Integrin Clustering Research Articles

KANK2 Links αVβ5 Focal Adhesions to Microtubules and Regulates Sensitivity to Microtubule Poisons and Cell Migration.

Integrins are heterodimeric glycoproteins that bind cells to extracellular matrix. Upon integrin clustering, multimolecular integrin adhesion complexes (IACs) are formed, creating links to the cell cytoskeleton. We have previously observed decreased cell migration and increased sensitivity to microtubule (MT) poisons, paclitaxel and vincristine, in the melanoma cell line MDA-MB-435S upon transfection with integrin αV-specific siRNA, suggesting a link between adhesion and drug sensitivity. To elucidate the underlying mechanism, we determined αV-dependent changes in IAC composition. Using mass spectrometry (MS)-based proteomics, we analyzed the components of isolated IACs of MDA-MB-435S cells and two MDA-MB-435S-derived integrin αV-specific shRNA-expressing cell clones with decreased expression of integrin αV. MS analysis showed that cells preferentially use integrin αVβ5 for the formation of IACs. The differential analysis between MDA-MB-435S cells and clones with decreased expression of integrin αV identified key components of integrin αVβ5 adhesion complexes as talins 1 and 2, α-actinins 1 and 4, filamins A and B, plectin and vinculin. The data also revealed decreased levels of several components of the cortical microtubule stabilization complex, which recruits MTs to adhesion sites (notably liprins α and β, ELKS, LL5β, MACF1, KANK1, and KANK2), following αV knockdown. KANK2 knockdown in MDA-MB-435S cells mimicked the effect of integrin αV knockdown and resulted in increased sensitivity to MT poisons and decreased migration. Taken together, we conclude that KANK2 is a key molecule linking integrin αVβ5 IACs to MTs, and enabling the actin-MT crosstalk that is important for both sensitivity to MT poisons and cell migration.

Integrin-Dependent Difference in Cell Adhesion and Force Exertion

Cell adhesion is mediated by various mechanosensitive integrin receptors. We show that α4β1 and α5β1 respectively mediate cell adhesion in a different manner. Unlike α5β1-RGD ligand binding, α4β1-LDVP ligand binding does not activate cell polarization and migration, lacking the focal adhesion which is a large elongated sub-cellular structure. In addition, integrin α5β1-mediated cell adhesion requires relatively high tension (>33 pN) on RGD ligand binding to activate cell adhesion/spreading, but α4β1-mediated adhesion requires much lower force (<12 pN) on LDVP ligand binding. The level of tension required can be altered by utilizing allosteric Fabs against β1 subunit to alter integrin conformational equilibria. When the extended-open conformation populates at higher population, the tension force required to support cell adhesion decreases. Furthermore, we monitored integrin clustering, integrin β1 activation, and molecular force transmission on ligands with high spaciotemporal resolution to show the dynamic molecular details of cell adhesion. In summary, we found that α4β1 mediates a simple manner of cell adhesion distinguished from typical α5β1 mediated adhesions.

Crucial Role for Endothelial Cell α2β1 Integrin Receptor Clustering in Collagen-Induced Angiogenesis.

Angiogenesis is a crucial mechanism of vascular growth and regeneration that requires biosynthesis and cross-linking of collagens in vivo and is induced by collagen in vitro. Here, we use an in vitro model in which apical Type I collagen gels rapidly induce angiogenesis in endothelial monolayers. We extend previous studies demonstrating the importance of the endothelial α2β1 integrin, a key collagen receptor, in angiogenesis by investigating the roles of receptor clustering and conformational activation. Immunocytochemical localization of α2β1 integrins in endothelial monolayers showed a concentration of integrins along cell-cell borders. After inducing angiogenesis with collagen, the receptors redistributed to apical cell surfaces, aligning with collagen fibers, which were also redistributed during angiogenesis. Levels of conformationally activated α2β1 integrins were unchanged during angiogenesis and undetected on endothelial cells binding collagen in suspension. We mimicked the polyvalency of collagen fibrils using antibody-coated polystyrene beads to cluster endothelial cell surface α2β1 integrins, which induced rapid angiogenesis in the absence of collagen gels. Clustering of αvβ3 integrins and PECAM-1 but not of α1 integrins also induced angiogenesis. Soluble antibodies alone had no effect. Thus, the angiogenic property of collagen may reside in its ability to ligate and cluster cell surface receptors such as α2β1 integrins. Furthermore, synthetic substrates that promote the clustering of select endothelial cell surface receptors mimic the angiogenic properties of Type I collagen and may have applications in promoting vascularization of engineered tissues. Anat Rec, 2019. © 2019 American Association for Anatomy.

