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Simple Summary: Cutaneous squamous cell carcinomas (SCCs) are the most common tumors in patients suffering from the genetic disorder epidermolysis bullosa (EB). These tumors frequently show aggressive growth, leading to metastatic disease and fatal outcomes. Therapeutic options in advanced stages are limited, and new drug targets are needed. The present study evaluated the immune microenvironment of cutaneous SCCs, which is assumed to favor local immunosuppression and lead to a more severe disease course in patients. The expression of several immune checkpoint molecules in tumor cells and cells in the tumor microenvironment was evaluated in EB-SCCs and compared with those in SCCs from immunocompetent and immunosuppressed patients. Our results show high expression of the immunosuppressive markers indoleamine 2,3-dioxygenase (IDO), PD-1, and programmed cell death ligand-1 (PD-L1) in tumor cells from dystrophic EB (DEB) patients. Cutaneous squamous cell carcinomas (SCCs) are a major complication of some subtypes of epidermolysis bullosa (EB), with high morbidity and mortality rates and unmet therapeutic needs. The high rate of endogenous mutations and the fibrotic stroma are considered to contribute to the pathogenesis. Patients with dystrophic EB (DEB) and Kindler EB (KEB) have the highest propensity for developing SCCs. Another patient group that develops high-risk SCCs is immunosuppressed (IS) patients, especially after organ transplantation. Herein, we interrogate whether immune checkpoint proteins and immunosuppressive enzymes are dysregulated in EB-associated SCCs as an immune resistance mechanism and compare the expression patterns with those in SCCs from IS patients, who frequently develop high-risk tumors and sporadic SCCs, and immunocompetent (IC) individuals. The expression of indoleamine 2,3-dioxygenase (IDO), programmed cell death protein-1 (PD-1), programmed cell death ligand-1 (PD-L1), T cell immunoglobulin and mucin-domain-containing protein-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), and inflammatory infiltrates (CD4, CD8, and CD68) was assessed via immunohistochemistry and semi-quantitative analysis in 30 DEB-SCCs, 22 KEB-SCCs, 106 IS-SCCs, and 100 sporadic IC-SCCs. DEB-SCCs expressed significantly higher levels of IDO and PD-L1 in tumor cells and PD-1 in the tumor microenvironment (TME) compared with SCCs from IC and IS individuals. The number of CD4-positive T cells per mm2 was significantly lower in DEB-SCCs compared with IC-SCCs. KEB-SCCs showed the lowest expression of the exhaustion markers TIM-3 and LAG-3 compared with all other groups. These findings identify IDO, PD-1, and PD-L1 to be increased in EB-SCCs and candidate targets for combinatory treatments, especially in DEB-SCCs. [ABSTRACT FROM AUTHOR]

Simple Summary: Neurons are biological wires that send signals not only across the brain, but throughout the body. Like almost all cells in the human body, neurons adhere to their surroundings through receptors known as integrins. As the nervous system develops, integrins become essential for neuronal wires, known as axons, to adhere within their environment and pull themselves forward as they grow towards their target. Following injury to the nervous system, the damaged axons must regrow towards their target to restore communication. The integrin receptor therefore plays an important role in this process of regeneration. Integrins mediate their effects through an intricate scaffold of proteins inside the cell, which must be coordinated by a specific central protein, called focal adhesion kinase. This review discusses the signalling pathway of integrin receptors and focal adhesion kinase, specifically in neurons, and how this signalling pathway mediates neuronal growth. We also discuss how integrins and focal adhesion kinase could contribute to neuronal repair following nervous system injury. Integrin receptors are essential contributors to neurite outgrowth and axon elongation. Activated integrins engage components of the extracellular matrix, enabling the growth cone to form point contacts, which connect the extracellular substrate to dynamic intracellular protein complexes. These adhesion complexes facilitate efficient growth cone migration and neurite extension. Major signalling pathways mediated by the adhesion complex are instigated by focal adhesion kinase (FAK), whilst axonal guidance molecules present in vivo promote growth cone turning or retraction by local modulation of FAK activity. Activation of FAK is marked by phosphorylation following integrin engagement, and this activity is tightly regulated during neurite outgrowth. FAK inhibition slows neurite outgrowth by reducing point contact turnover; however, mutant FAK constructs with enhanced activity stimulate aberrant outgrowth. Importantly, FAK is a major structural component of maturing adhesion sites, which provide the platform for actin polymerisation to drive leading edge advance. In this review, we discuss the coordinated signalling of integrin receptors and FAK, as well as their role in regulating neurite outgrowth and axon elongation. We also discuss the importance of the integrin–FAK axis in vivo, as integrin expression and activation are key determinants of successful axon regeneration following injury. [ABSTRACT FROM AUTHOR]

Integrin receptors enable cells to sense and respond to their chemical and physical environment. As a class of membrane receptors, they provide a dynamic, tightly regulated link between the extracellular matrix or cellular counter-receptors and intracellular cytoskeletal and signaling networks. They enable transmission of mechanical force across the plasma membrane, and particularly for cardiomyocytes, may sense the mechanical load placed on cells. Talins and Kindlins are two families of FERM-domain proteins which bind the cytoplasmic tail of integrins, recruit cytoskeletal and signaling proteins involved in mechano-transduction, and those which synergize to activate integrins, allowing the integrins to physically change and bind to extracellular ligands. In this review, we will discuss the roles of talin and kindlin, particularly as integrin activators, with a focus on cardiac myocytes.

