of proteins to control adhesion maturation, disassembly and cytoskeletal dynamics (Askari et al., 2009, Shattil et al., 2010). As the cell moves forward, nascent adhesions can undergo a maturation process to form focal complexes that are larger in size (∼0.5 μm), have longer lifetimes and depend upon non-muscle myosin II (NMII) for assembly (Choi et al., 2008). Increased tension induces the recruitment of mechano-sensory and signalling proteins such as vinculin and α-actinin, and the focal complexes can enlarge in size and show a more stable phenotype to become a focal adhesion (FA) (Choi et al., 2008). FAs display a more elongated morphology and wider size range (between 1 and 5 μm) and they progressively change their integrin composition from β3 in focal complexes to β1 integrins, which tend to localise more in the cell body. Proteins such as focal adhesion kinase (FAK), VASP and Src among others are recruited to mature adhesions and these molecules play a role in co-ordinating disassembly. In this process, as before, localised cell tension within substrate plays an essential role in maturation process (Fig. 1A and C) (Scales and Parsons, 2011, Vicente-Manzanares and Horwitz, 2011). Recent studies using super-resolution microscopy have provided new insight into the hierarchical structure of adhesions in cells on 2D surfaces, demonstrating the tight regulation of spatial localisation of focal adhesion proteins within the adhesive structure (Kanchanawong and Waterman, 2011) and the dependence of Rho-associated kinase and actomyosin contractile machinery-mediated tension for their maturation (Patla et al., 2010).
Podosomes and invadopodia are also adhesive structures but differ from FA by their ECM-degrading activity. They share with adhesion structures some of their core proteins, such as integrins, integrin activators and actin cytoskeleton connecting proteins, but they differ in their ultrastructural organisation, inner cytoskeletal and regulatory machinery organisation, and function (Albiges-Rizo et al., 2009). They usually have an actin core surrounded by an adhesive integrin ring formed by integrins and integrin-bound proteins, and they induce localised matrix degradation by recruitment and secretion of matrix metalloproteinases (MMP). Traditionally podosomes have been observed and studied primarily in cells of monocytic origin, whilst invadopodia are considered to be a feature of invasive cancer cells. It has recently been reported that podosomes are more labile structures, with a life-time in the range of minutes, whilst invadopodia can last for minutes and they can project further into the ECM (Baldassarre et al., 2006). Most of our knowledge regarding these structures is derived from fluorescently labelled gelatin degradation assays in 2D, and as such podosome organisation in 3D matrices is still not clear. Macrophages can form podosome-like structures either migrating in collagen and Matrigel 3D gels (Van Goethem et al., 2011), and cancer cells are able to form invadopodia-like structures in complex environments (Tolde et al., 2010). So although it is clear that many cells need to proteolytically degrade matrix in order to migrate through 3D matrices, the real nature of these ECM degrading-structure is still under debate (Li et al., 2010, Wolf and Friedl, 2009, Wolf et al., 2007).
As highlighted above, most of our knowledge about adhesive structures comes from studies performed on cells on rigid 2D surfaces, where the ECM proteins are located only one side of the cell. In vivo, cells are surrounded by ECM proteins and even if the key factors and processes mediating adhesion formation and turnover are probably similar, the difference in ECM structure leads to completely different cell behaviour and migratory strategies. There are two broad ECM assembly patterns in vivo in mammals. Basement membranes that are mainly composed of laminins and type IV collagen (Kalluri, 2003), are layered as a thin and flat surface with a dense network of ECM proteins, so epithelial and endothelial cells spread and remain anchored and polarised on this support. Other cells such as leucocytes or tumour cells can then migrate on top of this cell layer in a 2D fashion or undergo transmigration through it. The pores within the basement membrane are relatively small, and it is therefore believed that extracellular protease activity is required for cancer cell transmigration (Madsen and Sahai, 2010, Voisin et al., 2010). Interestingly, a recent report showed the presence of areas in endothelial basement membranes with lower protein concentration where leucocytes can migrate across without degradation due to their high deformability (Voisin et al., 2010). Once cells have passed the basement membrane they must navigate through stromal tissue. In the interstitial tissues, the main ECM protein is collagen type I which is predominantly arranged into fibrils with varying degrees of branching, pore size, stiffness and composition (Wolf et al., 2009). Cells can migrate through interstitial tissues collectively when they retain cell–cell junctions, or forming streams following the tracks present in the ECM (Friedl and Wolf, 2010, Ilina and Friedl, 2009). As a single cell, cells can adopt different strategies, adopting a rounded or more mesenchymal (elongated) phenotype. Cells with a rounded phenotype lack clear adhesion complexes and actin stress fibres, and their motility is usually independent of matrix degrading activity. These cells can adapt to the ECM topography and migrate either forming blebs in order to protrude through matrix pores or in an amoeboid way by protruding with an actin polymerisation-dependent pseudopod (Charras and Paluch, 2008). In both cases cell movement is provided by strong actomyosin contractility that propels the cell body (Lammermann and Sixt, 2009, Sanz-Moreno and Marshall, 2010). Cells migrating in a classically mesenchymal manner generally adopt an elongated morphology that is aligned with surrounding matrix fibres. The formation of strong adhesion sites within the matrix allows the generation of the contractility needed to induce the movement of the cell body, and their migration is generally dependent on their matrix-degrading capacity (Wolf et al., 2009). These general patterns are typically associated with certain cell types (leucocytes for rounded migration and fibroblasts for mesenchymal, among others) but have been shown to be interchangeable in response to ECM features, such as stiffness and pore size, matrix degrading capacity and cell signalling, especially in cancer cells (Sanz-Moreno et al., 2008, Sanz-Moreno and Marshall, 2009, Wolf et al., 2007, Zaman et al., 2006). This reflects the plasticity of this process and the complexities of interpreting data from different in vitro ECM models used to characterise adhesion and migration.
