The International Journal of Biochemistry & Cell Biology
ReviewRegulation of substrate adhesion dynamics during cell motility
Introduction
The adhesion of a cell to a substrate is a necessary requirement for it to spread and crawl. Studies using the technique of interference reflection microscopy (IRM) [1] were the first to show that cells do not attach uniformly to a surface but at specialised foci, the largest of which have been termed focal contacts or focal adhesions [2], [3]. From the interference patterns in the IRM images it was estimated that the cell to substrate separation at focal adhesions lies in the range of 10–15 nm. The same studies [2] revealed the general immobility of focal adhesions relative to the substrate, consistent with an adhesive function. And the adhesive nature of these foci was confirmed in experiments whereby cells were mechanically sheared from the surface on which they were grown: after such treatments, focal adhesion sites were left behind, isolated and still attached to the substrate [4], [5].
It is now well established that adhesion foci are complex molecular assemblies that link the extracellular matrix, via transmembrane matrix receptors (integrins) to the actin cytoskeleton [6], [7]. And the identification of component proteins of focal adhesions, starting with vinculin [8] and now numbering over 50 [7] has resulted in alternative tools to visualise adhesion sites in living and fixed cells. In particular, the possibility to tag adhesion site proteins with fluorescent probes, including green fluorescent protein (GFP) has allowed the detection in living cells of adhesion complexes below the resolution offered by the IRM method [9], [10], [11]. It has also become apparent that focal adhesions are only one of a few classes of adhesion complexes observed in spreading and migrating cells. In discussing what is known about the genesis and turnover of adhesion sites during cell movement we will highlight alternative strategies of adhesion site dynamics adopted by selected cell types to move. We will then survey the current ideas about the role microtubules play in determining cell polarity, through their influence on adhesion site dynamics. And finally comment will be made on the purported pathways signaling adhesion site formation and turnover.
Section snippets
Adhesion foci and the actin cytoskeleton
In discussing the types of adhesion complexes we first note that they are all exclusively coupled to the actin cytoskeleton; and second, that the different types of adhesion complex can be conveniently classified according to the assemblies of actin filaments with which they are associated. This implicit interrelationship between adhesion site genesis and actin cytoskeleton assembly necessitates a brief description of the actin filament subcompartments generated in spreading and moving cells.
Rho GTPases and adhesion complexes
Experiments involving the manipulation of starved cell models have established the Rho family of small GTPases as central players in the regulatory pathways signaling the assembly of the actin cytoskeleton and adhesion formation (reviewed in [22], [23]). Of those studies dealing with adhesion, one or other Rho GTPase was injected into starved cells and the adhesion patterns analysed either after fixation [17], [18] or directly in living cells [19]. In the present context we may note that in
Alternative strategies of adhesion formation in motile cells
So what types of adhesion dynamics are shown by moving cells? The early, pioneering investigations on the dynamics of the molecular components of substrate adhesions in living cells were restricted to the more prominent focal adhesions [29]. In more recent years, the development of more sensitive cameras, as well as of new fluorescent analogues of adhesion components, has opened the way for renewed analysis of the origin and turnover of adhesion sites [11], [19], [30], [31], [32], [33], [34],
Tension, adhesion and retraction
Chrzanowska-Wodnicka and Burridge [58] have shown that the formation of focal adhesions is dependent on the development of tension in the actin filament cytoskeleton, through actin–myosin interactions (reviewed in [58]). Likewise, focal complexes rely on actomyosin tension for their formation and integrity [19] and, like focal adhesions exert traction on the substrate [36]. The dependence of focal adhesion development on tension has been elegantly illustrated by the mechanical manipulation of
Cross-talk of microtubules with adhesion foci
In fibroblasts, the depolymerisation of microtubules leads to the depolarisation of cell shape [65], an increase in the contractility of the cytoskeleton [66] and an amplification in the size of focal adhesions [58]. This response is paralleled by the activation of RhoA [67], [68]. Conversely, the repolymerisation of microtubules following the disassembly is associated with the activation of Rac1 [69]. A direct correlation therefore exists between microtubule polymerisation dynamics and the
Rho GTPases and microtubule engagement
As already indicated, RhoA, Rac1 and Cdc42 act in three signal-transduction pathways regulating the assembly of actin stress fibre bundles, lamellipodia and filopodia respectively. Rho GTPases signal to diverse effectors to initiate a downstream response. Each of these GTPases act as a molecular switch, cycling between an active GTP-bound, and an inactive GDP-bound, state. Guanosine nucleotide exchange factors (GEFs) facilitate the exchange of GDP for GTP, and GTPase-activating proteins (GAPs)
Src
Different lines of evidence suggest that Src kinase activity is involved in regulating focal adhesion turnover. The v-Src temperature-sensitive mutant (as well as c-Src in its active conformation [95]) translocates to focal adhesions at the permissive temperature [96] and the kinase activity of v-Src leads to eventual focal adhesion disassembly upon phosphorylation and degradation of FAK. FAK-containing focal adhesions also grow faster in Src−/− cells in comparison with wild type cells [97],
Delivery of components to the cell front
Alternative ideas of how microtubules may influence cell polarity have been discussed by Nabi [108]. These hinge in the main on the delivery of membrane via vesicle traffic along microtubules. We cannot rigorously exclude the possibility that structural components of adhesions are delivered to or removed from adhesion sites by microtubules. In this connection, paxillin was found to bind alpha- and gamma-tubulin, as well as to co-localize with microtubule organising centres in lymphocytes [109].
Concluding remarks
Much has still to be learned about adhesion site dynamics during motility. Not least is the problem of adhesion site composition, which is far from complete [7], as well as the temporal association of the component molecules with adhesion sites. Here we are only just beginning to scratch the surface [79], [35], [114], [115]. Other questions include the localisation of regulators and regulatory complexes, which can only properly be defined in living cells, requiring probes and instrumentation
Acknowledgements
We thank Drs. Klemens Rottner and Kurt Anderson for providing figures and Drs. Benny Geiger, Clare Waterman-Storer, Torsten Wittmann and Frederic Bard for permission to cite unpublished work. Studies forming the background of this review were supported by grants from the Austrian Science Foundation.
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