Recent Advances and Prospects in the Research of Nascent Adhesions
Henning Stumpf, Andreja Ambriović-Ristov, Aleksandra Radenovic, Ana-Sunčana Smith
RRecent Advances and Prospects in theResearch of Nascent Adhesions
Henning Stumpf , Andreja Ambriović-Ristov , AleksandraRadenovic , and Ana-Sunčana Smith PULS Group, Institute for Theoretical Physics, Interdisciplinary Center for Nanostructured Films,Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Laboratory for Cell Biology and Signalling, Division of Molecular Biology, Ruđer Bošković Institute,Zagreb, Croatia Laboratory of Nanoscale Biology, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Group for Computational Life Sciences, Department of Physical Chemistry, Ruđer Bošković Institute,Zagreb, Croatia
July 28, 2020
Nascent adhesions are submicron transient structures promoting the earlyadhesion of cells to the extracellular matrix. Nascent adhesions typicallyconsist of several tens of integrins, and serve as platforms for the recruitmentand activation of proteins to build mature focal adhesions. They are alsoassociated with early stage signalling and the mechanoresponse. Despitetheir crucial role in sampling the local extracellular matrix, very little isknown about the mechanism of their formation. Consequently, there is astrong scientific activity focused on elucidating the physical and biochemicalfoundation of their development and function. Precisely the results of thiseffort will be summarized in this article.
Integrin-mediated adhesion of cells and the associated mechanosensing is of monumentalimportance for the physiology of nearly any cell type [1]. It often proceeds through thematuration of nascent adhesions (NAs), which are transient supramolecular assemblies, tofocal adhesions (FAs), connecting a cell to the extracellular matrix or another cell. NAstypically contain around 50 integrins [2], and show a high turnover rate, with lifetimesof a bit over a minute [3]. FAs, which arise upon the maturation of NAs by recruitment1 a r X i v : . [ q - b i o . S C ] J u l f numerous proteins to their cytoplasmic tails, form multimolecular integrin adhesioncomplexes. FAs are establishing the linkage between the extracellular matrix and theactin cytoskeleton [4]. However, another cytoskeletal element, microtubules, also play animportant role in adhesion and regulate the turnover of adhesion sites [5, 6].While the FAs have been studied extensively in the last decades [7–12], the smallercharacteristic size of ∼
100 nm diameter make NAs significantly more elusive [2]. Studiesof NAs require single molecule localization microscopy (SMLM) techniques to image belowthe diffraction limit of conventional light microscopy, and, furthermore, need to accountfor the short characteristic lifetime of few minutes [2]. The resulting scarcity of dataassociated with NAs makes the theoretical modeling very difficult.In our current understanding, the NA formation follows three major steps [13]. First,integrins activate, going from a state of low to high affinity through a conformationalchange. This can be induced by binding of activating proteins like talin or kindlin [2, 14–18], or by binding to a ligand [19]. In the second step, the integrins cluster into NAs,which show similar structures on substrates of different rigidities [2], and are not relianton myosin II (MII) activity [3, 20, 21]. Finally, these clusters are either disassembled, orthey mature into FAs and possibly further into fibrillar adhesions.Despite the efforts leading to our current understanding, the determinants of NAformation, turnover, or maturation, as well as their role in mechanosensing and signaling,are far from being fully resolved. However, the past two decades witnessed the emergenceof several novel optical imaging techniques, technological advances in protein engineeringand mass spectrometry analysis, as well as the expansion of theoretical modeling thatnow allow the investigation of protein organization of NAs at the nanoscale. Motivatedby these perspectives, we here attempted to recapture recent advances in the field, whileidentifying open questions which we believe will be addressed in future research.
