Structure and mechanics of integrin-based cell adhesion (2023)


Integrin-mediated cell adhesion plays a central role in tissue and organ formation and remodeling in multicellular organisms [1]. Integrins bind to extracellular matrix (ECM) proteins through their large ectodomain and attach to the cytoskeleton through their short cytoplasmic tails. These integrin-mediated connections on both sides of the plasma membrane are dynamically coupled; The cytoskeleton controls the affinity and avidity of the integrin ectodomain, thus modulating the ECM, and binding of the integrin to the ECM changes the shape and composition of the underlying cytoskeleton [2]. Forces generated by cytoskeletal contraction or ECM rigidity are exerted via integrins on the plasma membrane and contribute to conformational changes in these receptors and their anchor proteins, and to the nature of biochemical signaling. The composition and morphology of integrin-dependent adhesions (focal complexes/podosomes, focal adhesions, immunological synapses and fibrillar adhesions) vary with cell type, matrix and integrin and can change from one adhesion contact to another. In motile cells, high-affinity integrin-based focal complexes are formed in the bulging lamelepodia. The retraction of actin filaments by myosin motors exerts force on the initial ECM-integrin-cytoskeletal junctions, resulting in rapid assembly of anchor proteins that convert focal complexes into focal adhesions, with further contact developing large, stiffer matrices and thus directing cell movement [3 , 4]. Focal adhesions dissolve at the trailing edge, separating the ECM from the integrins, which are returned to the leading edge, creating new matrix interactions that allow cell progression. Focal adhesions can also turn into fibrillar adhesions, which alter the structure and stiffness of the ECM, thus contributing to tissue remodeling and repair.

In this communication, we investigate the structure of integrins in their non-geligand and geligand states, assess the conformational rearrangements associated with integrin activation, and describe the structures, conformations, and adhesion dynamics of key cytoskeletal anchoring proteins from which they transduce integrin signals from outside to inside. convey. . .

The 18 α-subunits and 8 β-subunits of integrins express 24 different receptors in mammals and are divided into two groups, one with an additional von Willebrand factor type A domain (vWFA) and the other without. (as αA or αI in integrins) in their α subunits. αA mediates the binding of divalent cations to extracellular ligands on integrins containing αA [5]. αA is a GTPase-like domain in which the catalytic apex site has been replaced by a conserved metal ion-dependent adhesion site (MIDAS) occupied by a divalent cation. αA naturally exists in two conformations: closed (low affinity) and open (high affinity) [6, 7]. The open form differs from the closed form by an inward movement of the n-terminal α1 helix, a rearrangement of the F strand/α7 loop (F/α7 loop), and a two-turn downward movement of the α7 - c- Helix terminals (reviewed in ref. [8]). These tertiary changes lead to rearrangements in the three surface loops that make up MIDAS, allowing MIDAS to be occupied by an acidic residue of an exogenous ligand that provides the sixth coordination site for the bound metal ion, replacing a water molecule. The closed and open states are in an equilibrium favoring the former with a ratio of ~10:1 [9]. Mutations that reshape the c-terminal α7 helix [10], favor its hydrophobic contacts with the central strand [11], or promote its downshift produce high or intermediate affinity states [12].

The crystal structure of the αA-deficient integrin ectodomain, αVβ3, was determined in the absence of ligand (in the presence of Ca).2+of manganese2+) and ligand states [13, 14, 15] (Figure 1a and b). αV consists of four domains: an N-terminal seven-lobed β-helix, an Ig-like thigh domain, and two large β-sandwich domains, Calf-1 and Calf-2. The β subunit has eight domains: an N-terminal cysteine-rich plexin-semaphore integrin (PSI) domain, with an Ig-like "hybrid" domain inserted in its C-terminal loop (the hybrid domain itself contains an αA-like the domain {the βA domain} inserted between the two β sheets). PSI is followed by four EGF-like domains and a novel membrane proximal tail domain (βTD). The helical and βA domains form a "head" structure, explaining the formation of the αβ heterodimer. Glycosylation sites in the helix may also contribute to the stability of the heterodimer [16]. The integrin head sits on top of the α-chain and β-chain "legs" formed by the αV subunit femoral and calf domains and the integrin PSI, hybrid, EGF1-4 and βTD domains, respectively. β3. subunit. An unexpected feature of the integrin structure is that the legs are bent at both "knees" (between the hybrid and Calf-1 domains in aV and between EGF1 and EGF2 in β3). The folded conformation is mainly stabilized by multiple contacts between the upper (PSI-hybrid-βA, EGF1) and lower (EGF2-4, βTD) leg domains of the β3 subunit. There are also multiple contacts of a mixed nature between the largely parallel lower leg domains of αV (calf domains) and β3 (EGF2-4, βTD).

The three-dimensional (3D) structural features of the non-ligand βA domain largely overlap with those of the non-ligand αA domain, but there are two small insertions between the BC and DD' strands, each responsible for their ligand-binding specificity . (via a specificity determination loop, SDL) and for their association (via a 3rd10helix) with the helical domain (Figure 2a). In some integrins, SDL also contributes to the formation of heterodimers. The βA MIDAS is released by a metal ion. Adjacent to MIDAS is an ADMIDAS staffed by a preferred Ca2+, which binds the tip of the activation-sensitive n-terminal α1 helix and the c-terminal F/α7 loop, stabilizing the ligand-free state. Five metal ions bind to the αV subunit, one in each of the four hairpin loops at the base of the helix and a fifth within Calf-1 very close to the α genus. The βA domain of Pactulous, a neutrophil-specific, nonmetal-binding, single-chain β2 subunit-like protein of unknown function, lacks SDL and critical residues in all three10Helix, MIDAS and ADMIDAS, explaining the inability of this βA domain to heterodimerize with integrin α subunits or to bind integrin ligands [17].