Focal adhesions are composed of transmembrane integrins, linking the extracellular matrix to the actomyosin cytoskeleton, via cytoplasmic proteins. Adhesion depends on the activation of integrins. Talin and kindlin proteins are intracellular activators of integrins that bind to β-integrin cytoplasmic tails. Integrin activation and clustering through extracellular ligands guide the organization of adhesion complexes. However, the roles of talin and kindlin in this process are poorly understood. To determine the contribution of talin, kindlin, lipids and actomyosin in integrin clustering, we used a biomimetic in vitro system, made of giant unilamellar vesicles, containing transmembrane integrins (herein aIIbβ3), with purified talin (talin-1), kindlin (kindlin-2, also known as FERMT2) and actomyosin. Here, we show that talin and kindlin individually have the ability to cluster integrins. Talin and kindlin synergize to induce the formation of larger integrin clusters containing the three proteins. Comparison of protein density reveals that kindlin increases talin and integrin density, whereas talin does not affect kindlin and integrin density. Finally, kindlin increases integrin-talin-actomyosin coupling. Our study unambiguously demonstrates how kindlin and talin cooperate to induce integrin clustering, which is a major parameter for cell adhesion. [ABSTRACT FROM AUTHOR]

Talin (herein referring to the talin-1 form), is a cytoskeletal adapter protein that binds integrin receptors and F-actin, and is a key factor in the formation and regulation of integrin-dependent cell-matrix adhesions. Talin forms the mechanical link between the cytoplasmic domain of integrins and the actin cytoskeleton. Through this linkage, talin is at the origin of mechanosignaling occurring at the plasma membrane-cytoskeleton interface. Despite its central position, talin is not able to fulfill its tasks alone, but requires help from kindlin and paxillin to detect and transform the mechanical tension along the integrin-talin-F-actin axis into intracellular signaling. The talin head forms a classical FERM domain, which is required to bind and regulate the conformation of the integrin receptor, as well as to induce intracellular force sensing. The FERM domain allows the strategic positioning of protein-protein and protein-lipid interfaces, including the membrane-binding and integrin affinityregulating F1 loop, as well as the interaction with lipid-anchored Rap1 (Rap1a and Rap1b in mammals) GTPase. Here, we summarize the structural and regulatory features of talin and explain how it regulates cell adhesion and force transmission, as well as intracellular signaling at integrin-containing cell-matrix attachment sites. [ABSTRACT FROM AUTHOR]

β2 integrins are expressed on all leukocytes. Precise regulation of the β2 integrin is critical for leukocyte adhesion and trafficking. In neutrophils, β2 integrins participate in slow rolling. When activated by inside–out signaling, fully activated β2 integrins mediate rapid leukocyte arrest and adhesion. The two activation pathways, starting with selectin ligand engagement and chemokine receptor ligation, respectively, converge on phosphoinositide 3-kinase, talin-1, kindlin-3 and Rap1. Here, we focus on recent structural insights into autoinhibited talin-1 and autoinhibited trimeric kindlin-3. When activated, both talin-1 and kindlin-3 can bind the β2 cytoplasmic tail at separate but adjacent sites. We discuss possible pathways for talin-1 and kindlin-3 activation, recruitment to the plasma membrane, and their role in integrin activation. We propose new models of the final steps of integrin activation involving the complex of talin-1, kindlin-3, integrin and the plasma membrane. [ABSTRACT FROM AUTHOR]

In multicellular organisms, cell adhesion is a pivotal physiological process which is essential for cell–cell communications, cell migration, and interactions with extracellular matrix. Integrins, a family of large hetero-dimeric type I membrane proteins, are known for driving cell adhesion functions. Among 24 different integrins, four β2 integrins, αL β2, αM β2, αX β2 and αD β2, are specific for cell adhesion and migration of leukocytes. Many cytosolic proteins interact with short cytosolic tails (CTs) of β2 and other integrins which are essential in bi-directional signaling processes. Further, phosphorylation of CTs of integrins regulates binding of intra-cellular proteins and signaling systems. In this review, recent advances in structures and interactions of multi-protein complexes of integrin tails, with a focus on β2 integrin, and cytosolic proteins are discussed along with a proposed future direction. [ABSTRACT FROM AUTHOR]

Neutrophils are the most abundant leukocytes in humans and are critical for innate immunity and inflammation. Integrins are critical for neutrophil functions, especially for their recruitment to sites of inflammation or infections. Integrin conformational changes during activation have been heavily investigated but are still not fully understood. Many regulators, such as talin, Rap1-interacting adaptor molecule (RIAM), Rap1, and kindlin, are critical for integrin activation and might be potential targets for integrin-regulating drugs in treating inflammatory diseases. In this review, we outline integrin activation regulators in neutrophils with a focus on the above critical regulators, as well as newly discovered modulators that are involved in integrin activation. [ABSTRACT FROM AUTHOR]