As their name suggests, integrins are cell adhesion proteins that are integral for numerousphysiological functions. Examples include cell gene expression and differentiation, cellmigration during embryonic development, immune response, or wound healing. Theysense the mechanical properties of the cell environment and provide signals necessaryfor cell survival, proliferation, and migration [11, 12, 22–24]. Moreover, integrins are aprominent target for medication [25, 26].In humans, this broad range of functionalities is maintained by integrins [27] builtfrom α - and β -subunits. Several combinations of α and β subunits are possible, aseither is not necessarily limited to a single partnered subunit to form a heterodimer [28, 29].They structurally form a headpiece and two legs [30]. However, only integrins assembledas heterodimers in the endoplasmic reticulum are expressed on the cell surface, thecomposition of which cannot be reliably predicted by the mRNA expression levels [29] ofintegrin subunits. Integrin expression can be regulated by modulating their internalizationand recycling, which contributes to the dynamic remodeling of adhesion [31].2s integrins bind to more than one ligand, the 24 heterodimers are broadly catego-rized by their ligand specificity into (i) Arg-Gly-Asp receptors, binding to fibronectin,fibrinogen, and thrombospondin, (ii) laminin receptors, (iii) collagen receptors, andfinally (iv) leukocyte-specific receptors binding to different cell surface receptors suchas intercellular adhesion molecule and some extracellular matrix proteins [19, 32, 33].Additional ligands relevant in the immunological context are the intercellular adhesionmolecules, immunoglobulin superfamily members present on inflamed endothelium, andantigen-presenting cells. Besides binding to a wealth of ligands, the specificity for theseinteractions is promiscuous, as one integrin binds multiple ligands. Furthermore, it isalso extremely redundant, as different integrins bind to the same ligand [32]. Theseproperties of integrins are essential for the interaction with the extracellular matrix andthe consequent mechanotransduction. However, they make integrin research challengingat the cellular level.Integrin binding and clustering provokes the formation of multimolecular integrinadhesion complexes recruited to their cytoplasmic tails. The composition of integrin ad-hesion complexes, termed adhesome, have been analysed from cells seeded on fibronectin,using different methods [34–38]. This led to the definition of a fibronectin-induced metaadhesome composed of over 2400 proteins which was further reduced to 60 core proteins,termed consensus adhesome [22]. Since integrins have no actin binding sites, they rely ona range of so-called adaptor proteins which bind to their cytoplasmic tails of integrinsand bridge to the cytoskeleton. There are four potential axes that link integrins toactin, namely (i) integrin-linked kinase-particularly interesting new cysteine-histidinerich protein-1-kindlin, (ii) focal adhesion kinase (FAK)-paxillin, (iii) talin-vinculin and(iv) α -actinin-zyxin-vasodilator-stimulated phosphoprotein [4, 22, 24, 39]. The consensusadhesome also contains signalling molecules such as kinases, phosphatases, guanine nu-cleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and GTPases [39].Talin, a well-known activator discussed below, also coordinates the microtubule cy-toskeleton at adhesion sites through the interaction with KN motif and kidney ankyrinrepeat-containing proteins [5, 6, 40, 41], which was shown to stimulate FA turnover [42].A lot of detail regarding integrin structure and interactions with adaptor protein isobtained from molecular dynamics simulations, which starting with the seminal works onconformational changes in activation [43], addressed integrin unfolding [44], differences inintegrin transmembrane domains (TMDs) [45], talin-integrin interactions also regardingthe surrounding lipids [46], and interactions with other proteins [47].Integrin involvement in pathological conditions is mostly the consequence of changes inthe expression, either up- or down-regulation. Prominent examples here are tumorigenesisbut also the response to chemo- or radiotherapy [11]. Therefore, integrin repertoire changesare an active target for drug development in tumours with potential to inhibit metastasis,as well as to overcame resistance to chemotherapy or radiotherapy. However, despiteconvincing experimental evidence that demonstrates the capacity of integrin inhibitorsand monoclonal antibodies to contribute to inhibition of cancer progression, metastasis, orboost therapeutic effects, no integrin-targeting drugs have been registered as anti-cancerdrug [11, 25, 48–51]. Integrins are, nonetheless, used as targets in the prevention ofblood clots during the opening of blood vessels in the heart [52], multiple sclerosis [53]3nd CrohnâĂŹs disease [54, 55]. Furthermore, since the accumulation of disorganizedextracellular matrix is modulated by several integrin heterodimers via activation of latenttransforming growth factor- β , the selected integrins are considered as promising therapeu-tic targets for