Review
Adhesion dynamics: Mechanisms and measurements

https://doi.org/10.1016/j.biocel.2008.04.008Get rights and content

Abstract

Adhesion to the extracellular matrix (ECM) is a fundamental requirement for survival, differentiation and migration of numerous cell types during both embryonic development and adult homeostasis. Different types of adhesion structures have been classified in different cell types or tissue environments. The best studied of these are focal adhesions which are found on a wide variety of cell types and will be the main focus of this review. Many years of research into the control of adhesion has yielded a wealth of information regarding the complexity of protein composition of these critical points of cell:ECM contact. Moreover, it has emerged that adhesions are not only highly ordered, but also dynamic structures under tight spatial control at the subcellular level to enable localised responses to extracellular cues. However, it is only in the last decade that the relative dynamics of these adhesion proteins have been closely studied. Here we provide an overview of the imaging strategies that have been developed and implemented to study the intricacies and hierarchy of protein turnover within focal adhesions. The caveats of employing these imaging techniques, as well as future directions will also be discussed.

Introduction

Cell adhesion to the extracellular matrix (ECM) is a fundamental requirement for normal embryonic development, adult homeostasis and immune function (for reviews see Reddig and Juliano, 2005, Wozniak et al., 2004). The cellular structures that mediate interactions with the ECM can take a number of different forms depending upon both the cell type and the tissue environment. The protein composition, localisation and proteolytic capabilities of these so-called adhesion complexes all contribute to the classification and function of the structure. One of the most commonly studied are focal adhesions that are present in a number of cell types typically of mesenchymal origin. Podosomes and invadopodia are also adhesion structures typically associated with sites of proteolytic degradation of ECM. Podosomes form spontaneously in cells of hematopoietic origin or in a number of other cell types upon stimulation with exogenous factors, whereas invadopodia are generally thought to be characteristic of tumour cells (for review see Linder, 2007). Hemidesmosomes are epithelial specific adhesion complexes and are required for epithelial cell attachment to stroma as well as control of cell differentiation and migration (for review see Litjens et al., 2006). Focal adhesions will be the main focus of this article, however many of the signalling components and technologies discussed here are also relevant to other types of adhesions.

More than 30 years of research has yielded a wealth of information regarding the various proteins and lipids that contribute to the formation of functional focal adhesion complexes. However, until recently, little was known about the dynamics controlling the hierarchy of protein recruitment or removal within these specialised structures. Advances in both fluorescence tagging and imaging technology have now made it possible to address complex questions about the dynamic behaviour of proteins within adhesions.

To date approximately 150 different proteins have been identified as participating in the control of adhesion formation, stability and dynamics (for review see Zaidel-Bar et al., 2007). Functional adhesions control not only the local signalling environment but also convey essential mechanical stability to provide the cell with support for tractional movement. The heterodimeric transmembrane receptor family of integrins form the main direct cell:ECM contact point (Hynes, 2002). Integrins are unique in their ability as receptors to bind ECM via the extracellular domain as well as recruiting cytoplasmic signalling proteins to enable formation of stable links to the actin cytoskeleton (Hynes, 2002). As such, integrins are an integral part of any stable adhesion complex and specificity of individual integrins for different ECM ligands provides a mechanism by which cells can adapt signalling responses to changes in ECM composition. Subsequent recruitment of actin-binding proteins to the cytoplasmic face of integrins provides both the scaffold and signalling platform from which a mature adhesion develops (Fig. 1). Talin, α-actinin and filamin are all examples of such linker proteins (Calderwood et al., 1999, Calderwood et al., 2001, Critchley et al., 1999). Each confer their own signalling properties and in turn lead to recruitment of key kinases and adaptor proteins such as focal adhesion kinase (FAK) and Src, which form the basis of the adhesion signalling cascade (Zaidel-Bar et al., 2004, Zamir and Geiger, 2001).

In addition to integrins, other types of transmembrane receptors have also been characterised as important regulators of signalling at focal adhesions. Syndecans are one such family of proteoglycans, which can bind directly to ECM and growth factors extracellularly as well as act in synergy with integrins in the recruitment of cytoplasmic signalling proteins to focal adhesion sites (for review see Morgan et al., 2007). Recent studies have demonstrated a specific role for syndecan-4 in the control of localised protein kinase C (PKC) recruitment to adhesion sites and subsequent downstream control of the small GTPase Rac in co-operation with α5β1 integrin (Bass et al., 2007, Lim et al., 2003, Mostafavi-Pour et al., 2003).