Cell adhesion manifests as cell linkages to neighboring cells and/or the extracellular matrix (ECM). Migfilin is a widely expressed adhesion protein. It comprises three LIM domains in the C-terminal region and one proline-rich sequence in the N-terminal region. Through interplay with its various binding partners, such as Kindlin-2, Filamin, vasodilator-stimulated phosphoprotein (VASP) protein and the transcription factor CSX, Migfilin facilitates the dynamic association of connecting actomyosin fibers, orchestrating cell morphogenetic movement and cell adhesion, proliferation, migration, invasion, differentiation and signal transduction. In this review, to further elucidate the functional contributions of and pathogenesis induced by Migfilin, we focused on the structure of Migfilin and the targets which it directly binds with. We also summarized the role of Migfilin and its binding partners in the progression of different diseases and malignancies. As a possible candidate for coordinating various cellular processes and because of its association with both the pathogenesis and progression of certain tumors, Migfilin likely has utility as a therapeutic target against multiple diseases in the clinic. [ABSTRACT FROM AUTHOR]

Cell adhesion is essential for the formation of organs, cellular migration, and interaction with target cells and the extracellular matrix. Integrins are large protein α/β-chain heterodimers and form a major family of cell adhesion molecules. Recent research has dramatically increased our knowledge of how integrin phosphorylations regulate integrin activity. Phosphorylations determine the signaling complexes formed on the cytoplasmic tails, regulating downstream signaling. α-Chain phosphorylation is necessary for inducing β-chain phosphorylation in LFA-1, and the crosstalk from one integrin to another activating or inactivating its function is in part mediated by phosphorylation of β-chains. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus receptor angiotensin-converting enzyme 2 (ACE2) and possible integrin coreceptors may crosstalk and induce a phosphorylation switch and autophagy. Integrins are transmembrane proteins involved in cell–cell and cell–matrix communication. Recent studies show how integrin phosphorylations regulate integrin activity. Phosphorylation of both integrin α- and β-chains are emerging as being critical for activity. Phosphorylation of integrin β-chains enables kindlin binding by the assistance of talin, resulting in cell adhesion. Crosstalk between integrins and other receptors may occur by phosphorylation switches. [ABSTRACT FROM AUTHOR]

Abstract Integrins are a family of transmembrane glycoprotein signaling receptors that can transmit bioinformation bidirectionally across the plasma membrane. Integrin αIIbβ3 is expressed at a high level in platelets and their progenitors, where it plays a central role in platelet functions, hemostasis, and arterial thrombosis. Integrin αIIbβ3 also participates in cancer progression, such as tumor cell proliferation and metastasis. In resting platelets, integrin αIIbβ3 adopts an inactive conformation. Upon agonist stimulation, the transduction of inside-out signals leads integrin αIIbβ3 to switch from a low- to high-affinity state for fibrinogen and other ligands. Ligand binding causes integrin clustering and subsequently promotes outside-in signaling, which initiates and amplifies a range of cellular events to drive essential platelet functions such as spreading, aggregation, clot retraction, and thrombus consolidation. Regulation of the bidirectional signaling of integrin αIIbβ3 requires the involvement of numerous interacting proteins, which associate with the cytoplasmic tails of αIIbβ3 in particular. Integrin αIIbβ3 and its signaling pathways are considered promising targets for antithrombotic therapy. This review describes the bidirectional signal transduction of integrin αIIbβ3 in platelets, as well as the proteins responsible for its regulation and therapeutic agents that target integrin αIIbβ3 and its signaling pathways.

Integrin-mediated cell–extracellular matrix (ECM) interactions play crucial roles in a broad range of physiological and pathological processes. Kindlins are important positive regulators of integrin activation. The FERM-domain-containing kindlin family comprises three members, kindlin-1, kindlin-2 and kindlin-3 (also known as FERMT1, FERMT2 and FERMT3), which share high sequence similarity (identity >50%), as well as domain organization, but exhibit diverse tissue-specific expression patterns and cellular functions. Given the significance of kindlins, analysis of their atomic structures has been an attractive field for decades. Recently, the structures of kindlin and its β-integrin-bound form have been obtained, which greatly advance our understanding of the molecular functions that involve kindlins. In particular, emerging evidence indicates that oligomerization of kindlins might affect their integrin binding and focal adhesion localization, positively or negatively. In this Review, we presented an update on the recent progress of obtaining kindlin structures, and discuss the implication for integrin activation based on kindlin oligomerization, as well as the possible regulation of this process. [ABSTRACT FROM AUTHOR]