fibrosis [56]. Besides integrin up- or down-regulation, integrin mutationsare also associated with some diseases like junctional epidermolysis bullosa, caused bymutations in either integrin subunit of integrin α β forming hemidesmosomes or integrin α , which pairs with β , forming FAs [57, 58]. Furthermore, integrin related diseasesmay also be caused by impaired activation as observed on platelets and leukocytes [59].Integrins are also involved in bacterial [60] and viral infections, either in attachmentor internalisation [61], thus representing possible target molecules to combat infectiousdiseases.Most integrin-related research, nevertheless, involves studies of mature adhesions. Thephysiological role of NAs is thus typically discussed in the context of the physiology of thesesuperstructures. However, with the recently initiated debate that NAs may themselvesact as signaling platforms, new perspectives in targeting NA-associated processes emerges.However, harnessing these possibilities requires detailed knowledge of the sensory role ofNAs, their dynamic behavior, and their regulation, which are all still poorly understood. Activation is the first step in the formation of NAs and is associated both with an integrinaffinity change and the binding of integrins to extracellular ligands [62]. Activation as aterm is also used to signify the switch to the extended-open (EO) conformation, which iswith the bent-closed (BC) and the extended-closed states, one of three major integrinconformations [63]. The changes of conformation may be introduced by thermodynamicfluctuations [64], but the switch is often induced by the very association of integrins withligands, adaptor proteins or Mn . Generally, each conformation has a specific affinityfor ligands [65], although all three conformations may be specific to one or more ligands.Often though, the activated EO state is the one with the highest binding affinity [66].For example, prior to activation, the BC state is the most common conformation of α β in the K562 chronic myelogenous leukemia cell line, making up for around 99.76 % of the population [66]. Simultaneously, the extended-closed and EO states contributewith . and . , respectively. However, α β and α β in the EO state havea 4000–6000 fold and a 600–800 fold higher affinity for a ligand compared to the BCstate [67]. Notably, these affinities are measured for ligands in solution, where they donot induce integrin clustering [15].In the environment of the plasma membrane, however, these affinities may changeconsiderably due to the coupling to the membrane [68–71]. Namely, the membrane, by itselasticity and fluctuations can change significantly the affinity of a bond, and can induceswitches from low to high affinity states, even without changing the actual conformationof the proteins binding the ligands [72, 73]. This mechanism of regulation of affinity wasoriginally suggested by theoretical modeling [70], and was demonstrated for a variety ofmembrane associated ligand-receptor pairs [71, 74]. However, its relevance for integrinbinding remains to be shown explicitly, although it should be particularly relevant for the4ormation of NAs. Preliminary hints for the role of these mechanism come from mimeticliposome or bilayer model systems. In the absence of adaptor proteins, activation ofintegrins was here successfully achieved by Mn [75, 76]. The later was necessary toenable binding to Arg-Gly-Asp [77], which was however, very sensitive. Mn , however,may induce integrin conformations that can be different from a physiological ones [78],which may contribute to the low binding yield, but cannot account for the observedvariability of the data.In cellular systems, the activation may be induced by the binding to ligands [19, 79]but also by adaptor proteins [80, 81], for example, by talin [15, 16, 79]. Actually, alreadytalin head domain (THD) was found to be enough to activate integrins [82]. Furthermore,THD was found to synergize with the kindlin in integrin activation [80, 83, 84], which incombination may promote binding to multivalent ligands [14]. However, there seems to bea competition between talin and kindlin, as the overexpression of kindlin-1 and kindlin-2can both enhance and reduce integrin activation by THD, depending on the integrintype [85]. In other cases, kindlin overexpression showed only a small effect compared toTHD [14, 78, 81, 84, 86].Figure 1: Current picture of integrin activation and clustering. ( A ) Fluctuations betweenthe closed and open integrin activation state are stabilized by binding of intra-cellular talin, or extracellular ligands. Although kindlin also plays a role, talinshows the strongest effect in stabilizing the open state in early adhesions. ( B )Initial liganded integrins create a region with increased binding probability, bymembrane and glycocalyx deformation. Clustering is further amplified by mul-tivalent ligands, dimerizing adaptor proteins, established scaffolding structuresand TMD interactions.Binding of ligands allows for anchoring of integrins and the exertion of forces by theactomyosin network, but also by the fluctuations of the membrane (Figure 1A). Theconsequence