Observations from very early studies on adhesion structures in fixed cells identified the presence of different types of adhesions within a single cell at any one time (Izzard and Lochner, 1980). On two-dimensional substratum in vitro, focal contacts are distributed across the ventral surface of the cell in an asymmetric fashion and are generally associated with or related to morphologically distinct actin structures. Based on subsequent detailed analyses, largely of fibroblast and epithelial cells, these adhesions have been classified into three main groups (Fig. 1): focal complexes (FC), focal adhesions (FA) and fibrillar adhesions (FB) (Geiger et al., 2001). Focal complexes are small, transient structures, which are usually seen immediately behind the leading edge of spreading or migrating cells. These adhesions can support nascent filopodial growth and lamellipodial actin networks and are considered to be involved in the ‘sampling’ of the ECM environment prior to formation of more stable contacts. Focal adhesions are larger, more mature structures, which are in some cases formed from the maturation of FCs and are located across the base of an adherent cell. FAs contain multiple signalling and actin-binding proteins responsible for providing mechanical stability and enabling tractional forces to be transmitted from cell to ECM and vice versa. Fibrillar adhesions have been described in three-dimensional matrix systems or in cells plated on 2D complex ECM and are thought to be derived from a subset of FA (Cukierman et al., 2001, Cukierman et al., 2002). These are long, highly stable complexes that run parallel to bundles of fibronectin in vivo and as such are highly enriched in tensin and active α5β1 integrin and indeed are sites of localised matrix deposition and fibronectin fibrillogenesis beneath the cell (Pankov et al., 2000, Zamir et al., 2000).

Recent detailed analyses of the distribution of well-characterised adhesion proteins have revealed differences in concentration and post-translational modifications of these proteins between different adhesion types. For example, the more transient FC do not contain zyxin (Zaidel-Bar et al., 2004) whereas highly stable FB do not contain β3 integrin or phosphorylated (active) FAK (Zaidel-Bar et al., 2004). The functional relevance of these differences in molecular composition has yet to be determined but it is likely that distinct populations of proteins will convey distinct mechanical properties to each adhesion. Future studies on the dynamics of multiple proteins within individual cells will be required to provide clues as to how relative levels of the same groups of proteins can locally dictate adhesion formation and type.

Although each type of adhesion has a different molecular composition and lifespan, a common series of events has to occur before each can be formed. This starts with the engagement of an integrin receptor with the ECM. Upon binding, the integrin will begin the process of activation; a conformational change that allows the receptor to recruit proteins to its cytoplasmic face. For activation to be completed, the cytoskeletal protein talin is required to bind the β-subunit of the integrin, assisting the receptor in its conformational change (Wegener et al., 2007). Once the conformational change has occurred, activated receptors cluster, thus providing a platform for future adhesion formation. Early studies of adhesions in fixed cells revealed the wide variety of proteins recruited to integrin clusters (Miyamoto et al., 1995a, Miyamoto et al., 1995b). However, only recent studies of protein kinetics in live cells have begun to reveal the complex hierarchy of protein recruitment and release within adhesions. This molecular switching is largely dependent upon protein–protein interactions and conformational changes but also upon the local environment within the cell. This is particularly relevant to adhesions within polarised migratory cells whereby complexes forming at the front of the cell exhibit different kinetics and reduced stability compared to those at the rear (Broussard et al., 2007, Ridley et al., 2003, Schwartz and Horwitz, 2006). The ability to spatially dictate the behaviour of adhesions during migration is vital for determining both polarity and speed of motility.

Formation of adhesions is dependent on the conformation, binding motifs and signalling domains contained within adhesion proteins. Not all constituents of an adhesion are able to bind the integrin directly and so a hierarchical ‘chain’ is established whereby proteins such as talin, paxillin and filamin, can act as linkers that bind the integrin and subsequently recruit other proteins. Talin binds to vinculin, another actin-binding protein, through various motifs in the unfolded protein (Brakebusch and Fassler, 2003). This association leads to recruitment of α-actinin to adhesions and also to induction of vinculin binding to paxillin. In addition to integrins and vinculin, paxillin can also bind FAK, recruiting the kinase to adhesions and triggering autophosphorylation at the tyrosine 397 residue (Mitra and Schlaepfer, 2006). This not only activates FAK, but also creates a binding site for the SH2 domain of Src family kinases. The interaction with Src kinases further phosphorylates FAK at several other residues found throughout the molecule that allows it to achieve maximal kinase activity. The scaffold of phospho-proteins formed provides a base for talin, vinculin and filamin to bind F-actin, which then leads to the production of bundles of actin filaments known as stress fibres under the influence of RhoGTPases (Burridge and Wennerberg, 2004, Ridley et al., 2003). A number of studies have suggested a direct role for actin stress fibres in regulating activation of adhesion proteins through mediating increased mechanical forces (reviewed in Evans and Calderwood, 2007). These force-driven signalling changes act to further stabilise an adhesion once it is assembled.