The structure of the linked αVβ3 ectodomain was determined after diffusion of the high affinity cyclic pentapeptide cilengitide containing the prototypical Arg-Gly-Asp (cRGD) sequence into preformed integrin crystals in the presence of 5 mM Mn.2+. RGD inserts into a gap between the helical and βA domains, with the R and D side chains in exclusive contact with the helical and βA domains, respectively, bringing these two domains closer together [14] . The D-ligand contacts a metal ion-studded MIDAS in a manner strikingly similar to that previously seen in αA.

The activation of the inward movement of the α1 helix and the rearrangement of the F/α7 loop observed in ligated αA are also observed in ligated βA, resulting in similar, though much less dramatic, changes in the three surface loops coordinated by MIDAS , as if as well as minor changes to the SDL loop. . The metal coordination in ADMIDAS changes significantly so that the α1 helix to the F/α7 loop is no longer blocked, allowing activation movements in both. The rearrangement of the F/α7 loop is accompanied by a loss of contact between an invariant Asn339 at the apex of the α7 helix and the Ser334 residue of the F/α7 loop, mitigating the activating effect of an Asn339Ala mutation. on integrins could explain. β2 and β3. 18]. The inward movement of α1 carries ADMIDAS with it, bringing it closer to MIDAS and helping to stabilize the new metal ion in MIDAS. A third MIDAS ion stabilizer site, LIMBS (liassociated with grandMetroet alBVerbotenSite) are 6 Å apart in the linked structure. The two-helix downward movement of the α7-helix observed in ligated αA is not present in the ligated βA fold. However, in the RGD-linked αIIbβ3 main crystal structure from which the bone domains (thigh/calf 1-2 and EGF1-4/βTD domains) were amputated [19], a down-movement of one helix of the α7-helix was observed, coupled to a large outward displacement of the hybrid domain relative to βA, separating the integrin knees by ~70 Å (Fig. 2a).

Rearrangement of the F/α7 loop with mutation-inserted disulfides designed to link the M335 residue of the F/α7 loop to the V332 F chain residue, or deletion of an α7 loop, regardless of the position of the deletion for high affinity integrins. 20, 21]. The choice of M335 and V332 was based on a modeled high-affinity βA conformation in which the predicted Ca distances between these two residues are ~3.4 Å, compared to ~10 Å in the unliganded and ligated folded structures. However, this distance between Cα remained at ∼10 Å (actually it increased to a Cɛ-CG2 distance of 13 Å) in the high-affinity boneless αIIbβ3 structure [19], suggesting that the generated high-affinity state is caused caused by unknown tertiary changes. In the absence of direct structural data, conclusions about the influence of artificial disulphides on protein conformation should be drawn with caution. It is also noteworthy that the tertiary switch generated by this artificial disulfide did not express true stretch-responsive epitopes on the respective full-length integrin; Minnesota2+More ligands were needed [20]. Similar results were found for αLβ2 when αA was stabilized in the high affinity state [22].

The interpretation of the above structural and biochemical findings remains the subject of lively debate [8]. By analogy with the high affinity αA state, a switchblade model proposed that the head structure with the large hybrid oscillation (~80°) represents the high affinity state of βA (although the high affinity state in αA is characterized by a two- versus one-helix flip in βA; a one-helix downward flip in αA resulted in an intermediate affinity state). Constraints imposed by the folded bone domains in the structure occupied by the folded ligand likely prevented the hybrid from swinging outward, which is necessary to pull down the α7 helix and switch βA to high affinity. To create the necessary space for hybrid wobbling, the model proposes that the integrin must first fully extend its knees to convert the flexion into a linear asymmetric conformation, as described on two-dimensional (2D) EM images [23]. Full true elongation also reduces the steric hindrance of ligand binding across the plasma membrane expected in the folded conformation. An alternative model, the Schloss [24], integrates structural features in βA that are absent in αA (absence of a metal ion in MIDAS in the unliganded state, presence of an ADMIDAS strategically enclosing the conformationally sensitive α1 helix and the F/ loop) . α7 and the proximity of the new βTD to the βA and hybrid domains, which are not accounted for in the Razor model), suggesting that it is possible to model the elastic deformation of the F/α7 loop with only small quaternary changes in the activate βTD associations with βA and possibly with hybrid domains. This move disrupts ADMIDAS and triggers the activation changes in the α1 helix and F/α7 loop if α7 is not moved down one turn. The bound ligand provides the energy for the hybrid oscillation, which can be associated with different degrees of genu expansion, which can be related to the magnitude and nature of the extraneous mechanochemical signals generated. While hybrid oscillations and knee reflex responses are recognized conformational changes in both models, they are considered necessary for activation in the Razor model, but are a hallmark of outside-in signaling in the latching model. Experimental evidence supporting the locking model was reviewed, including the derivation of the 3D EM structure of the folded ectodomain stably bound to a physiological ligand (see below) [8]. βTD/βA contact interruptionand the Ortit activated on a β2 integrin [25] but not on a β3 integrin [26]. Differences in the relative contribution of the βTD contact to βA (van der Waals contact in β3) and to the hybrid domain may partly explain these differences (the βTD/βA interface in the dormant state is likely to be more robust in β2 versus β3 due to the longest contact loop in β2). Further structural analysis is needed to determine the activating role of these contacts in other integrins.