Word games and molecular recognition – The creative control of endothelial cell function

In this paper we postulate that figures of language and structural features, which are characteristic of poetic writings and word games, have direct correlates in the cell/cell and cell/matrix recognition processes. We shall discuss the particular example of endothelial cells engagement into angiogenesis (the making of new blood vessels in adult vertebrates)—an activity that requires intense extracellular matrix (ECM) remodeling and Cell Adhesion Molecules (CAMs) rearrangement—to argue that angiogenesis controls are organized into semiotic dimensions. CAMs attach cells to the ECM; they are mainly composed of integrins (transmembrane receptors) that bind selectively to different ECM components. After ECM binding, the cytoplasmic tails of integrins within the cell will begin to interact with a wide range of recruited factors that, in turn, regulate integrin clustering in the cell membrane. These recruited factors also activate signaling pathways that link activated integrins to later microfilament system remodeling during cell migration. Ultimately CAMs work as functional protein networks—as the adaptor molecules in an organic code, the Adhesion Code—controlling cell migration/proliferation transition through the continual rearrangement of both ECM adhesion and Actin polymerization. Additionally, orchestrating the transition from clonal growth to differentiation, the interaction between CAMs and ECM components will trigger specific Signal Transduction pathways. We shall examine some attempts to conceptualize “somatic cell function” in the recent specialized literature, which introduces the notion of hierarchic organization into levels i.e. molecular, sub-cellular and cellular and describes an informational flow of increasing complexity versus decreasing number of entities through the levels. Beyond the syntactic level—the specific recognition of discrete ECM motifs by integrin heterodimers—we shall examine the semantic and pragmatic levels at which the composition and architecture of multimolecular complexes will dictate cell recognition events and cell fate decisions. These higher-level codes will be compared to the dynamics of the word games of Lewis Carroll, e.g. “doublets” and “magic squares.” In such creative games words are subjected to synthetic transformations that have to conform to semantic rules, but are ultimately constrained by meaning, as concrete pragmatics. This gives us a model for analyzing angiogenesis control as a word game.

Mechanosensitivity Occurs along the Adhesome’s Force Train and Affects Traction Stress

Herein, we consider the process of force development along the adhesome within cell focal adhesions. Our model adhesome consists of the actin cytoskeleton-vinculin-talin-integrin-ligand-extracellular matrix-substrate force train. We specifically consider the effects of substrate stiffness on the force levels expected along the train and on the traction stresses they create at the substrate. We find that significant effects of substrate stiffness are manifest within each constitutive component of the force train and on the density and distribution of integrin/ligand anchorage points with the substrate. By following each component of the force train, we are able to delineate specific gaps in the quantitative descriptions of bond survival that must be addressed so that improved quantitative forecasts become possible. Our analysis provides, however, a rational description for the various levels of traction stresses that have been reported and of the effect of substrate stiffness. Our approach has the advantage of being quite clear as to how each constituent contributes to the net development of force and traction stress. We demonstrate that to provide truly quantitative forecasts for traction stress, a far more detailed description of integrin/ligand density and distribution is required. Although integrin density is already a well-recognized important feature of adhesion, our analysis places a finer point on it in the manner of how we evaluate the magnitude of traction stress. We provide mechanistic insight into how understanding of this vital element of the adhesion process may proceed by addressing mechanistic causes of integrin clustering that may lead to patterning.