is a dynamic change of the free energy of BC, extended-closed and EOstates. The importance of this effect was highlighted in the model of Li and Springer [87],who find a sigmoidal dependence of activation probability with respect to the applied5orce, where activation here signifies binding of both ligand and adaptor protein. Thisresulted in full activation of all integrins over few pN for a wide range of adaptor proteinconcentrations, permitting a quick response to mechanical stimuli. Comparable behaviourwas found in fibroblasts, which reinforce early integrin adhesions ≤ under load bybinding additional integrins [88].The response of integrins to force has to be considered also in the context of the catch-bond effect. Namely, unlike slip bonds, which subject to force show an increase in theunbinding rate [68], catch bonds are stablized by force [69]. So far, in the case of integrins,both behaviors were found for different force regimes, which lead to the introduction ofthe term catch-slip bond. The latter was observed for example for α β [89], α β [90],and α L β [91]. In the case of α β , catch bond formation seemed to involve the headpiece,but not integrin extension [89].Other distinct mechanisms that, similarly to catch bonds, strengthen integrin attach-ments in a force dependent manner are cyclic mechanical reinforcement [92], and theso called dynamic catch [93]. In cyclic mechanical reinforcement, an increase of bondlifetime occurs over several loading-unloading cycles. In dynamic catch, the force responseis regulated synergistically by the of binding of an additional co-receptor to form atrimolecular complex with the integrin and the common ligand. In mimetic, actin freesystems, cyclic application of force also resulted in bond-strengthening [77], which wasshown to emerge from a thermodynamic response of the ensemble. Application of apulling force [94] induced a regrouping of bonds from sparse configurations to clusters inwhich cooperative response is allowed (strengthening each bond in average), and a newthermodynamic state is established [77]. Interestingly, recent work showed that even inafter the links with the cytoskeleton are fully established, most integrins existed in thestate of near-mechanical equilibrium [95].This complex behavior of integrins is cast into two major activation models, the so-called inside-out, induced by cytoplasmic factors, and the outside-in, where the activationresults from binding to extracellular ligands [65]. However, in the formation of NAs, thetime-sequence of binding events is not yet fully established, and it is not clear to whatextend the cell relies on either method in order to form such complex structures as NAs. Clustering of integrins (Figure 1B), with and without the help of adaptor proteins andindependent of F-actin [15] and MII activity [3], builds the second step of NA formation.Understanding of this process is greatly facilitated by the emergence of super-resolutionmicroscopy techniques. The latter provide optical images with spatial resolutions belowthe diffraction limit of light of the order of ∼
100 nm [96]. Therefore, it should be possibleto resolve the dynamic nanoscale organization of NAs and the force transduction acrossindividual components within FAs. However, quantitative investigations of NAs, are still6acking. The main reason is that existing SMLM techniques require cluster analysis tools,which have been developed for relatively simple cases, such as membrane protein clusterswithout strong heterogeneity in size, shape, and density [97, 98]. Several studies haveaddressed this by designing novel approaches to investigate the inner architecture of NAsand FAs, such as one based on the expectation-maximization of a Gaussian mixtures [99].So far, however, various nanoscale distributions have been observed for integrins.Clusters as small as – integrins were reported using electron microscopy [100], whileclusters observed in SMLM range from tens to hundreds of molecules. Some of thefirst application of SR techniques yielded
100 nm large NAs, containing on average 50integrins [2]. This data is contrasted by a more recent work with improved expectation-maximization of a Gaussian mixtures method used on the photoactivated localizationmicroscopy (PALM) data, when it was determined that FAs cover areas between . and µ m . Using expectation-maximization of a Gaussian mixtures, localization uncertainties,an important and unavoidable aspect of any SMLM experiment, could be correctedshowing that the assemblies contained to localizations, and exhibited strongeccentricities [99]. Notably, most existing SMLM clustering methods ignored this effect,which can lead to substantial overestimation of the size of identified localization structures.While the dynamic behavior of NAs is still an open problem, it is nevertheless clearthat clusters allow for quick rebinding after bond failure [64, 70], and the control overmaturation or disassembly [101]. Furthermore, clusters could serve as platforms forrigidity sensing [102], however, it is still unclear which point in the process of NA assemblycorresponds to the onset of signaling.In the absence of detailed