It has not been established that the crystal structure of an intact αA-containing integrin accurately positions αA in the integrin head: the αA domain is inserted between sheets 2 and 3 of the helical domain on the same side that interacts with the αA domain. βA domain. We proposed that αA acts as an endogenous ligand for βA: an immutable Glu at the flexible C-terminus of the α7 helix could coordinate the MIDAS metal in the βA domain when αA is in the high affinity open state . In this “ligand-relay” model [27], active βA can bias the conformational balance of αA in favor of the high-affinity state, which can then bind exogenous ligands through its own MIDAS (Figure 2b). Extensive biochemical studies now support this model [8]. High affinity αA-containing integrins generated by activation of mutations within the βA domain or by activation of the metal ion Mn2+They have been shown in many studies to express true and hybrid stretch responsive epitopes (see e.g. refs [18, 21]) in the absence of a physiological ligand. These findings led to the development of the switchblade model, largely based on studies of αA-containing integrins. However, αA-missing integrins with activating mutations in the βA domain only express such epitopes in the presence of a physiological ligand. The ligand-relay model may provide the correct explanation for these results. The high affinity βA in αA-containing integrins is rapidly occupied by the covalently bound αA ligand and therefore expresses wobble/stretch epitopes, consistent with the locking model. Therefore, the introduction of an αA domain into a subset of integrins not only results in the respective integrins being able to recognize glutamate rather than aspartate residues in integrin-binding ligands [7], but also predisposes them to accept that subset of extended conformations. easier. in response to inside-out activation in the absence of an extrinsic physiological ligand (Figure 2b).

Transmission MS and single-particle image analysis combined with molecular modeling or tomography were used to characterize the structures of negatively stained [28, 29, 30•] fibronectin-bound and unligated integrin ectodomains alone or in combination with stretch-sensitive monoclonal antibodies (mAb). to investigate. .) [23] and frozen full-length hydrated integrins [31]. Preliminary biophysical studies of the αVβ3 ectodomain by gel filtration revealed a calculated Stokes radius of 57 Å in the presence of 1 mM Ca.2+, a metal ion known to stabilize several membrane-bound integrins, including αVβ3 in its low-affinity state, and has been used to generate the ligandless folded αVβ3 structure [14]. 1 mM manganese exchange2+for about2+increased the Stokes radius to 60 Å and further to 64 Å by adding a high affinity c-RGD ligand, leading to the conclusion that the ligand and Mn2+induce the genu-linear conformation.

Negative staining MS analysis of two-dimensional mean projections of apparently curved, hand-picked particles (in ca2+) and linear (in Mn2+) form the ectodomain, leading to the conclusion that Mn2+induces a blade-like genu elongation, which could explain the observed enlargement of the Stokes radius. In the presence of cRGD, a greater proportion of the linear form was observed even in Ca2+. In a randomized follow-up conical tilt study, the structure of manually selected images of the negatively stained α5β1 head was depicted, which also contains the femoral domains (thigh, hybrid and PSI) at two tilt angles [29]. The three-dimensional reconstruction revealed that the hybrid domain occupied the same acute angle to βA found in the unligated folded X-ray structure in the presence of Ca.2+of manganese2+This shows that the acute angle observed throughout the ectodomain is not caused by head-to-bone contacts. In the presence of the c-RGD ligand, an approximately 80° oscillation of the hybrid domain relative to PA was observed, indicating that the ligand induces or stabilizes the hybrid oscillation. These results were extended in an electron tomography study on negatively stained, manually selected, detergent extracted, full-length human αIIbβ3 bound to RGD peptide and imaged at 23 tilt angles. This study also showed an oscillation of the hybrid domain in an extended integrin conformation, although it was not possible to determine whether the conformation was linear as the mean values ​​showed no density for the bone domains [32]. 2D projections of integrin negatively stained ectodomains without αA or with αA showed images consistent with at least three conformations: a bent state and presumably linear states with and without the hybrid wobble [23]. Two extension-sensitive mAbs bound to the lower domains of the β branch of the linear conformation regardless of whether the hybrid domain was swung in or out, demonstrating that genu and hybrid movements are not sequentially linked. Examination of published 2D images of integrin ectodomains [23, 28] shows that it is difficult to confirm linearity, in part due to the lack of clearly visible density for different bone domains.

3D particle reconstruction of the negatively stained αVβ3 ectodomain in its unligated state (in 1 mM Ca2+) and stably bound to a five-domain fibronectin III ligand in solution (in 0.2 mM Mn).2+) showed that both structures assume a compact state; only a minority of the particles took on a more extended shape. The compact conformation closely resembled the folded X-ray structure and allowed molecular coupling with a moderate oscillation of the hybrid domain of 11 ± 4° [30]. The Vogel group used a molecular dynamic stress analysis of αVβ3 to model what happens when force is applied to the integrin ligand binding site when it binds to domain 10 of fibronectin III [33].••]. The model suggests that only a 20° hybrid oscillation (due to changes in the α1 and α7 helices of the βA domain) is sufficient to transform the integrin head into the high affinity conformation, consistent with previous studies [30 ].].

The finding that the bent conformation stably binds the physiological ligand is inconsistent with the previously presented 2D imaging studies of full-length ectodomains or detergent-extracted ligated full-length integrin. lower manganese2+The concentration used to form the complex, the sample and model bias, and the use of a single angle to visualize particles were the main arguments [34] to support the positive results of Adair et al. [30] to refute]. Manganese concentration2+However, its use was sufficient to form a stable ectodomain-ligand complex in solution, an accepted definition of the high-affinity state of integrin. Sample bias was reduced by using an automated selection routine, in addition to manual selection used exclusively in imaging studies of full-length ectodomains or structures [23, 28, 32]. The model bias was derived from the inability to observe the density in genus β in the unliganded state (genus β was not included in the search model). However, the lack of known density perception is found in several techniques and may be due to flexibility. For example, the density of fibronectin III domain 9 is absent in the 3D images of α5β1 without legs [29], and several domains of the legs were not visible in the tomography study [32]. The genus β is visible in the fibronectin-bound state [30], despite using the same search model, suggesting that ligand binding stabilizes this region. We also tried to bias the datasets collected by the automatic particle selection routine using the genu-linear model [13]; Yet neither view resembled linear integrin conformation. In addition, we subjected a number of particles to unmodeled alignment, classification and averaging using the SPIDER software, with identical results. Third, we imaged the particles at only one angle, because their random orientation on the carbon lattice allowed full coverage of the Euler angles without the need for tilting. The 3D image reconstruction was performed using a projection adjustment algorithm, which is typical when particles are randomly oriented. Conversely, the random cone reconstruction algorithm works best when the particles have a preferred orientation on the lattice. Finally, the importance of Adair's 3D study is importantet al.[30] is that a bent conformation can stably bind physiological ligands in solution, inviting alternative models to instinctively expand to explain the induction of a competent (high affinity) ligand steady-state. The difficulty in evaluating conformational structures of large modular proteins from manual selection of 2D image sets, where differences may be due to particles with different structures or different orientations, suggests that an additional analysis step of 3D reconstruction of the imaged particles may be necessary. are. . in these cases.