Integrin nanoclusters can bridge thin matrix fibres to form cell-matrix adhesions.

Integrin-mediated cell-matrix adhesions are key to sensing the geometry and rigidity of extracellular environments and influence vital cellular processes. In vivo, the extracellular matrix is composed of fibrous arrays. To understand the fibre geometries that are required for adhesion formation, we patterned nanolines of various line widths and arrangements in single, crossing or paired arrays with the integrin-binding peptide Arg-Gly-Asp. Single thin lines (width ≤30 nm) did not support cell spreading or formation of focal adhesions, despite the presence of a high density of Arg-Gly-Asp, but wide lines (>40 nm) did. Using super-resolution microscopy, we observed stable, dense integrin clusters formed on parallel (within 110 nm) or crossing thin lines (mimicking a matrix mesh) similar to those on continuous substrates. These dense clusters bridged the line pairs by recruiting activated but unliganded integrins, as verified by integrin mutants unable to bind ligands that coclustered with ligand-bound integrins when present in an active extended conformation. Thus, in a fibrous extracellular matrix mesh, stable integrin nanoclusters bridge between thin (≤30 nm) matrix fibres and bring about downstream consequences of cell motility and growth.

Dynamics of Mechanosensitive Nascent Adhesion Formation

Cellular migration is a tightly regulated process that involves actin cytoskeleton, adaptor proteins, and integrin receptors. Forces are transmitted extracellularly through protein complexes of these molecules, called adhesions. Adhesions anchor the cell to its substrate, allowing it to migrate. In Chinese hamster ovary cells, three classes of adhesion can be identified: nascent adhesions (NAs), focal complexes, and focal adhesions, ranked here ascendingly based on size and stability. To understand the dynamics and mechanosensitive properties of NAs, a biophysical model of these NAs as colocalized clusters of integrins and adaptor proteins is developed. The model is then analyzed to characterize the dependence of NA area on biophysical parameters that regulate the number of integrins and adaptor proteins within NAs through a mechanosensitive coaggregation mechanism. Our results reveal that NA formation is triggered beyond a threshold of adaptor protein, integrin, or extracellular ligand densities, with these three factors listed in descending order of their relative influence on NA area. Further analysis of the model also reveals that an increase in coaggregation or reductions in integrin mobility inside the adhesion potentiate NA formation. By extending the model to consider the mechanosensitivity of the integrin bond, we identify mechanical stress, rather than mechanical load, as a permissive mechanical parameter that allows for noise-dependent and independent NA assembly, despite both parameters producing a bistable switch possessing a hysteresis. Stochastic simulations of the model confirm these results computationally. This study thus provides insight into the mechanical conditions defining NA dynamics.

Integrins as biomechanical sensors ofthe microenvironment.

Integrins, and integrin-mediated adhesions, have long been recognized to provide the main molecular link attaching cells to the extracellular matrix (ECM) and to serve as bidirectional hubs transmitting signals between cells and their environment. Recent evidence has shown that their combined biochemical and mechanical properties also allow integrins to sense, respond to and interact with ECM of differing properties with exquisite specificity. Here, we review this work first by providing an overview of how integrin function is regulated from both a biochemical and a mechanical perspective, affecting integrin cell-surface availability, binding properties, activation or clustering. Then, we address how this biomechanical regulation allows integrins to respond to different ECM physicochemical properties and signals, such as rigidity, composition and spatial distribution. Finally, we discuss the importance of this sensing for major cell functions by taking cell migration and cancer as examples.