Focal adhesions (FAs) are integrin‐containing protein complexes regulated by a network of hundreds of protein–protein interactions. They are formed in a spatiotemporal manner upon the activation of integrin transmembrane receptors, which is crucial to trigger cell adhesion and many other cellular processes including cell migration, spreading and proliferation. Despite decades of studies, a detailed molecular level understanding on how FAs are organized and function is lacking due to their highly complex and dynamic nature. However, advances have been made on studying key integrin activators, talin and kindlin, and their associated proteins, which are major components of nascent FAs critical for initiating the assembly of mature FAs. This review will discuss the structural and functional findings of talin and kindlin and their immediate interaction network, which will shed light upon the architecture of nascent FAs and how they act as seeds for FA assembly to dynamically regulate diverse adhesion‐dependent physiological and pathological responses. [ABSTRACT FROM AUTHOR]

Integrins are heterodimeric transmembrane receptors that connect the extracellular matrix environment to the actin cytoskeleton via adaptor molecules through assembly of a range of adhesion structures. Recent advances in biochemical, imaging and biophysical methods have enabled a deeper understanding of integrin signalling and their associated regulatory processes. The identification of the consensus integrin-based 'adhesomes' within the last 5 years has defined common core components of adhesion complexes and associated partners. These approaches have also uncovered unexpected adhesion protein behaviour and molecules recruited to adhesion sites that have expanded our understanding of the molecular and physical control of integrin signalling. [ABSTRACT FROM AUTHOR]

ABSTRACT: Integrins are cell surface receptors that form the link between extracellular matrix molecules of the cell environment and internal cell signalling and the cytoskeleton. They are involved in several processes, e.g. adhesion and migration during development and repair. This review focuses on the role of integrins in axonal regeneration. Integrins participate in spontaneous axonal regeneration in the peripheral nervous system through binding to various ligands that either inhibit or enhance their activation and signalling. Integrin biology is more complex in the central nervous system. Integrins receptors are transported into growing axons during development, but selective polarised transport of integrins limits the regenerative response in adult neurons. Manipulation of integrins and related molecules to control their activation state and localisation within axons is a promising route towards stimulating effective regeneration in the central nervous system. [ABSTRACT FROM AUTHOR]

The Kindlins are FERM domain proteins comprising three members (Kindlin-1, -2 and -3) which are evolutionarily conserved. Kindlins bind with β-integrin cytoplasmic tails and execute broad biological functions including directed cell migration, proliferation, differentiation and survival. In light of more and more evidence point to the importance of Kindlin family members in normal development and human diseases especially in cancers, we aim to portrait the profile of Kindlins in the regulation of embryonic development and cancer progression. We first summarize all the known binding proteins for individual member of Kindlin family. We then outline the Kindlin-regulated signaling pathways including Wnt/β-catenin, TGFβ, EGFR, and Hedgehog signalings. Furthermore, we descript the pivotal role of Kindlins in embryonic development in detail with notions that Kindlin-1 is highly expressed in endo/ectodermal originated tissues, Kindlin-2 is highly expressed in mesoderm-derived tissues and Kindlin-3 is highly expressed in mesoderm- and ectoderm-derived tissues. Deregulation of Kindlins is generally reported in cancers from different organs. We also briefly descript the role of Kindlins in other diseases. Finally, we update the recent understanding of how Kindlins are regulated and modified as well as the degradation mechanism of Kindlins, respectively. [ABSTRACT FROM AUTHOR]

Integrin activation is essential for creating functional transmembrane receptors capable of inducing downstream cellular effects such as cell migration, cell spreading, neurite outgrowth and axon regeneration. Integrins are bidirectional signalling molecules that mediate their effects by 'inside--out' and 'outside--in' signalling. This review will provide a detailed overview of integrin activation focusing on intracellular activation in neurons and discussing direct implications in the regulation of neurite outgrowth and axon regeneration. [ABSTRACT FROM AUTHOR]

Kindlins play an important role in supporting integrin activation by cooperating with talin; however, the mechanistic details remain unclear. Here, we show that kindlins interacted directly with paxillin and that this interaction could support integrin αIIbβ3 activation. An exposed loop in the N-terminal F0 subdomain of kindlins was involved in mediating the interaction. Disruption of kindlin binding to paxillin by structure-based mutations significantly impaired the function of kindlins in supporting integrin αIIbβ3 activation. Both kindlin and talin were required for paxillin to enhance integrin activation. Interestingly, a direct interaction between paxillin and the talin head domain was also detectable. Mechanistically, paxillin, together with kindlin, was able to promote the binding of the talin head domain to integrin, suggesting that paxillin complexes with kindlin and talin to strengthen integrin activation. Specifically, we observed that crosstalk between kindlin-3 and the paxillin family in mouse platelets was involved in supporting integrin αIIbβ3 activation and in vivo platelet thrombus formation. Taken together, our findings uncover a novel mechanism by which kindlin supports integrin αIIbβ3 activation, which might be beneficial for developing safer anti-thrombotic therapies. [ABSTRACT FROM AUTHOR]