Adhesion disassembly occurs at different rates across the base of a migrating cell. Focal contacts at the leading edge disassemble very rapidly to allow continuous protrusion, whereas focal adhesions under the cell body or at the rear are much slower. FAK has been shown to be a vital regulator of adhesion disassembly at the leading edge of motile fibroblasts through activation of p190RhoGAP resulting in decreased active Rho and increased active Rac (Schober et al., 2007). This suppresses myosin-dependent tension at the leading edge and promotes Rac-dependent adhesion instability. As well as FAK and Src, members of the PKC family of kinases have also been shown to be important binding partners in the regulation of signalling events at focal adhesions (Ivaska et al., 2003) and potentially during adhesion disassembly. PKCα has been shown to bind directly to the cytoplasmic domains of both integrins and syndecans where it can control the traffic of β1 integrin and subsequent signalling events to protrusion and migration (Bass et al., 2007, Lim et al., 2003, Ng et al., 1999, Parsons et al., 2002). PKCɛ has also been shown to be an important regulator of integrin endocytosis and traffic during motility (Ivaska et al., 2002, Ivaska et al., 2005). In addition, the protease Calpain has been shown to cleave talin at adhesion sites leading to more rapid disassembly rates through additional downstream control of paxillin, vinculin and zyxin (Franco et al., 2004).

Microtubules also play a key role in the co-ordination of adhesion disassembly through a number of proposed distinct mechanisms. A number of live imaging studies have revealed microtubule tips can repeatedly target adhesions both at the leading edge and rear of migrating cells (Kaverina et al., 1998, Kaverina et al., 1999, Krylyshkina et al., 2003). This targeting drives adhesion disassembly through a combination of local Arg mediated inhibition of Rho activation, calpain-mediated proteolysis of proteins at the contact site and FAK-dependent endocytosis (Bhatt et al., 2002, Ezratty et al., 2005, Peacock et al., 2007).

Although our knowledge of these structures is by no means complete, recent advances in imaging techniques have allowed us to generate a better understanding of focal adhesion composition and the dynamics of proteins within them. A number of the most commonly used techniques are discussed below.

Section snippets

Studying adhesion protein localisation and dynamics in living cells

Recent advances in the generation of spectral monomeric variants of green fluorescent protein (GFP) tag have made it possible to visualise multiple proteins expressed within a single cell at the same time (for reviews see Shaner et al., 2005, Shaner et al., 2007). This has resulted in the accelerated development of novel techniques to analyse protein distribution, dynamics and interactions in both cells and tissues. The application of such techniques in recent years to unravel the complexity of

Studies of protein interactions in adhesion

Adhesions are complex structures containing a large number of highly dynamic proteins and lipids, many of which require direct interaction with a network of molecules in order to be correctly localised (Zaidel-Bar et al., 2007). It is therefore important to measure both dynamics of protein recruitment and protein–protein interactions within these structures in order to provide a more comprehensive dissection of temporal and molecular control of adhesion dynamics. The most commonly used

Imaging adhesions in 3D and in vivo

Recent publications have highlighted differences in structures and potentially function of adhesions in cells plated within a complex three-dimensional environment as compared to two-dimensional standard tissue culture substratums (Cukierman et al., 2001, Cukierman et al., 2002). Increasing numbers of studies are now also employing some form of three-dimensional (3D) matrix to study adhesion formation in a more physiological system. Similarly, considerable efforts have recently been made to

Future directions and concluding remarks

The last decade has seen an explosion of new techniques to analyse protein function, dynamics, localisation and interactions at sites of cell adhesion. With both microscopy companies and biologists alike now investing considerable time and effort into developing new imaging systems, the next decade also looks set to bring new and exciting technology to the adhesion and migration field. The expansion of data and software sharing sites online are also providing invaluable resources to

Acknowledgements

The authors would like to thank Gareth Jones and Soren Prag for critical reading of the manuscript. DW is funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and MP is funded by a Royal Society University Research Fellowship.

References (118)

  • S.T. Lim et al.

    Direct binding of syndecan-4 cytoplasmic domain to the catalytic domain of protein kinase C alpha (PKC alpha) increases focal adhesion localization of PKC alpha

    J Biol Chem

    (2003)
  • S. Linder

    The matrix corroded: podosomes and invadopodia in extracellular matrix degradation

    Trends Cell Biol

    (2007)
  • J. Lippincott-Schwartz et al.

    Fluorescent proteins for photoactivation experiments

    Methods Cell Biol

    (2008)
  • S.H. Litjens et al.

    Current insights into the formation and breakdown of hemidesmosomes

    Trends Cell Biol

    (2006)
  • P.S. Maddox et al.

    Spinning disk confocal microscope system for rapid high-resolution, multimode, fluorescence speckle microscopy and green fluorescent protein imaging in living cells

    Methods Enzymol

    (2003)
  • S.K. Mitra et al.

    Integrin-regulated FAK-Src signaling in normal and cancer cells

    Curr Opin Cell Biol

    (2006)
  • T. Nakamura et al.