Multiscale model of integrin adhesion assembly

The ability of adherent cells to form adhesions is critical to numerous phases of their physiology. The assembly of adhesions is mediated by several types of integrins. These integrins differ in physical properties, including rate of diffusion on the plasma membrane, rapidity of changing conformation from bent to extended, affinity for extracellular matrix ligands, and lifetimes of their ligand-bound states. However, the way in which nanoscale physical properties of integrins ensure proper adhesion assembly remains elusive. We observe experimentally that both β-1 and β-3 integrins localize in nascent adhesions at the cell leading edge. In order to understand how different nanoscale parameters of β-1 and β-3 integrins mediate proper adhesion assembly, we therefore develop a coarse-grained computational model. Results from the model demonstrate that morphology and distribution of nascent adhesions depend on ligand binding affinity and strength of pairwise interactions. Organization of nascent adhesions depends on the relative amounts of integrins with different bond kinetics. Moreover, the model shows that the architecture of an actin filament network does not perturb the total amount of integrin clustering and ligand binding; however, only bundled actin architectures favor adhesion stability and ultimately maturation. Together, our results support the view that cells can finely tune the expression of different integrin types to determine both structural and dynamic properties of adhesions.

Bond tension on neutrophil LFA-1 regulates membrane calcium flux from the outside-in

Abstract Recruitment of neutrophils in appropriate numbers at vascular sites of inflammation is critical for innate immune surveillance. LFA-1 adhesive bonds that support neutrophil deceleration and arrest under fluid shear, also function to mechanotransduce signals that spatially localize calcium flux necessary to guide transendothelial migration. We set out to characterize how bond tension acting on high-affinity LFA-1/ICAM-1 bonds transduce intracellular signaling in a manner proportional to the shear force acting on adherent cells. A sequential set of events beginning with a shift to high affinity of LFA-1 that binds ICAM-1, a buildup of tension, and assembly of adaptor proteins that terminate in spatial linkage to the CRAC channel Orai-1. Utilizing microfluidic vascular mimetic flow chamber, we observed that force catalyzed integrin clustering and calcium flux in a Kindlin-3 dependent manner. Kindlin-3 has a dual role as a mechanical anchor that links to adaptors that assemble Orai-1 mediated calcium influx and as a support for LFA-1 membrane clustering. RACK1 deficient neutrophils retained the capacity for integrin clustering but lacked calcium signaling. Utilizing a molecular platform that regulates the force acting on LFA-1 bonds we determined the characteristic bond tension necessary to transduce signaling. Forces between 33 and 54 pN were sufficient to support LFA-1 binding to Kindlin-3 and promote integrin clustering. As this characteristic force completed a macromolecular complex constituted of RACK1/Orai1 signaling complex competent to transduce calcium influx. We identify a molecular circuit catalyzed by a well-defined force that may represent the necessity to assemble Kindlin-3 to intracellular LFA-1 domains.

Integrin-Mediated Macrophage Adhesion Promotes Lymphovascular Dissemination in Breast Cancer

SummaryLymphatic vasculature is crucial for metastasis in triple-negative breast cancer (TNBC); however, cellular and molecular drivers controlling lymphovascular metastasis are poorly understood. We define a macrophage-dependent signaling cascade that facilitates metastasis through lymphovascular remodeling. TNBC cells instigate mRNA changes in macrophages, resulting in β4 integrin-dependent adhesion to the lymphovasculature. β4 integrin retains macrophages proximal to lymphatic endothelial cells (LECs), where release of TGF-β1 drives LEC contraction via RhoA activation. Macrophages promote gross architectural changes to lymphovasculature by increasing dilation, hyperpermeability, and disorganization. TGF-β1 drives β4 integrin clustering at the macrophage plasma membrane, further promoting macrophage adhesion and demonstrating the dual functionality of TGF-β1 signaling in this context. β4 integrin-expressing macrophages were identified in human breast tumors, and a combination of vascular-remodeling macrophage gene signature and TGF-β signaling scores correlates with metastasis. We postulate that future clinical strategies for patients with TNBC should target crosstalk between β4 integrin and TGF-β1.