Notably, the deduced conformations of the negatively stained integrins only roughly resemble the 20 Å 3D structure of full-length unliganded αIIbβ3 determined by cryo-electron microscopy and single-particle image reconstruction [31].]. In the cryoEM structure, the integrin head appears to be easily accessible for ligand binding and is not sterically hindered by the plasma membrane, as often shown in drawings where the Calf-2 domain is perpendicular to the plane of the plasma membrane. It remains to be elucidated whether some of the multiple conformations described from 2D images of negatively stained full-length ectodomains and integrins are artifacts caused by desiccation or by the non-physiological solutions used, as recently in head domains. of myosin was observed. filaments [35]. Alternatively, the presence of the transmembrane and cytoplasmic domains may constrain the conformational space in full-length integrins.

Additional information on structural changes in high affinity integrins has been obtained by biophysical techniques such as FRET, using a small integrin ligand as donor and a probe inserted into a lipophilic membrane as acceptor [36, 37•]. For α4β1, the shortest distance from the membrane binding site to the ligand was ~50 Å between resting Mn and Mn.2+-activated integrins or reductant-activated integrins and 25 Å between resting and chemokine-activated integrins. Since the theoretically fully extended length of the integrin is >200 Å [13], it is difficult to explain this distance in terms of a linearized knife-shaped conformation, even if native integrins do not emerge orthogonally from the membrane [31].]. These results support previous studies showing that mAbs in the head and leg domains of the platelet integrin αIIbβ3 continue to compete even after platelets are activated with saturated concentrations of multiple agonists known to convert integrin to high affinity [38]. A recent visualization of αIIbβ3 using atomic force microscopy suggests what can be achieved in the future by analyzing the structure and dynamic changes in native integrins [39].].

Integrins are often expressed in cells exposed to mechanical stress, such as muscle or endothelial cells under flow conditions, and this stress is known to have profound effects on cell behavior and phenotype [40]. Short cytoplasmic integrin tails play a key role in switching the ectodomain to the ligand-competent state and subsequently translate conformational changes in the ligand-bound receptor into mechanical and biochemical signals through interactions with cytoskeletal, adapter and signaling proteins. The process of integrin activation and signaling through cytoplasmic domains has gained more definition in recent years, including details on the structures of the cytoskeletal proteins talin1 [41, 42••], tensin [43] and vinculin [44, 45, 46] . . , α-actinin [47, 48] and filamin [49••], along with some of their interactions with cytoplasmic integrin domains and with each other [47, 50].

The actin-binding protein talin plays an essential role in activating integrins from within [51], early coupling of extracellular ligand-bound integrins to the cytoskeleton [52], and in enhancing integrin-cytoskeletal binding through the recruitment of other proteins such as paxillin. , vinculin, α-actinin, tensin and zixin [53]. Talin is found along with high-affinity integrins in short-lived focal adhesion complexes at the tips of prominent lamellapodia very early before other cytoskeletal proteins are detected [3]. Recruitment of talin to the plasma membrane near target integrins occurs in part through an association with RIAM, a target of the small G protein Rap1; Rap1 itself is activated by PKC or Rap1-GEF and recruited to the plasma membrane in response to receptor activation by chemokines or growth factors [54, 55]. RIAM, PIP2 (56) and calpain (57), among others, unmask the integrin binding site in the trilobal FERM domain (4.1, ezrin, radixin and moeisin) of the talin head. The structure of the phosphotyrosine-binding β-sandwich (PTD) F3 lobe of FERM in complex with the cytoplasmic tail of the integrin β3 tail has been determined [41, 42••]. An NPxY motif of β3 forms a reverse loop, inserting tyrosine (747 in β3) into a hydrophobic pocket in F3, an interaction stabilized by a tryptophan (W739 in β3). Residues upstream of NPxY form a β-strand that strengthens the S5-7 β-sheet of talin F3 [41] (Figure 3). F3 also makes a second upstream hydrophobic interaction with the membrane proximal α-helical segment of cytoplasmic β3, opposite β strands S1-2 and S6-7 of the F3 lobe. Lysine residues in the flexible S1-S2 loop of F3 are predicted to contact the cytosolic surface of the plasma membrane (Figure 3). Modeling the structure of the cytoplasmic F3/β3 complex from a previous NMR structure of the cytoplasmic αIIb/β3 complex [58] suggests that this second membrane-proximal binding by F3-Taline is a salt bridge between the cytosolic α- and interrupts β-tails (R995). or D723). , Figure 3) [59], which normally stabilizes the dormant state and provides a kinetic pathway for activation from the inside out. Mutagenesis studies [42••] and the loss of fluorescence resonance energy transfer (FRET) between the CFP and YFP fused α/β cytoplasmic tails at their c-termini [60]. However, the degree of cytoplasmic tail separation required to induce inside-out integrin activation is unclear. Although a gap of >100 Å was suggested in the FRET study [60], altered tail orientation and/or the presence of at least the trilobed FERM domain of talin could disrupt FRET without actual gap. The novel structure also explains the inability of other PTB domains that interact with the NPxY motif but not the membrane-proximal helix of the β3 tail to activate integrins.