Kindlins and talins are integrin-binding proteins that are critically involved in integrin activation, an essential process for many fundamental cellular activities including cell-matrix adhesion, migration, and proliferation. As FERM-domain-containing proteins, talins and kindlins, respectively, bind different regions of β-integrin cytoplasmic tails. However, compared with the extensively studied talin, little is known about how kindlins specifically interact with integrins and synergistically enhance their activation by talins. Here, we determined crystal structures of kindlin2 in the apo-form and the β1- and β3-integrin bound forms. The apo-structure shows an overall architecture distinct from talins. The complex structures reveal a unique integrin recognition mode of kindlins, which combines two binding motifs to provide specificity that is essential for integrin activation and signaling. Strikingly, our structures uncover an unexpected dimer formation of kindlins. Interrupting dimer formation impairs kindlin-mediated integrin activation. Collectively, the structural, biochemical, and cellular results provide mechanistic explanations that account for the effects of kindlins on integrin activation as well as for how kindlin mutations found in patients with Kindler syndrome and leukocyte-adhesion deficiency may impact integrin-mediated processes. [ABSTRACT FROM AUTHOR]

Integrins require an activation step prior to ligand binding and signaling. How talin and kindlin contribute to these events in non-hematopoietic cells is poorly understood. Here we report that fibroblasts lacking either talin or kindlin failed to activate β1 integrins, adhere to fibronectin (FN) or maintain their integrins in a high affinity conformation induced by Mn2+. Despite compromised integrin activation and adhesion, Mn2+ enabled talin- but not kindlin-deficient cells to initiate spreading on FN. This isotropic spreading was induced by the ability of kindlin to directly bind paxillin, which in turn bound focal adhesion kinase (FAK) resulting in FAK activation and the formation of lamellipodia. Our findings show that talin and kindlin cooperatively activate integrins leading to FN binding and adhesion, and that kindlin subsequently assembles an essential signaling node at newly formed adhesion sites in a talin-independent manner.

Phorbol diester PMA (phorbol 12-myristate 13-acetate) is a well-known promoter of tumor progression. PMA also regulates cell adhesion by several mechanisms including conformational activation of integrins and integrin clustering. Here, PMA was shown to induce lamellipodia formation and reorganization of the adhesion sites as well as actin and vimentin filaments independently of integrin preactivation. To further analyze the mechanism of PMA action, the protein composition in the α1β1 integrin/collagen IV adhesion sites was analyzed by mass spectrometry and proteomics. In four independent experiments we observed the reduced recruitment of vimentin in relation to integrin α1 subunit. This was in full agreement with the fact that we also detected the retraction of vimentin from cell adhesions by confocal microscopy. Furthermore, the accumulation of kindlin-2 into cell adhesions was significantly increased after PMA treatment. Kindlin-2 siRNA inhibited cell spreading as well as the formation of actin fibrils and cell adhesions, but did not prevent the effect of PMA on lamellipodia formation. Thus, kindlin-2 recruitment was considered to be a consequence rather than the primary cause for the loss of connection between vimentin and the adhesion sites. [ABSTRACT FROM AUTHOR]

We describe a strategy for exploring the function of protein-protein interactions in striated muscle in vivo. We describe our experience using this strategy to study the interaction of UNC-112 (kindlin) with PAT-4 (integrin linked kinase). Random mutagenesis is used to generate a collection of mutants that are screened for lack of binding or gain of binding using a yeast 2-hybrid assay. The mutant proteins are then expressed in transgenic C. elegans to determine their ability to localize in the sarcomere. We emphasize two advantages of this strategy: (1) for studying the interaction of protein A with protein B, when protein A can interact with multiple proteins, and (2) it explores the function of an interaction rather than the absence of, or reduced level of, a protein as can be obtained with null mutants or knockdown by RNAi. We propose that this method can be generalized for studying the meaning of a protein-protein interaction in muscle for any system in which transgenic animals can be generated and their muscles can be imaged.

Mutations in the FERMT1 gene, encoding the focal adhesion protein kindlin-1 underlie the Kindler syndrome ( KS), an autosomal recessive skin disorder with a phenotype comprising skin blistering, photosensitivity, progressive poikiloderma with extensive skin atrophy, and propensity to skin cancer. The FERMT1 mutational spectrum comprises gross genomic deletions, splice site, nonsense, and frameshift mutations, which are scattered over the coding region spanning exon 2-15. We now report three KS families with mutations affecting the promoter region of FERMT1. Two of these mutations are large deletions (∼38.0 and 1.9 kb in size) and one is a single nucleotide variant (c.- 20A>G) within the 5′ untranslated region ( UTR). Each mutation resulted in loss of gene expression in patient skin or cultured keratinocytes. Reporter assays showed the functional relevance of the genomic regions deleted in our patients for FERMT1 gene transcription and proved the causal role of the c.- 20A>G variant in reducing transcriptional activity. [ABSTRACT FROM AUTHOR]