Citrullination of fibronectin alters integrin clustering and focal adhesion stability promoting stromal cell invasion

The extracellular matrix (ECM) microenvironment is increasingly implicated in the instruction of pathologically relevant cell behaviors, from aberrant transdifferentation to invasion and beyond. Indeed, pathologic ECMs possess a panoply of alterations that provide deleterious instructions to resident cells. Here we demonstrate the precise manner in which the ECM protein fibronectin (FN) undergoes the posttranslational modification citrullination in response to peptidyl-arginine deiminase (PAD), an enzyme associated with innate immune cell activity and implicated in systemic ECM-centric diseases, like cancer, fibrosis and rheumatoid arthritis. FN can be citrullinated in at least 24 locations, 5 of which reside in FN's primary cell-binding domain. Citrullination of FN alters integrin clustering and focal adhesion stability with a concomitant enhancement in force-triggered integrin signaling along the FAK-Src and ILK-Parvin pathways within fibroblasts. In vitro migration and in vivo wound healing studies demonstrate the ability of citrullinated FN to support a more migratory/invasive phenotype that enables more rapid wound closure. These findings highlight the potential of ECM, particularly FN, to “record” inflammatory insults via post-translational modification by inflammation-associated enzymes that are subsequently “read” by resident tissue fibroblasts, establishing a direct link between inflammation and tissue homeostasis and pathogenesis through the matrix.

Unique arrangement of bone matrix orthogonal to osteoblast alignment controlled by Tspan11-mediated focal adhesion assembly

During tissue construction, cells coordinate extracellular matrix (ECM) assembly depending on the cellular arrangement. The traditional understanding of the relationship between the ECM and cells is limited to the orientation-matched interaction between them. Indeed, it is commonly accepted that the bone matrix (collagen/apatite) is formed along osteoblast orientation. Nonetheless, our recent findings are contrary to the above theory; osteoblasts on nanogrooves organize formation of the bone matrix perpendicular to cell orientation. However, the precise molecular mechanisms underlying the orthogonal organization of bone matrix are still unknown. Here, we show that mature fibrillar focal adhesions (FAs) facilitate the perpendicular arrangement between cells and bone matrix. The osteoblasts aligned along nanogrooves expressed highly mature fibrillar FAs mediated by integrin clustering. Microarray analysis revealed that Tspan11, a member of the transmembrane tetraspanin protein family, was upregulated in cells on the nanogrooved surface compared with that in cells on isotropic, flat, or rough surfaces. Tspan11 silencing significantly disrupted osteoblast alignment and further construction of aligned bone matrix orthogonal to cell orientation. Our results demonstrate that the unique bone matrix formation orthogonal to cell alignment is facilitated by FA maturation. To the best of our knowledge, this report is the first to show that FA assembly mediated by Tspan11 determines the direction of bone matrix organization.

Clustering of integrin \u03b2 cytoplasmic domains triggers nascent adhesion formation and reveals a protozoan origin of the integrin-talin interaction

Integrins and integrin-dependent cell-matrix adhesions are essential for a number of physiological processes. Integrin function is tightly regulated via binding of cytoplasmic proteins to integrin intracellular domains. Yet, the complexity of cell-matrix adhesions in mammals, with more than 150 core adhesome proteins, complicates the analysis of integrin-associated protein complexes. Interestingly, the evolutionary origin of integrins dates back before the transition from unicellular life to complex multicellular animals. Though unicellular relatives of metazoa have a less complex adhesome, nothing is known about the initial steps of integrin activation and adhesion complex assembly in protozoa. Therefore, we developed a minimal, microscope-based system using chimeric integrins to investigate receptor-proximal events during focal adhesion assembly. Clustering of the human integrin β1 tail led to recruitment of talin, kindlin, and paxillin and mutation of the known talin binding site abolished recruitment of this protein. Proteins indirectly linked to integrins, such as vinculin, migfilin, p130CAS, or zyxin were not enriched around the integrin β1 tail. With the exception of integrin β4 and integrin β8, the cytoplasmic domains of all human integrin β subunits supported talin binding. Likewise, the cytoplasmic domains of integrin β subunits expressed by the protozoan Capsaspora owczarzaki readily recruited talin and this interaction was based on an evolutionary conserved NPXY/F amino acid motif. The results we present here validate the use of our novel microscopic assay to uncover details of integrin-based protein-protein interactions in a cellular context and suggest that talin binding to integrin β cytoplasmic tails is an ancient feature of integrin regulation.