The PTB domain of the actin-binding protein tensin binds to the β-tail in a manner similar to talin, but with lower affinity; The positively charged tyrosine pocket explains why the interaction is indifferent to tyrosine phosphorylation [43]. Tensin recruitment is critical for the formation of fibrillar adhesions [2]. The crystal structure of the Ig-like domain 21 (IgFLN21) of the actin cross-linking homodimer filamin 1 in complex with the integrin β-tail shows that the ser/thr-rich distal membrane segment of the β-tail contains an elongated β-strand, interacts with the C and D strands of IgFLN21. The binding interface extends to the NPxY binding site of the talin head [49••], preventing simultaneous binding of talin and filamin to the β-tail of the same integrin molecule. This competition for filamin binding may negatively regulate talin-induced integrin activation and may explain the known inhibitory role of filamin in cell migration [61].

(Video) Cell Adhesion Molecules | Structure and Types

Although the binding of F3 to the proximal β3 tail of the integrin is sufficient to bring the ectodomain into the ligand-competent state, it is not involved in the binding of the integrin to the cytoskeleton in focal complexes [62]; A second interaction between the talin bar domain, composed of multiple amphipathic helix bundles, and the liganded integrin β tails appears to be necessary [63]. The structural basis of this interaction between integrin and talin rod is unknown; The multivalent ligand and force exerted by Rho-activated actomyosin motors on early matrix-integrin talin complexes may expose a β3 tail binding site for the talin rod [52, 64]. The adapter protein paxillin, incorporated into early focal complexes along with talin, can bind talin to the cytoplasmic tail of α-integrin (see below), increasing the resistance of matrix-integrin-talin complexes to mechanical stress. [65] This could explain why paxillin is often found in slow-moving protrusions of the membrane.

The actin and talin-binding protein vinculin and the focal adhesion tyrosine kinase FAK are then incorporated into the focal adhesion complexes, strengthening the contacts between the ECM and the cytoskeleton via integrin. Cryptic vinculin binding sites (VBS) exposed to the talin rod [46], in concert with talin-bound actin [66], activate vinculin by destabilizing the auto-inhibitory head-to-tail intramolecular interaction [67]. Cocrystal and NMR structures of talin-derived VBS peptides complexed with the D1 helix bundle subdomain (Vh1) of the vinculin head reveal the insertion and replacement of VBS helix 1 of D1 [50, 68]. Increased force exerted by actomyosin contractility at the adhesion site likely releases more talin VBS (up to 11 sites), leading to increased vinculin recruitment. The vinculin tail domain also interacts with leucine-rich LD motifs of paxillin [69]. Both interactions likely contribute to the transformation of focal complexes in focal adhesions. FAK plays an important role in signaling networks in focal contacts [70]. It is recruited to ECM-bound integrins through the interaction of its c-terminal four-helical FAT domain with paxillin LD motifs [71, 72]. FAK also binds to the integrin's β-tail via the F1 lobe in its n-terminal FERM domain, an interaction that destabilizes the autoinhibitory state, allowing activation of FAK by Src [73]. This interaction has not been structurally characterized. FAK is also involved in the recruitment of the p130Cas adapter protein, a primary mechanical force sensor [74].••] (see below).

Recruitment of the α-actinin homodimer to form an actin package is a later event in focal adhesion formation [3, 75]. A cryoEM study suggests that the region between the spectrin-like α-actinin repeats R1 and R2 in the core α-actinin domain (consisting of four repeats of the tandem triple helix bundle) is already β-tailed from the proximal helical integrin interacts complexed with the Talin head, presumably by linking the side of the helix opposite the β-tail [76]. Mechanical tension exerted by integrin ligation imparted to the R4 repeat of α-actinin likely causes helix 3 to swing out of the triple-helical bundle. The now accessible helix inserts into the D1 subdomain of vinculin in a manner similar to talin, but with an inverted orientation relative to talin, resulting in marked structural activation changes in vinculin [47, 68]. Recruitment of zyxin through a biochemically but not structurally defined n-terminal interaction with α-actinin R2/3 repeats [77] enhances Arp2/3-independent actin assembly in focal adhesions through interactions with the Ena/VASP family of proteins [ 78].

The cytoplasmic tail of the α-integrin is not a passive player in integrin activation or signaling. An interaction between talin and the cytoplasmic tail of αIIb has been described [79], but has not yet been structurally characterized. In addition, at least one cytosolic protein, the calcium-containing EF-hand and integrin-binding protein 1 (CIB1), interacts with hydrophobic residues in the membrane-proximal region of the cytoplasmic tail of αIIb [80] and disrupts the binding of talin to a tail peptide of aIIb. [81] so that it could act as a negative regulator of talin-induced integrin activation. The α4 (and α9) subunits of liganded integrins also strengthen shear-formed integrin-cytoskeletal bonds [65] by binding indirectly to talin via the cytoskeletal adapter paxillin. The structures of paxillin in complex with talin or the α4 integrin tail have not been determined. The α4-paxillin interaction is inhibited by phosphorylation of the leading edge α4 tail by PKA type I anchored to the α4 tail [82]. This abrogates Paxillin-mediated inhibition of Rac, allowing vectorization of lamellar podia [83]. The association of a second Rap1 effector, RAPL, with the cytoplasmic αL tail (CD11a) may cooperate with or possibly independently of talin bound to the β2 tail to activate and assemble integrins [84]. Integrin clustering also induces T cell protein tyrosine phosphatase (TCPTP) activation through its association with the cytoplasmic tail of integrin α1; TCPTP-mediated dephosphorylation of the EGF receptor associated with integrin clusters inhibits anchorage-independent cell proliferation [85].

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    (Video) Cell adhesion molecules/ Cadherins/integrins/IgSF/selectins
  • Vinculin transfers high-level integrin strains that are dispensable for focal adhesion formation.