microscopy studies, even the necessary conditions for theformation of these meta-stable aggregates are unclear. Some studies report that integrinactivation is indispensable for clustering [15], promoting the nucleation of new struc-tures [16]. These results are contrasted by experimental findings that show both activeand inactive integrin nanoclusters in FAs [103], obtained using extended state specificantibodies that co-localized with talin, kindlin-2 and vinculin. The existence of inactiveclusters could suggest the affinity for ligands in the inactive states is sufficiently largeto promote nucleation of domains, although with smaller probability than in the activestate. Alternatively, one could conclude that ligand binding is not necessary for clustering,although it is possible that ligand bound states preceded cluster formation.Further scenarios suggest a link between integrin activation and clustering mediatedby lateral interactions between tails of TMD [100, 104, 105]. However, limited size ofNAs [2, 106] requires further regulation of such interactions. Moreover, the necessaryactivation energy between TMDs also seems too high to overcome without help [105]. Insimulations, fewer integrins cluster when the lateral integrin interactions are weak [107].In addition, the TMD could not drive the clustering in Mn activated integrins, withoutligands present [15].With mobile ligands, on the other hand, Mn activated integrins formed small adhesiondomains, which significantly increased in size if integrins themselves were maintaininglateral mobility prior to the establishment of bonds [77]. In this case the clustering ofbound integrins was mediated by the deformed membrane. The nature and magnitude ofthese forces could be clearly elucidated [108], and were proposed to play an important role7n the cluster nucleation and growth [70]. Given that these types of interactions are notprotein specific, they should also be seen in other binding systems. Indeed, correlationsin membrane dynamics and topography with cell spreading reported recently in severalstudies of cell adhesions [109–111], and systematically in reconstituted passive systemsbased on giant unilamellar vesicles [112], including those involving integrins [75–77].However, this mechanism remains to be directly confirmed for integrins in the cellularcontext.Figure 2: Different theoretical models of NA formation, or parts thereof. ( A ) TMDinteractions between neighbouring integrins as well as clustered ligands cancooperatively increase clustering further by guiding close-by integrins to freeligand binding sites [113]. ( B ) Membrane and glycocalyx deformations cancreate regions with enhanced binding probabilities, catching diffusing freeintegrins [71]. ( C ) In a rate equation model of the NA formation, the NA sizeis defined only by adaptor protein concentration, which are stabilized throughpresent integrins, by reducing their unbinding rates [114]. The honeycomb gridas well as the hexagonal adaptor proteins shape and size are for visualizationonly.Most of membrane-related mechanisms include implicitly the existence of the cellularglycocalyx [112, 115], which was indeed found to play an important role in integrinclustering [116]. The compression and consequent expulsion of glycocalyx, by the formationof the initial bond primes the surroundings for further interactions. Concomitantly themembrane deforms towards the ligand [108], creating a microenvironment in which theadditional bonds have a much higher likelihood to form [71, 74] (figure 2B). The tension onthe bond furthermore increases their lifetimes [89], in a synergistic fashion. These effectscan be further strengthened by membrane thermal [117] and active fluctuations [118],which adds to the portfolio of forces acting on NAs, the latter being regulators of adhesionformation [20, 87, 88].While the interplay between these many factors contributing to the NA formation inits early stages is not yet fully understood, there is a consensus that integrin activationincreases binding to anchored, clustered ligands. A strong increase in the number ofspreading cells was found for a basic pattern of 4 ligands at ∼
60 nm distance comparedto 3 ligands at the same density [119]. Similar effects are well captured by theoretical8orks. Simulations can also account for the interplay between different integrin types,as demonstrated on the example the competition between ligand binding and clusteringof β and β [107]. Similarly, the effect of closely spaced multivalent ligands was alsocaptured in simulations in which clusters of more than two integrins can form throughdimerization, if the interactions are weak [113], allowing transient dimer interactions withswitching partners. A similar result was confirmed by an agent-based model [120], wherelarge agglomerates of ligands provide largest integrin clusters.Besides ligands, a number of adaptor proteins have been involved in cluster forma-tion. The most prominent examples are kindlin [2, 14] and talin (particularly its headdomain) [2, 15, 16, 80], that have been already implicated in integrin activation [14, 121–123]. Their association to integrins then of