Integrin function is regulated by activation involving conformational changes that modulate ligand-binding affinity and downstream signaling. Activation is regulated through inside-out signaling which is controlled by many signaling pathways via a final common pathway through kindlin and talin, which bind to the intracellular tail of beta integrins. Previous studies have shown that the axon growth inhibitory molecules NogoA and chondroitin sulfate proteoglycans (CSPGs) inactivate integrins. Overexpressing kindlin-1 in dorsal root ganglion (DRG) neurons activates integrins, enabling their axons to overcome inhibitory molecules in the environment, and promoting regeneration in vivo following dorsal root crush. Other studies have indicated that expression of the talin head alone or with kindlin can enhance integrin activation. Here, using adult rat DRG neurons, we investigate the effects of overexpressing various forms of talin on axon growth and integrin signaling. We found that overexpression of the talin head activated axonal integrins but inhibited downstream signaling via FAK, and did not promote axon growth. Similarly, co-expression of the talin head and kindlin-1 prevented the growth-promoting effect of kindlin-1, suggesting that the talin head acts as a form of dominant negative for integrin function. Using full-length talin constructs in PC12 cells we observed that neurite growth was enhanced by the expression of wild-type talin and more so by two ‘activated’ forms of talin produced by point mutation (on laminin and aggrecan–laminin substrates). Nevertheless, co-expression of full-length talin with kindlin did not promote neurite growth more than either molecule alone. In vivo, we find that talin is present in PNS axons (sciatic nerve), and also in CNS axons of the corticospinal tract. [ABSTRACT FROM AUTHOR]

Background Kindlins (FERMT) are cytoplasmic proteins required for integrin (ITG) activation, leukocyte transmigration, platelet aggregation and thrombosis. Characterization of kindlins and their association with atherosclerotic plaques in human(s) is lacking. Methods and results Exploratory microarray (MA) was first performed followed by selective quantitative validation of robustly expressed genes with qRT-PCR low-density array (LDA). In LDA, ITGA1 (1.30-fold, p = 0.041) and ITGB3 (1.37-fold, p = 0.036) were upregulated in whole blood samples of patients with coronary artery disease (CAD) compared to healthy controls. In arterial plaques, both robustly expressed transcript variants of FERMT3 (MA: 5.90- and 3.4-fold; LDA: 3.99-fold, p < 0.0001 for all) and ITGB2 (MA: 4.81- and 4.92-fold; LDA: 5.29-fold, p < 0.0001 for all) were upregulated while FERMT2 was downregulated (MA: −1.61-fold; LDA: −2.88-fold, p < 0.0001 for both). The other integrins (ITGA1, ITGAV, ITGB3, ITGB5) were downregulated. All these results were replicated in at least one arterial bed. The latter FERMT3 transcript variant associated with unstable plaques (p = 0.0004). FERMT3 correlated with M2 macrophage markers and in hierarchical cluster analysis clustered with inflammatory and macrophage markers, while FERMT2 correlated with SMC-rich plaque markers and clustered with SMC markers. In confocal immunofluorescence analysis, FERMT3 protein colocalized with abundant CD68-positive cells of monocytic origin in the atherosclerotic plaques, while co-localization of FERMT3 with HHF35 indicative of smooth muscle cells was low. Conclusions Kindlin-3 (FERMT3) is upregulated in atherosclerotic, especially unstable plaques, mainly in cells of monocytic origin and of M2 type. Simultaneous upregulation of ITGB2 suggests a synergistic effect on leukocyte adherence and transmigration into the vessel wall. [ABSTRACT FROM AUTHOR]

Integrins mediate leukocyte accumulation to the sites of inflammation, thereby enhancing their potential as an important therapeutic target for inflammatory disorders. Integrin activation triggered by inflammatory mediators or signaling pathway is a key step to initiate leukocyte migration to inflamed tissues; however, an appropriately regulated integrin deactivation is indispensable for maintaining productive leukocyte migration. While typical integrin antagonists that block integrin activation target the initiation of leukocyte migration, a novel class of experimental compounds has been designed to block integrin deactivation, thereby perturbing the progression of cell migration. Current review discusses the mechanisms by which integrin is activated and subsequently deactivated by focusing on its structure-function relationship. [ABSTRACT FROM AUTHOR]

Studies on the mechanism of integrin inside-out activation have been focused on the role of β-integrin cytoplasmic tails, which are relatively conserved and bear binding sites for the intracellular activators including talin and kindlin. Cytoplasmic tails for α-integrins share a conserved GFFKR motif at the membrane-proximal region and this forms a specific interface with the β-integrin membrane-proximal region to keep the integrin inactive. The α-integrin membrane-distal regions, after the GFFKR motif, are diverse both in length and sequence and their roles in integrin activation have not been well-defined. In this study, we report that the α-integrin cytoplasmic membrane-distal region contributes to maintaining integrin in the resting state and to integrin inside-out activation. Complete deletion of the α-integrin membrane-distal region diminished talin-and kindlin-mediated integrin ligand binding and conformational change. A proper length and suitable amino acids in α-integrin membrane-distal region was found to be important for integrin inside-out activation. Our data establish an essential role for the α-integrin cytoplasmic membrane-distal region in integrin activation and provide new insights into how talin and kindlin induce the high-affinity integrin conformation that is required for fully functional integrins. [ABSTRACT FROM AUTHOR]