Kindlin‐3 recruitment to the plasma membrane in neutrophils precedes high affinity integrin activation

Integrin‐mediated adhesion of circulating leukocytes to the blood vessel wall starts by arrest from rolling. Activation of integrins by inside‐out signaling involves conformational changes from an inactive bent conformation to an extended conformation (E+) with high affinity for ligand binding (H+). The cytoplasmic protein kindlin‐3 is critically required for leukocyte adhesion. Mutations of kindlin‐3 cause leukocyte adhesion deficiency type III (LAD‐III) syndrome, characterized by severe bleeding (a platelet adhesion defect) and recurring infections (a neutrophil adhesion defect). The clinical significance of kindlin‐3 has been extensively studied; however, the molecular mechanism by which kindlin‐3 regulates the β2 integrin activation is unknown. Kindlin‐3 can bind the β2 integrin cytoplasmic tail at a site distinct from the talin‐1 binding site. It remains unclear whether kindlin‐3 affects individual integrin conformation and/or integrin clustering. Here we monitored the spatiotemporal dynamics of kindlin‐3 and β2 integrin conformation changes during neutrophil rolling, arrest and spreading under flow (6 dyn/cm2) using high‐resolution quantitative dynamic footprinting (qDF) microscopy in combination with an integrin conformation‐specific antibody reporting assay and kindlin‐3 fusion reporters. We find that kindlin‐3 is recruited to the plasma membrane during neutrophil adhesion prior to induction of H+ β2 integrin conformation, indicating that kindlin‐3 is required for H+ integrin conformation. Indeed, deletion of kindlin‐3 in neutrophil‐like HL‐60 cells completely abolishes high affinity β2 integrin. Together, our data suggest that kindlin‐3 is permissive for the high affinity β2 integrin conformation.Support or Funding InformationThis work was supported by funding from the National Institutes of Health, USA (NIH, P01‐HL078784).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Platelet Integrin αIIbβ3: Mechanisms of Activation and Clustering; Involvement into the Formation of the Thrombus Heterogeneous Structure

Glycoproteins IIb-IIIa (GPIIb-IIIa), also known as αIIbβ3 integrins, are key platelet adhesion receptors. These molecules are the most abundant (over 10 000 copies per cell) transmembrane receptors playing a crucial role in thrombus formation by promoting platelet aggregation. Integrins need to undergo activation and transit to high-affinity state for their ligands – fibrinogen, fibrin, and von Willebrand factor (VWF) – in order to form bonds between platelets. Activation of integrins is mediated by a set of various messengers through intracellular signalization. Integrins αIIbβ3, like other integrins, are capable of reverse signal transmission inside the cell, called “outside-in” signaling. Recent studies have shown heterogeneity of the thrombus structure and the existence of a stable and dense inner core and a fluid-like loose shell. Since platelet aggregation is provided by integrin-mediated interactions, one can suggest that it is the features of integrin activation and clustering that strongly influence the formation of thrombus architecture. This work is intent on systematizing recent data concerning activation and functioning of platelet integrins αIIbβ3 and searching for correlations between thrombus heterogeneity and the state of integrins on the platelets surface.

Regulation of cell migration by α4 and α9 integrins.

Integrins are heterodimeric transmembrane receptors that play an essential role in enabling cells to sense and bind to extracellular ligands. Activation and clustering of integrins leads to the formation of focal adhesions at the plasma membrane that subsequently initiate signalling pathways to control a broad range of functional endpoints including cell migration, proliferation and survival. The α4 and α9 integrins form a small sub-family of receptors that share some specific ligands and binding partners. Although relatively poorly studied compared with other integrin family members, emerging evidence suggests that despite restricted cell and tissue expression profiles, these integrins play a key role in the regulation of signalling pathways controlling cytoskeletal remodelling and migration in both adherent and non-adherent cell types. This review summarises the known shared and specific roles for α4 and α9 integrins and highlights the importance of these receptors in controlling cell migration within both homeostatic and disease settings.