course promotes binding to ligands andclustering. Furthermore, both kindlin [124, 125] and talin [126] have a capacity for dimer-ization. For example, talin rod, which, using its integrin binding site, can rescue clusteringin talin depleted cells [2]. However, the efficiency of talin rod fragments was found to beinferior to the full length talin [16]. This points to a possible synergy between dimerizationand binding the monovalent, and even more so multivalent ligands (figure 2A).These potentially complex stoichiometries are, however, a challenge for SMLM. Complexphoto-physics of interacting fluorophores can lead to over-counting of molecules at a givenlocation [127]. This complicates the accurate determination of protein stoichiometries fromthe data. It is, however, possible to estimate the number of labelled-proteins contained ina single cluster [128–131], but more accurate quantifications are needed and their accuracydemands further validation. A recently developed supervised machine-learning approachto cluster analysis can be an interesting candidate to cope effectively with NAs sampleheterogeneity [132]. It was successfully applied on data of the C-terminal Src kinase andthe adaptor PAG in primary human T cell immunological synapses [132], but was not yettested for talin-integrin complexation or even more generally on NA data.Talin rod has an additional property important for the formation of NAs, namely itpossesses a binding site for vinculin. Vinculin is a cytoskeletal protein with binding sites,besides talin, for actin, α -actinin, and lipids and it is usually associated with the forcetransmission. However, recently it was found that the talin rod domain is available tovinculin in a force-independent manner upon the release of talin autoinhibition [133, 134].This would suggest that vinculin could play a role in NAs even before the cytoskeletalforces are involved.The integration of vinculin could be facilitated by PI(4,5)P . This phospholipidregulates talin-integrin interactions at the level of the membrane. Its association with the β units opens a binding site for the integrin on the talin head, hence controlling the talinauto-inhibition. Furthermore, the PI(4,5)P interaction with the integrin creates a saltbridge toward the membrane that prevents the close interactions of the α and β subunits.Therefore, the integrin remains in an activated, clustering-competent state [15, 16, 133].Consistently with these findings, sequestering of PI(4,5)P diminishes the formation ofclusters [15].One more protein strongly investigated in the context of integrin-clustering is α -actinin [13]. It is microfilament protein necessary for the attachment of actin. Both positiveand negative effects were demonstrated for β and β integrins, respectively [47, 135],9hich could relate to an integrin crosstalk strategy [136]. However, its role in clusteringis still debated [121].This large number of molecular players involved through various interactions poses asignificant challenge for comprehending the formation of NAs. A promising approachthat can address this diversity is theoretical modeling based on rate equations [137–141].An early attempt focused purely on the talin-PIP2 interaction and is therefore limitedin scope [142]. However, more recently, emulating the formation of entire NAs has beenattempted [114]. In the latter case, a higher emphasis is set on the crosslinking function ofadaptor proteins (figure 2C). The model reproduces some features found in experiments,for example the limited area of NA, even at high integrin density. It also predictsunliganded clusters. Actually, the model defines NAs as plaques of adaptor proteins andnot as integrin clusters. Integrins stabilize the plaque but are not required, which allowsfor the possibility of preclustering adaptor proteins in the absence of integrins. Clustering of NAs into FAs or disassembly is the last step in the NA life time. Thefate of NAs depends on the cell type, protein composition, and mechanical propertiesof the substrate [8], as well as the attachment to the actin cytoskeleton and both MIIisoforms [143].Most NAs disassemble when the lamellipodium moves past them [3] following one ofseveral competing ways of NA disassembly, as discussed in the literature [144]. Particularlywell studied is the role of the non-receptor tyrosine kinase FAK that is known to regulateadhesion disassembly [145], possibly through talin proteolysis [146]. In addition, FAKmight be inhibited at the leading edge of the lamellipodium through interactions withArp2/3, a protein complex that is related to the actin branching [147]. Because theregulation of rearrangements of the actin cytoskeleton is crucial for filopodia extension [148]and lamellipodia formation [149], FAK is implicated in the spatial control of the advanceof the leading edge and the NA disassembly. This regulation is facilitated by the bindingof FAK to paxillin, which is also recruited by kindlin [18, 150, 151], to further control theadhesion turnover [152, 153]. Interestingly enough, vinculin, can impede the FAK-paxillininteraction [154], while FAK may play a role in recruiting talin to NA sites [146, 155],as