Kindlins are essential FERM-domain-containing focal adhesion (FA) proteins required for proper integrin activation and signaling. Despite the widely accepted importance of each of the three mammalian kindlins in cell adhesion, the molecular basis for their function has yet to be fully elucidated, and the functional differences between isoforms have generally not been examined. Here, we report functional differences between kindlin-2 and -3 (also known as FERMT2 and FERMT3, respectively); GFP-tagged kindlin-2 localizes to FAs whereas kindlin-3 does not, and kindlin-2, but not kindlin-3, can rescue α5β1 integrin activation defects in kindlin-2- knockdown fibroblasts. Using chimeric kindlins, we show that the relatively uncharacterized kindlin-2 F2 subdomain drives FA targeting and integrin activation. We find that the integrin-linked kinase (ILK)-PINCH-parvin complex binds strongly to the kindlin-2 F2 subdomain but poorly to that of kindlin-3. Using a point-mutated kindlin-2, we establish that efficient kindlin-2-mediated integrin activation and FA targeting require binding to the ILK complex. Thus, ILK-complex binding is crucial for normal kindlin-2 function and differential ILK binding contributes to kindlin isoform specificity. [ABSTRACT FROM AUTHOR]

The kindlin/fermitin family includes three proteins involved in regulating integrin ligand-binding activity and adhesion. Loss-of-function mutations in kindlins1 and 3 have been implicated in Kindler Syndrome and Leukocyte Adhesion Deficiency III (LAD-III) respectively, whereas kindlin2 null mice are embryonic lethal. Post translational regulation of cell–cell and cell-ECM adhesion has long been presumed to be important for morphogenesis, however, few specific examples of activation-dependent changes in adhesion molecule function in normal development have been reported. In this study, antisense morpholinos were used to reduce expression of individual kindlins in Xenopus laevis embryos in order to investigate their roles in early development. Kindlin1 knockdown resulted in developmental delays, gross malformations of the gut and eventual lethality by tadpole stages. Kindlin2 morphant embryos displayed late stage defects in vascular maintenance and angiogenic branching consistent with kindlin2 loss of function in the mouse. Antisense morpholinos were also used to deplete maternal kindlin2 protein in oocytes and eggs. Embryos lacking maternal kindlin2 arrested at early cleavage stages due to failures in cytokinesis. Kindlin3 morphant phenotypes included defects in epidermal ciliary beating and partial paralysis at tailbud stages but these embryos recovered eventually as morpholino levels decayed. These results indicate a remarkably diverse range of kindlin functions in vertebrate development. [ABSTRACT FROM AUTHOR]

We describe a strategy for exploring the function of protein-protein interactions in striated muscle in vivo. We describe our experience using this strategy to study the interaction of UNC-112 (kindlin) with PAT-4 (integrin linked kinase). Random mutagenesis is used to generate a collection of mutants that are screened for lack of binding or gain of binding using a yeast 2-hybrid assay. The mutant proteins are then expressed in transgenic C. elegans to determine their ability to localize in the sarcomere. We emphasize two advantages of this strategy: (1) for studying the interaction of protein A with protein B, when protein A can interact with multiple proteins, and (2) it explores the function of an interaction rather than the absence of, or reduced level of, a protein as can be obtained with null mutants or knockdown by RNAi. We propose that this method can be generalized for studying the meaning of a protein-protein interaction in muscle for any system in which transgenic animals can be generated and their muscles can be imaged. [ABSTRACT FROM AUTHOR]

The article discusses a study on the role of FERMT1 gene mutations in the secretion of cytokines mediating local inflammation and fibrosis in Kindler syndrome (KS). The study showed that the lack of intracellular kindlin-1 in epidermal keratinocytes resulted to changes in the dermis. It found that kindlin-1-deficient keratinocytes upregulated the expression of cytokines including interleukin-20 (IL-20), IL-24 and transforming growth factor-β2 (TGF-β2) in response to cell stress.

Glioblastomas are characterized by an intense local invasiveness that limits surgical resection. One mechanism by which glioma cells enforce their migration into brain tissue is reorganization of tumour associated extracellular matrix (ECM). Collagen XVI is a minor component of connective tissues. However, in glioblastoma tissue it is dramatically upregulated compared to the ECM of normal cortex. The aim of this study is to delineate tumour cell invasion and underlying mechanisms involving collagen XVI by using a siRNA mediated collagen XVI knockdown model in U87MG human glioblastoma cells. Knockdown of collagen XVI resulted in decreased invasiveness in Boyden chamber assays, and in a reduction of focal adhesion contact numbers per cell. Gene expression was upregulated for protocadherin 18 and downregulated for kindlin-1 and -2. Proliferation was not affected while flow cytometric analysis demonstrated reduced β1-integrin activation in collagen XVI knockdown cells. We suggest that in glioblastoma tissue collagen XVI may impair the cell-cell interaction in favour of enhancement of invasion. The modification of the β1-integrin activation pattern through collagen XVI might be a molecular mechanism to further augment the invasive phenotype of glioma cells. Elucidating the underlying mechanisms of glioma cell invasion promoted by collagen XVI may provide novel cancer therapeutic approaches in neurooncology. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: The kindlins represent a class of focal adhesion proteins implicated in integrin activation. They comprise three evolutionarily conserved members, kindlin-1, kindlin-2 and kindlin-3, that share considerable sequence and structural similarities. The kindlins have a bipartite FERM (four point one protein, ezrin, radixin, moesin) domain interrupted by a pleckstrin homology domain and can bind directly to various classes of integrins as well as participate in inside–out integrin activation. They are encoded by three different genes, namely KIND1 (FERMT1; chromosome 20p12.3), KIND2 (FERMT2; chromosome 14q22.1) and KIND3 (FERMT3; chromosome 11q13.1). Loss-of-function mutations in KIND1 and KIND3 cause Kindler syndrome and leukocyte adhesion deficiency-III syndrome, respectively, although no human disease has yet been associated with KIND2 gene pathology. In this review, we focus on the cellular functions of the kindlins and their clinical relevance. [Copyright &y& Elsevier]