    2023, Biophysical Journal

    Focal adhesions (FA) transmit force and mediate mechanotransduction between cells and matrix. Previous studies showed that the power of integrin is essential in regulating fatty acid formation. Since vinculin is an important FA protein involved in the transmission of integrin stress, this work examines the relationship between integrin stress (force), vinculin (protein), and FA formation (structure) through manipulation of integrin tension, force display, and vinculin inactivation ( KO). . . Two DNA-based instruments for integrin strains are used: Strain Gauge Strap (TGT) and Integrating Strain Sensor (ITS), where TGT limits integrin strains by engineering design.TTol(stress tolerance) and ITS by integrin stress across theTTolfluorescence value. The results show that the large AF (area >1MetroMetro2) were co-formed on the TGT surfaceTTolof 54 pN, but not in those with lower onesTTolValues. Time series analysis of FA formation shows that focal complexes (area <0.5MetroMetro2) appeared on all TGT surfaces 20 min after cell seeding, but only matured on TGT with oversized FAsTTolof 54 pN. Next, we tested FA formation in vinculin KO cells on TGT surfaces. Surprisingly, theTTolThe TGT required for the formation of large fatty acids is drastically reduced to 23 pN. To investigate the cause, we visualized integrin stress in wild-type and vinculin KO cells using ITS. The results showed that integrin strains in FA of wild-type cells often activate ITSTTolof 54 pN. However, with vinculin KO, integrin voltages in the FAs decreased and failed to activate 54 pN ITS. Integrin stem strength signal intensities reported by 33 and 43 pN ITS were also significantly reduced by vinculin KO, suggesting that vinculin is essential for and involved in high-quality integrin strain transfer. Intermediate level integrin in FA. However, vinculin-transmitted strains with high integrin content are not required for AF production.

  • Toll-like receptor 3 in cardiovascular disease

    2022, heart, lungs and circulatory system

    Toll-like receptor 3 (TLR3) is an important member of the Toll-like receptor (TLR) family of innate immune responses that plays a critical role in regulating the immune response, promoting immune cell maturation and differentiation, and the involvement which this is due to the response of pro-inflammatory factors. TLR3 is activated by pathogen-associated molecular patterns and damage-associated molecular patterns that support the pathophysiology of many inflammation-related diseases. A growing body of research has confirmed that TLR3, as an important innate immune agent, is involved in the onset and development of cardiovascular disease (CVD) by regulating the transcription and translation of several cytokines, which influence the physiological structure and functioning of resident cells. being influenced. in the cardiovascular system, including vascular endothelial cells, vascular smooth muscle cells, cardiomyocytes, fibroblasts, and macrophages. Dysfunction and structural damage of vascular endothelial cells and proliferation of vascular smooth muscle cells are key factors in the development of vascular diseases such as pulmonary arterial hypertension, atherosclerosis, myocardial hypertrophy, myocardial infarction, ischemia/reperfusion injury and heart failure. Cardiomyocytes, fibroblasts and macrophages are now implicated in the development of cardiovascular disease. Therefore, the purpose of this review was to review the most recently published research on TLR3 in CVD and to discuss the current understanding of the possible mechanisms by which TLR3 contributes to CVD. Although TLR3 is an emerging field, it has strong treatment potential as an immunomodulator and deserves further investigation for its clinical implementation.

  • Alteration of key cell signaling pathways in cancer

    2022, Understanding Cancer: From Basics to Therapy

    A cancer cell undergoes numerous characteristic changes that allow it to evade the mechanisms that control its survival, proliferation, migration, angiogenesis and apoptosis. This evasion of mechanisms is due to changes in the signaling pathways. When a proto-oncogene becomes an oncogene or the tumor suppressor gene is turned off, signaling pathways that promote tumor growth are activated. The cause of cancer can be better understood by examining the signaling pathways that primarily influence cancer progression. In this chapter, we review the major signaling pathways that influence the process of tumor progression, their mechanism of action and drug targets.

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Featured Articles (6)

  • research article

    Effects of extension areas and single cell aspect ratios on chondrocyte dedifferentiation

    Biomaterials, Volume 35, Number 25, 2014, pp. 6871-6881

    Chondrocyte dedifferentiation is common in culture and significantly impairs the restorative efficacy of cartilage repair. The current study investigates the effect of initial cell shapes on chondrocyte vitro. The cell shape was controlled with a unique technique for microstructuring materials. Using this technique, a series of cell-adherent peptide arginine glycine aspartate (RGD) microarrays were generated in a persistent, non-staining polyethylene glycol (PEG) hydrogel. After culturing rat-derived chondrocytes on the microstructured surfaces, the cell shapes were fitted to the geometries of adherent microislands with predefined diameters (10, 15, 20 and 30 μm) for rounded shapes and aspect ratios (1, 1, 2 , 1.5 ) adjusted. 2, 4 and 6) for elliptical cross trainers. After 10 days, collagen II staining identified normal chondrocytes and dedifferentiated cells down to the individual cells in microislets. In addition, gene expression of collagen II, collagen I, aggrecan and SOX9 was detected using qRT-PCR. The statistical results indicated that chondrocyte dedifferentiation was more likely with larger sizes and higher aspect ratios. The conclusions are maintained under normoxic conditions (21% OR).2) in hypoxie (5% O2) the atmosphere.