well as Arp2/3 [146]. As FAK also plays an important role in signalling [147], it isdifficult to unravel the precise dynamical interactions in NAs.Another simple way to dissolve NAs is by offering soluble ligands [15]. The latter eitherexhibit a lateral pressure on the NA site or they compete for the integrin upon stochasticunbinding [156]. Since the 3D affinity of soluble ligands is always larger than the 2Daffinity of surface-confined ligands, the cluster becomes unstable.Alternative to disassembling is the sequential maturation of NAs into FAs, and insome cases, to centrally positioned fibrillar adhesions enriched in tensin and integrin α β [157]. The inhomogeneous structure of these assemblies were observed already overa decade ago using PALM for imaging FA proteins [158]. Just one year later, Shroffet al. [159] used dual-color PALM to determine the ultrastructural relationship betweendifferent pairs of FA proteins. The consensus today is that integrins bind via talin and10ther adaptor proteins with the actin cytoskeleton, allowing MII generated forces toact on the clusters [160]. Under force the bonds strengthen, and tilt in the directionof the retrograde actin flow [161]. The reinforcement of integrin assemblies is furtherpromoted by the recruitment of vinculin [162], the crosslinking by myosins [163], and theexposure of force-dependent cryptic binding sites [164–166] that allow for the attachmentof other adhesome proteins. Finally, through mature adhesions, force is propagated fromthe extracellular matrix to the actin cytoskeleton over the unfolding talin that permitsvinculin binding [167], resulting in a strong signalling cascade and mechanoresponse alongthe adhesome, that regulates a number of physiological processes in cells. As presented in the above discussion, many molecular players contributing to the formationof NAs have been identified, and their mutual interplay have been established, althoughfurther investigation of specific interactions is necessary. For example, very little isknown about the crosstalk between integrins of the same and different types, recentlyreported for early adhesions [88, 136, 168]. However, new research avenues in studyingmolecular interactions in NAs can emerge only from advances in the development of robustquantitative colocalization analysis [169]. This needs to be accompanied by progressin genome editing and novel protein labelling strategies that could enable quantitativesuper-resolution microscopy. So far, only very few colocalizations of active integrinswith talin and kindlin [103] and vinculin and talin [170] could be observed. This canbe either a technical issue, associated with protein expression, probe photophysics, andthe limited choice of labeling pairs and fluorophores. It could also point to unknownintegrin regulators [103], and hidden interactions that remain to be revealed. For thispurpose, molecular dynamics simulations will become increasingly important as theyprovide unmatched details in competing binding interactions [105]. With appropriatelevel of coarse-graining larger complexes and slower structural changes are becomingwithin reach of molecular dynamics simulations, which now can explicitly address integrinactivation and clustering.Probably, however, the most acute issue is the spatiotemporal evolution of NAs and therole of complex stoichiometries. Namely, the dynamics of NAs is subject of intense debateas the constitutive clusters could be either stationary or showing stochastic transientimmobilization [103]. This problem is very closely related to the sensory capacity ofNAs and the onset of signaling, that are equally ununderstood. Resolution of these openquestions requires new techniques that can deal with the fast molecular turnover withinNAs. However, this is still a significant challenge for the single molecule localizationmicroscopy [171] such as PALM [158], stochastic optical reconstruction microscopy [172]or super-resolution optical fluctuation imaging [173]). First promising insights into thedynamics of NAs, nevertheless were provided by the single particle tracking PALM [174],which revealed integrin cycling between free diffusion and immobilization, while transientinteraction with talins promoted integrin activation and immobilization [175]. Further11tudies of integrin dynamics will require techniques such that can operate at micro second-time scales with precision of molecules located few nanometers apart. An exampleof such a method is minimal photon fluxes nanoscopy [176], although other approachesare starting to appear and will need to be employed in the research of NAs.Another challenge in studies of NAs is the impact of force. Although not strictlyrelated to actomyosin activity, forces on integrin complexes arise due to the spacialconfinement of the molecular players and result in load-dependent competition for bindingpartners. Different sources of forces may play a role in NA formation, prior to theirmaturation into FAs. Specifically, the glycocalyx and the membrane are anticipated togenerate relatively strong tensions and direct stochastic forces on the individual integrinsand the clusters [87, 88, 116, 