Abstract: The integrin family of heterodimeric cell adhesion molecules exists in both low- and high-affinity states, and integrin activation requires binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to membrane-proximal sequences in the β-integrin cytoplasmic domain. However, it has recently become apparent that the kindlin family of FERM domain proteins is also essential for talin-induced integrin activation. FERM domains are typically composed of F1, F2, and F3 domains, but the talin FERM domain is atypical in that it contains a large insert in F1 and is preceded by a previously unrecognized domain, F0. Initial sequence alignments showed that the kindlin FERM domain was most similar to the talin FERM domain, but the homology appeared to be restricted to the F2 and F3 domains. Based on a detailed characterization of the talin FERM domain, we have reinvestigated the sequence relationship with kindlins and now show that kindlins do indeed contain the same domain structure as the talin FERM domain. However, the kindlin F1 domain contains an even larger insert than that in talin F1 that disrupts the sequence alignment. The insert, which varies in length between different kindlins, is not conserved and, as in talin, is largely unstructured. We have determined the structure of the kindlin-1 F0 domain by NMR, which shows that it adopts the same ubiquitin-like fold as the talin F0 and F1 domains. Comparison of the kindlin-1 and talin F0 domains identifies the probable interface with the kindlin-1 F1 domain. Potential sites of interaction of kindlin F0 with other proteins are discussed, including sites that differ between kindlin-1, kindlin-2, and kindlin-3. We also demonstrate that F0 is required for the ability of kindlin-1 to support talin-induced αIIbβ3 integrin activation and for the localization of kindlin-1 to focal adhesions. [Copyright &y& Elsevier]

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Aims: Leiomyosarcomas (LMS) are malignant neoplasms composed of cells that exhibit distinct smooth muscle differentiation. The molecular and cytogenetic features of LMS are complex and no consistent aberrations have been reported to date. Mitogen inducible gene-2 (Mig-2), kindlin and migfilin are recently identified cell–matrix adhesion proteins. The aim was to determine the expression and distribution of these proteins in human smooth muscle tumours of somatic soft tissue. Methods and results: Immunohistochemistry was performed on a human LMS tissue microarray and on sections of human leiomyomas (LM) and normal smooth muscle. Migfilin was barely detectable in normal smooth muscle cells, whereas increased levels of migfilin were observed in the majority of LM and LMS. Furthermore, the cytoplasmic level of migfilin was strongly associated with higher tumour grades. Additionally, the cytoplasmic levels of migfilin and Mig-2 were correlated with each other, suggesting an association between the two in the cytoplasm. Kindlin was expressed in normal smooth muscle, LM and LMS, and its level did not correlate with tumour grade. Conclusions: Our results suggest a role for cytoplasmic migfilin in the progression of LMS and identify cytoplasmic migfilin as a potentially important biological marker for human LMS progression. [ABSTRACT FROM AUTHOR]

Links between the plasma membrane and the actin cytoskeleton are essential for maintaining tissue integrity and for controlling cell morphology and behavior. Studies over the past several decades have identified dozens of components of such junctions. One of the most recently identified is migfilin, a widely expressed protein consisting of an N-terminal filamin-binding domain, a central proline-rich domain and three C-terminal LIM domains. Migfilin is recruited to cell-matrix contacts in response to adhesion and colocalizes with β-catenin at cell-cell junctions in epithelial and endothelial cells. Migfilin also travels from the cytoplasm into the nucleus, a process that is regulated by RNA splicing and calcium signaling. Through interactions with multiple binding partners, including Mig-2, filamin and VASP, migfilin links the cell adhesion structures to the actin cytoskeleton. It regulates actin remodeling, cell morphology and motility. In nuclei, migfilin interacts with the cardiac transcriptional factor CSX/NKX2-5 and promotes cardiomyocyte differentiation. It probably functions as a key regulator both at cell adhesion sites and nuclei, coordinating multiple cellular processes, and is implicated in the pathogenesis of several human diseases. [ABSTRACT FROM AUTHOR]