    Monitoring spatio-temporal regulation of Ras and Rho GTPase with GFP-based FRET probes

    Methods

    (2005)
  • D.W. Piston et al.

    Fluorescent protein FRET: the good, the bad and the ugly

    Trends Biochem Sci

    (2007)
  • A. Ponti et al.

    Computational analysis of F-actin turnover in cortical actin meshworks using fluorescent speckle microscopy

    Biophys J

    (2003)
  • M. Prasad et al.

    Cellular and molecular mechanisms of border cell migration analyzed using time-lapse live-cell imaging

    Dev Cell

    (2007)
  • J.P. Robinson

    Principles of confocal microscopy

    Methods Cell Biol

    (2001)
  • H. Schneckenburger

    Total internal reflection fluorescence microscopy: technical innovations and novel applications

    Curr Opin Biotechnol

    (2005)
  • M.A. Schwartz et al.

    Integrating adhesion, protrusion, and contraction during cell migration

    Cell

    (2006)
  • B.L. Sprague et al.

    FRAP analysis of binding: proper and fitting

    Trends Cell Biol

    (2005)
  • M.C. Adams et al.

    Signal analysis of total internal reflection fluorescent speckle microscopy (TIR-FSM) and wide-field epi-fluorescence FSM of the actin cytoskeleton and focal adhesions in living cells

    J Microsc

    (2004)
  • N. Anilkumar et al.

    Interaction of fascin and protein kinase Calpha: a novel intersection in cell adhesion and motility

    EMBO J

    (2003)
  • D. Axelrod

    Total internal reflection fluorescence microscopy in cell biology

    Traffic

    (2001)
  • M.D. Bass et al.

    Syndecan-4-dependent Rac1 regulation determines directional migration in response to the extracellular matrix

    J Cell Biol

    (2007)
  • I.R. Bates et al.

    Investigating membrane protein dynamics in living cells

    Biochem Cell Biol

    (2006)
  • E. Betzig et al.

    Imaging intracellular fluorescent proteins at nanometer resolution

    Science

    (2006)
  • A. Bhatt et al.

    Regulation of focal complex composition and disassembly by the calcium-dependent protease calpain

    J Cell Sci

    (2002)
  • A. Bianco et al.

    Two distinct modes of guidance signalling during collective migration of border cells

    Nature

    (2007)
  • C. Brakebusch et al.

    The integrin-actin connection, an eternal love affair

    EMBO J

    (2003)
  • J.A. Broussard et al.

    Asymmetric focal adhesion disassembly in motile cells

    Curr Opin Cell Biol

    (2007)
  • C.M. Brown

    Fluorescence microscopy—avoiding the pitfalls

    J Cell Sci

    (2007)
  • C.M. Brown et al.

    Probing the integrin-actin linkage using high-resolution protein velocity mapping

    J Cell Sci

    (2006)
  • D.A. Calderwood et al.

    Increased filamin binding to beta-integrin cytoplasmic domains inhibits cell migration

    Nat Cell Biol

    (2001)
  • P.J. Campagnola et al.

    Second-harmonic imaging microscopy of living cells

    J Biomed Opt

    (2001)
  • D.M. Chudakov et al.

    Photoswitchable cyan fluorescent protein for protein tracking

    Nat Biotechnol

    (2004)
  • C.G. Coates et al.

    Optimizing low-light microscopy with back-illuminated electron multiplying charge-coupled device: enhanced sensitivity, speed, and resolution

    J Biomed Opt

    (2004)
  • D.R. Critchley et al.

    Integrin-mediated cell adhesion: the cytoskeletal connection

    Biochem Soc Symp

    (1999)
  • E. Cukierman et al.

    Taking cell-matrix adhesions to the third dimension

    Science

    (2001)
  • E. Cukierman et al.

    Cell interactions with three-dimensional matrices

    Curr Opin Cell Biol

    (2002)
  • G. Danuser et al.

    Quantitative fluorescent speckle microscopy of cytoskeleton dynamics

    Annu Rev Biophys Biomol Struct

    (2006)
  • G.A. Dunn et al.

    Cell motility under the microscope: Vorsprung durch Technik

    Nat Rev Mol Cell Biol

    (2004)
  • A. Egner et al.

    Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment

    J Microsc

    (2002)
  • E.A. Evans et al.

    Forces and bond dynamics in cell adhesion

    Science

    (2007)
  • E.J. Ezratty et al.

    Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase

    Nat Cell Biol

    (2005)
  • T.J. Filler et al.

    Reflection contrast microscopy (RCM): a forgotten technique?

    J Pathol

    (2000)
  • S.J. Franco et al.

    Calpain-mediated proteolysis of talin regulates adhesion dynamics

    Nat Cell Biol

    (2004)
  • Cited by (0)

    View full text