  • research article

    Response of a wound coil to mechanical force: global stability and local crack formation

    Biophysical Journal, Volume 105, Issue 4, 2013, Pages 951-961

    Coiled coils are major structural motifs composed of two or more amphipathic structuresAPropellers rotate in a super spiral. These motifs are found in a variety of proteins, including motor proteins and structural proteins, known to convey mechanical stress. We analyze atomically detailed simulations of spiral cracking under load with milestones. Milestoneing is an approach that captures the key features of the process in a network and quantifies the kinetics and thermodynamics. A segment of 112 remains of theB-The myosin S2 domain was subjected to constant magnitude (0-200 pN) and constant direction tensile forces in molecular dynamics simulations. Twenty 20 ns forward simulations at different load levels revealed that initial single-residue breakage events (Ψ > 90°) at loads < 100 pN were accompanied by rapid refolding without propagation of unfolding within or between the coils. Only the initial unfolding events at the highest strain (200 pN) regularly propagated along and between the helices. Analysis of hydrophobic interactions and hydrogen bonding between helices revealed no significant variation with charge. Developmental events mainly occurred near E929, a charged residue at a hydrophobic position of the heptad repeat. A landmark network analysis of cracking at E929 found that the mean time to first twist ranged from 20 ns (200 pN) to 80 ns (50 pN), which is approximately 20 times the mean time to first twist of an isolated helix with the same sequence matches .

  • research article

    Statistical Mechanics of an Elastic Hinged Membrane: Static Profile and Correlations

    Biophysical Journal, volume 116, number 2, 2019, pages 283-295

    The relationship between thermal fluctuations and the mechanical response of a free membrane has been extensively studied both theoretically and experimentally. However, it is considerably more difficult to understand this relationship for locally anchored membranes by proteins. Because the coupling of the membrane to the cellular cytoskeleton, to the extracellular matrix and to other internal structures is crucial for the regulation of a number of cellular processes, it is of great importance to understand the role of tethering. In this manuscript, we consider a single protein (elastic spring with a finite rest length) that fixes a membrane patterned in Monge meter. First, we determine the Green's function for the system and supplement this approximation by calculating the modal coupling coefficients for the plane wave and orthonormal fluctuation expansion modes. We then create a set of tools for numerical studies and analysis of an attached membrane system. . Furthermore, we investigate the static correlations between free and attached membrane, as well as membrane shape and show that all three are interdependent and exhibit identical long-range behavior characterized by correlation length. Interestingly, the latter shows a non-monotonic behavior as a function of membrane voltage. It is important to emphasize that the use of these relationships allows the experimental determination of the elastic parameters of the fixation. Finally, we calculate the interaction potential between two attachment sites and show that even without membrane deformation, the attachments are subject to an attractive force due to changes in membrane fluctuations.

    (Video) Cell Adhesion
  • research article

    On activation of αIIbβ3 integrin: outside-in and inside-out pathways

    Biophysical Journal, Volume 105, Number 6, 2013, pp. 1304-1315

    IntegrinaAIIbB3 is a member of the integrin family of transmembrane proteins found in the plasma membrane of platelets. integrinAIIbBIt is well known that 3 regulates the thrombotic process through activation on its cytoplasmic side by talin and interaction with soluble fibrinogen. Three sets of interactions are also reported to maintain members of the integrin family in the inactive state, including a set of salt bridges on the cytoplasmic side of the integrin transmembrane domain.A- JB-Subunits known as inner membrane junction, hydrophobic packing of some transmembrane residues on the extracellular side between theA- JB-Subunits known as the outer membrane junction, and the main interacting group of theBA domain (located in theBroot domain of the subunit) with theBTD (proximal to the plasma membrane in theBsubunit). However, the molecular details of this key interacting group, as well as the events leading up to the group's detachment, are unknownBDT jBA domains have remained obscure. In this study, we used molecular dynamics models to take a comprehensive outside-in and inside-out approach and study how integrinAIIbB3 is activated. First, we show that the interaction of talin with the membrane proximal and distal regions of the integrin's cytoplasmic transmembrane domains significantly loosens inner membrane closure. Talin also interacts with an additional salt bridge (R734-E1006) that facilitates integrin activation through integrin cleavage.A- JBsubunits. The second part of our study classifies three types of interactions between RGD peptides and extracellular integrin domains.AIIbB3. Finally, we show that the interaction of the RGD sequence can activate Arg integrin through the main interaction group between K350 inBA domain and S673/S674 on theBTD.

  • research article

    Glycosylation and regulation of integrins in cancer

    Trends in Cancer, Volume 4, Issue 8, 2018, pp. 537-552

    Integrins are transmembrane receptors that coordinate the interactions between the extracellular matrix (ECM) and cells, as well as cell-cell interactions, signaling, gene expression and cell function. Abnormality of integrin function is one of the most well-known cancer mechanisms. Integrin activity is strongly influenced by glycans through glycosylation events and mediating glycan-mediated interactions. Glycans represent a class of ubiquitous biomolecules that exhibit extraordinary complexity and diversity in both structure and function. They are widely expressed on both the ECM and the cell surface and play a critical role in mediating cell proliferation, survival and metastasis in cancer. The purpose of this review is to provide an overview of how both glycosylation of integrins and the interaction of integrins with the cancer glycomicroenvironment may regulate cancer progression.

  • research article

    Force-regulated conformational change of integrin αVβ3

    Matrix Biology, vols. 60-61, 2017, pp. 70-85

    Integrins mediate cell adhesion to the extracellular matrix and transmit signals bidirectionally across the membrane. α integrinVB3It has been shown to play an essential role in tumor metastasis, angiogenesis, hemostasis and phagocytosis. Integrins can adopt several conformations, including the bent and elongated conformations of the ectodomain, which regulate integrin functions. Using a biomembrane force probe, we characterized the bending and non-bending conformational changes of a single α.VB3Integrins on living cell surfaces in real time. We measured the probabilities of conformational changes, rates and rates of conformational transitions, and the dynamic balance between the two conformations that were drag-regulated, ligand-dependent and altered by point mutations. These results shed light on how αVB3It behaves like a molecular machine and how its physiological function and molecular structure are linked at the level of single molecules.

    (Video) Focal Adhesions : Focal Adhesion Proteins
(Video) Cadherins | e-cadherin and n-cadherin

Copyright © 2007 Elsevier B.V. All rights reserved.