177]. The understanding of these effects relies on thedevelopment of force sensors [95], and techniques which combine the force applicationwith superresolution microscopy. Furthermore, given the intrinsically non-equilibrium andnoisy setting, theoretical support in formulating and validating the appropriate hypothesison the role of confinement is necessary.A particularly useful tool in the research of integrin adhesion so far have been functional-ized substrates [75, 119, 178, 179]. Manipulation with their stiffness, spatial coordinationand mobility of binders allowed to provide mechanical cues which could be exploited toresolve the response of different cell models. Based on this long standing success, it isexpected that patterned substrate will continue to play an important role in studies ofNAs. Especially interesting should be their combination with specifically designed cellmodels that express different types of integrins on the plasma membrane surface.Furthermore, these substrates could be very successfully combined with reconstitutedsystems. The latter serve as an ideal bridge between the biological complexity andtheoretical modeling. Reconstituted systems, typically based on giant lipid vesicles, wereinstrumental in elucidating the role of mechanical properties of binders, as the role of thereceptor and ligand density and mobility in the cell recognition process [112]. Furthermore,vesicle-substrate adhesion was successfully used to study the physical mechanisms thatregulate ligand-receptor binding, including the role of stochastic membrane deformations,fluctuations and composition, as well the steric repulsion role of the glycocalyx [177].However, the simplicity of these assemblies may not represent the appropriate biologicalcomplexity of NAs. To circumvent that issue, more recently droplet-stabilized giant unil-amellar vesicles were designed that can be sequentially loaded with talin and kindlin [180].These systems show great potential for the studies of NAs, and could be used to drivethe development and validation of theoretical models and simulations used to describethe growth process.At the current stage, most theoretical models that attempt to capture the formation ofNAs account for the molecular complexity of the system, but capture the bio-mechanicalcontext only implicitly, if at all. There is also another class of modes that is capable ofresolving the stochastic nature of NA formation and the forces acting on the bonds withrelatively high level of detail, but they are nearly void of molecular information. Futureefforts are likely to bring closer these two distinct families of approaches, with the aim ofproviding a more reliable foundation that is required to capture the development of NAsin a predictive manner. 12inally, it is a hope that the lessons learned in the studies of NAs may be useful inthe context of other integrin-based structures. For example, in hemidesmosomes, linkedto the internal keratin intermediate filament network, α β integrins mediate adhesionof epithelial cells to the underlying basement membrane [58]. In reticular adhesions,which serve to maintain the attachment of cells to the extracellular matrix during mitoticrounding and division, αVβ integrins, clathrin and endocytic adaptors also form adhesivecomplexes [181–185]. Currently, it is not known whether these different types of adhesionhave precursor structures analogous to NAs. However, it is highly likely that tools,methods and approaches developed in studies of NAs may prove to be useful in thesepotentially different settings.In closing, we strongly believe that a jointly advances in superresolution microscopy,the development of model systems and technology for manipulations of proteins, as wellas theoretical approaches is required to further propel our understanding in molecularmechanisms of integrin organization, stoichiometry and dynamics at the nanoscale. Thiswill not only allow us to rationalize the observed phenomena, but also gain importantconcepts and tools that can be used to resolve the physiological role of integrin basedstructures, but can be further applied beyond the NA research. Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial orfinancial relationships that could be construed as a potential conflict of interest.
Author Contributions
HS conceived and wrote the first draft of the manuscript under the supervision of ASS.All authors contributed to the writing of the final paper.
Funding
ASS and HS thank the joint German Science Foundation and the French National ResearchAgency project SM 289/8-1, AOBJ: 652939, and the Excellence Cluster: Engineering ofAdvanced Materials at FAU Erlangen for support. AAR thanks the Croatian ScienceFoundation (Grant IP-2019-04-1577 to AA-R). We also acknowledge support by the DFGResearch Training Group 1962, Dynamic Interactions at Biological Membranes.
Acknowledgments
AR acknowledges the support of the Max Planck-EPFL Center for Molecular Nanoscienceand Technology. 13 eferences [1] Jenny Z. Kechagia, Johanna Ivaska, and Pere Roca-Cusachs. Integrins as biome-chanical sensors of the microenvironment.
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