Adhesion complex visualisation in 3D environments is still at an early stage, and as such the composition, dynamics and regulation of these structures are still unclear. Adhesion structures in 3D have thus far been visualised in fibroblast and cancer cell lines in cell derived matrices (CDM), collagen gels and fibrin gels (Cukierman et al., 2001, Deakin and Turner, 2011, Friedl et al., 1998, Hakkinen et al., 2011, Kubow and Horwitz, 2011). However, it is clear that the factors determining their formation, regulation and stability still need to be clarified. Furthermore, recent studies have highlighted important technical issues regarding adhesion visualisation in cells in 3D (Fraley et al., 2010, Fraley et al., 2011, Kubow and Horwitz, 2011). Factors such as levels of ectopically expressed-fluorescently tagged focal adhesion proteins, matrix stiffness (higher when the cells are closer to the rigid substrate where the gel sits on), and the fibrillar structure and nature (e.g., native collagen vs. pepsin-treated) can have a significant effect on adhesion formation. Thus, the controversy raised by the Wirtz and Horwitz labs clearly demonstrated that imaging adhesions in 3D environments is highly dependent upon the model used, underlining the technical issues that must be considered when embarking on such studies. Moreover, in cells that undergo integrin-dependent migration through a 3D matrix, it would appear that adhesion structures are generally much smaller than on 2D surfaces, and detectable instead as punctate clusters of integrins and talin (Deakin and Turner, 2011). In certain conditions matrix characteristics (such as composition, compliance, stiffness and porosity), could provide enough support for the adhesion structure to induce a local increase in tension that allows recruitment of mechanosensitive proteins such as vinculin, and connection with the cytoskeleton (Harunaga and Yamada, 2011). This process, analogous to adhesion maturation in 2D would vary according to cell type matrix characteristics and features (Fig. 1A and B) (Doyle et al., 2009, Harunaga and Yamada, 2011).
The study of adhesion structures on cells embedded in ECM matrix is a challenging and still largely unexplored field. Recent advances in cell imaging techniques have provided very powerful tools to deep in this subject and many questions remain to be answered and/or clarified. In this context, the establishing robust models and conditions for the study of adhesion formation and dynamics in 3D is essential. Below is a summary of the most widely used models currently in use for the study of cell motility in 3D supports in vitro (also see Table 1 for summary).
Section snippets
Basement membrane-based gels
One of the most widely used models is based on reconstituted basement membrane-based gels (Matrigel™). Matrigel is composed of collagen type IV, laminin and proteoglycans and is produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (Kalluri, 2003). Matrigel forms a non-fibrous dense mesh-work gel to which, or within which, epithelial and mesenchymal cells can adhere, spread and migrate on 2D surfaces through engaging integrins α2β1, α3β1, α6β1 (Hakkinen et al., 2011, Huttenlocher and
Current strategies for imaging cells in 3D environments
Until relatively recently, the model systems described above to study cells in, or on, 3D scaffolds were generally used as functional end-point assays rather than a means to study detailed cell adhesion events in living cells. A significant barrier to this analysis has been the considerable technical challenges associated with detailed analysis of adhesions in cells in 3D vs. 2D. The very nature of 3D scaffolds makes the application of classical, simple biochemical assays to analyse cell
Future perspectives
With a range of techniques currently being developed and used successfully for studying cell matrix adhesions within an in vitro setting, the next steps are towards in vivo analysis of adhesion formation. The last decade has seen an upward trajectory of new techniques for the analysis of protein function, dynamics, localisation and interactions at sites of cell adhesion. Considerable efforts are being made to develop new microscopy platforms and the next decade looks set to also bring new and
Acknowledgements
Research in our lab is funded by the Royal Society (University Research Fellowship to MP), the Basque Government, Spain (Research Staff Training Fellowship to AJ) and the Medical Research Council (grant MR/J000641/1 to MP).
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