What is the role of integrin in cell cell adhesion? ›

Integrin-mediated cell adhesion plays a central role in the formation and remodeling of tissues and organs in multicellular organisms [1]. Integrins bind extracellular matrix (ECM) proteins through their large ectodomain, and engage via their short cytoplasmic tails the cytoskeleton.

What is the structure and function of integrins? ›

Integrins are large, membrane-spanning, heterodimeric proteins that are essential for a metazoan existence. All members of the integrin family adopt a shape that resembles a large “head” on two “legs,” with the head containing the sites for ligand binding and subunit association.

What is integrin adhesion? ›

Integrin-based adhesions support cell movement via indirect linkages to the actin cytoskeleton. 1. Integrins are transmembrane α and β subunit comprised receptors for distinct extracellular matrix (ECM) proteins.

What is the mechanism of action of integrin? ›

Mechanism of integrin ligand binding and conformational states. Integrins bind cell-surface ligands to promote cellular interactions with the ECM and with other cells in the transduction of complex signals that modulate many cellular processes, such as adhesion, migration, and differentiation.

What is cell adhesion and why is it important? ›

Cell–cell adhesion determines the polarity and the physiological function of cells within tissues. On every cell, adhesion molecules facilitate interactions within the cell microenvironment that consist of other cells and the extracellular matrix.

What is the principle of cell adhesion? ›

Cells adhere to each other and to the extracellular matrix through cell-surface proteins called cell adhesion molecules (CAMs)—a category that includes the transmembrane adhesion proteins we have already discussed. CAMs can be cell-cell adhesion molecules or cell-matrix adhesion molecules.

Which statement best describes the structure of integrins? ›

Which statement BEST describes the structure of integrins? Integrins are heterodimers composed of α and β subunits.

How do integrins bind? ›

With their extracellular head region, most integrins bind extracellular matrix (ECM) glycoproteins such as laminins and collagens in basement membranes or connective tissue components like fibronectin. Others bind counterreceptors on neighboring cells, bacterial polysaccharides, or viral coat proteins.

What are the properties of integrin? ›

Integrins are transmembrane heterodimers composed of α and β subunits that have a large extracellular domain that binds to specific ECM proteins and short cytoplasmic tails that interact with the actin cytoskeleton and associated proteins.

What are the 4 adhesion structures in cells? ›

There are four major superfamilies or groups of CAMs: the immunoglobulin super family of cell adhesion molecules (IgCAMs), Cadherins, Integrins, and the Superfamily of C-type of lectin-like domains proteins (CTLDs).

What are the different types of cell adhesion? ›

There are at least five groups of cell adhesion molecules: integrins, selectins, adhesion molecules belonging to the immunoglobulin superfamily, cadherins, and the CD44 family.

What happens when integrin is activated? ›

Integrin activation encompasses both changes in affinity of individual integrins due to conformational changes and avidity increases due to integrin clustering (3-5). Precise regulation of integrin activation is particularly important in controlling platelet aggregation through integrin αIIbβ3 (6).

Does integrin cause cell movement? ›

Integrin-containing adhesions function as signaling centers orchestrating a network of signaling pathways that mediate cell migration.

What is the main function of integrins for immune cells? ›

Integrins are transmembrane adhesion receptors that mediate cell–cell and cell–extracellular matrix adhesion and also induce bidirectional signalling across the cell membrane to regulate cell proliferation, activation, migration and homeostasis.

What do integrins regulate? ›

Integrins are the adhesion molecules and transmembrane receptors that consist of α and β subunits. After binding to extracellular matrix components, integrins trigger intracellular signaling and regulate a wide spectrum of cellular functions, including cell survival, proliferation, differentiation and migration.

What is cell adhesion in simple terms? ›

Cell adhesion is the process by which cells interact and attach to neighbouring cells through specialised molecules of the cell surface.

What causes loss of cell to cell adhesion? ›

Loss of Cell-Cell Contact Is Induced by Integrin-Mediated Cell-Substratum Adhesion in Highly-Motile and Highly-Metastatic Hepatocellular Carcinoma Cells.

How can I improve my cell adhesion? ›

Many biomolecules are used to modify material surface to improve adhesion. For example, planting adhesion-active proteins on scaffolds, such as type I collagen, vitreous laminin, FN, and laminin, can significantly promote cell adhesion.

What are the 3 main stages in cell adhesion? ›

The cell adhesion cascade and signaling events in vivo involve three basic steps: selectin-mediated rolling, chemokine-triggered activation, and integrin-dependent arrest [43].

What influences cell adhesion? ›

The dominant influence of cell adhesion area could be explained by cellular uptake capacity and DNA synthesis activity through the formation of FAs, cytoskeletal mechanics, and YAP/TAZ nuclear localization.

What are the materials for cell adhesion? ›

Cells adhere to surfaces through adhesion proteins (i.e. fibronectin, collagen, laminin, vitronectin) using specific cell receptors, called integrins, attached to the cell membrane.

Which one of the following is true about the function of integrins? ›

Which of the following is correct about integrins? Explanation: The correct answer is that integrins anchor the cell to the extracellular matrix (ECM) and relay signals from the ECM to the cell.

What is an example of an integrin? ›

Integrins can regulate the receptor tyrosine kinase signaling by recruiting specific adaptors to the plasma membrane. For example, β1c integrin recruits Gab1/Shp2 and presents Shp2 to IGF1R, resulting in dephosphorylation of the receptor.


1. Integrin Biology | Dr Yoshikazu Thakada
(KU Padasala)
2. Cell adhesion molecules in immune system
3. Cell Adhesion
(Biology Brainery)
4. Focal Adhesion Organization
(Introduction to Mechanobiology)
5. Cell–Extracellular Matrix Mechanobiology
6. Cell Adhesion Molecules: Cadherin, Selectin, IgSF, Integrin | CELL BIOLOGY CSIR NET Lifescience


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