Biochemical Ligand Density Regulates Yes-Associated Protein Translocation in Stem Cells through Cytoskeletal Tension and Integrins.

Different tissue types are characterized by varying stiffness and biochemical ligands. Increasing substrate stiffness has been shown to trigger Yes-associated protein (YAP) translocation from the cytoplasm to the nucleus, yet the role of ligand density in modulating mechanotransduction and stem cell fate remains largely unexplored. Using polyacrylamide hydrogels coated with fibronectin as a model platform, we showed that stiffness-induced YAP translocation occurs only at intermediate ligand densities. At low or high ligand densities, YAP localization is dominated by ligand density independent of substrate stiffness. We further showed that ligand density-induced YAP translocation requires cytoskeleton tension and αVβ3-integrin binding. Finally, we demonstrate that increasing ligand density alone can enhance osteogenic differentiation regardless of matrix stiffness. Together, the findings from the present study establish ligand density as an important parameter for modulating stem cell mechanotransduction and differentiation, which is mediated by integrin clustering, focal adhesion, and cytoskeletal tension.

Kindlin Is Mechanosensitive: Force-Induced Conformational Switch Mediates Cross-Talk among Integrins

Mechanical stresses directly regulate the function of several proteins of the integrin-mediated focal adhesion complex as they experience intra- and extracellular forces. Kindlin is a largely overlooked member of the focal adhesion complex whose roles in cellular mechanotransduction are only recently being identified. Recent crystallographic experiments have revealed that kindlins can form dimers that bind simultaneously to two integrins, providing a mechanistic explanation of how kindlins may promote integrin activation and clustering. In this study, using the newly identified molecular structure, we modeled the response of the kindlin2 dimer in complex with integrin β1 to mechanical cytoskeletal forces on integrins. Using molecular dynamics simulations, we show that forces on integrins are directly transmitted to the kindlin2 dimerization site, resulting in a shift in an R577-S550/E553 interaction network at this site. Under force, R577 on one protomer switches from interacting with S550 to forming new hydrogen bonds with E553 on the neighboring protomer, resulting in the strengthening of the kindlin2 dimer in complex with integrin β1. This force-induced strengthening is similar to the catch-bond mechanisms that have previously been observed in other adhesion molecules. Based on our results, we propose that the kindlin2 dimer is mechanosensitive and can strengthen integrin-mediated focal adhesions under force by shifting the interactions at its dimerization sites.

Fever Promotes T Lymphocyte Trafficking via a Thermal Sensory Pathway Involving Heat Shock Protein 90 and α4 Integrins

Fever is an evolutionarily conserved response that confers survival benefits during infection. However, the underlying mechanism remains obscure. Here, we report that fever promoted T lymphocyte trafficking through heat shock protein 90 (Hsp90)-induced α4 integrin activation and signaling in Tcells. By inducing selective binding of Hsp90 to α4 integrins, but not β2 integrins, fever increased α4-integrin-mediated Tcell adhesion and transmigration. Mechanistically, Hsp90 bound to the α4 tail and activated α4 integrins via inside-out signaling. Moreover, the N and C termini of one Hsp90 molecule simultaneously bound to two α4 tails, leading to dimerization and clustering of α4 integrins on the cell membrane and subsequent activation of the FAK-RhoA pathway. Abolishment of Hsp90-α4 interaction inhibited fever-induced Tcell trafficking todraining lymph nodes and impaired the clearance of bacterial infection. Our findings identify the Hsp90-α4-integrin axis as a thermal sensory pathway that promotes T lymphocyte trafficking and enhances immune surveillance during infection.