Protein Talin Research Articles

Precise coordination of cell-ECM adhesion is essential for efficient melanoblast migration during development.

Melanoblasts disperse throughout the skin and populate hair follicles through long-range cell migration. During migration, cells undergo cycles of coordinated attachment and detachment from the extracellular matrix (ECM). Embryonic migration processes that require cell-ECM attachment are dependent on the integrin family of adhesion receptors. Precise regulation of integrin-mediated adhesion is important for many developmental migration events. However, the mechanisms that regulate integrin-mediated adhesion in vivo in melanoblasts are not well understood. Here, we show that autoinhibitory regulation of the integrin-associated adapter protein talin coordinates cell-ECM adhesion during melanoblast migration in vivo Specifically, an autoinhibition-defective talin mutant strengthens and stabilizes integrin-based adhesions in melanocytes, which impinges on their ability to migrate. Mice with defective talin autoinhibition exhibit delays in melanoblast migration and pigmentation defects. Our results show that coordinated integrin-mediated cell-ECM attachment is essential for melanoblast migration and that talin autoinhibition is an important mechanism for fine-tuning cell-ECM adhesion during cell migration in development.

Phosphoinositides regulate force-independent interactions between talin, vinculin, and actin.

Focal adhesions (FA) are large macromolecular assemblies which help transmit mechanical forces and regulatory signals between the extracellular matrix and an interacting cell. Two key proteins talin and vinculin connecting integrin to actomyosin networks in the cell. Both proteins bind to F-actin and each other, providing a foundation for network formation within FAs. However, the underlying mechanisms regulating their engagement remain unclear. Here, we report on the results of in vitro reconstitution of talin-vinculin-actin assemblies using synthetic membrane systems. We find that neither talin nor vinculin alone recruit actin filaments to the membrane. In contrast, phosphoinositide-rich membranes recruit and activate talin, and the membrane-bound talin then activates vinculin. Together, the two proteins then link actin to the membrane. Encapsulation of these components within vesicles reorganized actin into higher-order networks. Notably, these observations were made in the absence of applied force, whereby we infer that the initial assembly stage of FAs is force independent. Our findings demonstrate that the local membrane composition plays a key role in controlling the stepwise recruitment, activation, and engagement of proteins within FAs.

Anti-Adhesion Behavior from Ring-Strain Amine Cyclic Monolayers Grafted on Silicon (111) Surfaces

In this manuscript, a series of amine tagged short cyclic molecules (cyclopropylamine, cyclobutylamine, cyclopentylamine and cyclohexylamine) were thermally grafted onto p-type silicon (111) hydride surfaces via nucleophilic addition. The chemistries of these grafting were verified via XPS, AFM and sessile droplet measurements. Confocal microscopy and cell viability assay was performed on these surfaces incubated for 24 hours with triple negative breast cancer cells (MDA-MB 231), gastric adenocarcinoma cells (AGS) endometrial adenocarcinoma (Hec1A). All cell types had shown a significant reduction when incubated on these ring-strain cyclic monolayer surfaces than compared to standard controls. The expression level of focal adhesion proteins (vinculin, paxilin, talin and zyxin) were subsequently quantified for all three cell types via qPCR analysis. Cells incubate on these surface grafting were observed to have reduced levels of adhesion protein expression than compared to positive controls (collagen coating and APTES). A potential application of these anti-adhesive surfaces is the maintenance of the chondrocyte phenotype during in-vitro cell expansion. Articular chondrocytes cultured for 6 days on ring strained cyclopropane-modified surfaces was able to proliferate but had maintained a spheroid/aggregated phenotype with higher COL2A1 and ACAN gene expression. Herein, these findings had help promote grafting of cyclic monolayers as an viable alternative for producing antifouling surfaces.

Basigin Associates with Integrin in Order to Regulate Perineurial Glia and Drosophila Nervous System Morphology.

The Drosophila nervous system is ensheathed by a layer of outer glial cells, the perineurial glia, and a specialized extracellular matrix, the neural lamella. The function of perineurial glial cells and how they interact with the extracellular matrix are just beginning to be elucidated. Integrin-based focal adhesion complexes link the glial membrane to the extracellular matrix, but little is known about integrin's regulators in the glia. The transmembrane Ig domain protein Basigin/CD147/EMMPRIN is highly expressed in the perineurial glia surrounding the Drosophila larval nervous system. Here we show that Basigin associates with integrin at the focal adhesions to uphold the structure of the glia-extracellular matrix sheath. Knockdown of Basigin in perineurial glia using RNAi results in significant shortening of the ventral nerve cord, compression of the glia and extracellular matrix in the peripheral nerves, and reduction in larval locomotion. We determined that Basigin is expressed in close proximity to integrin at the glial membrane, and that expression of the extracellular integrin-binding domain of Basigin is sufficient to rescue peripheral glial compression. We also found that a reduction in expression of integrin at the membrane rescues the ventral nerve cord shortening, peripheral glial compression, and locomotor phenotypes, and that reduction in the integrin-binding protein Talin can partially rescue glial compression. These results identify Basigin as a potential negative regulator of integrin in the glia, supporting proper glial and extracellular matrix ensheathment of the nervous system.SIGNIFICANCE STATEMENT The glial cells and extracellular matrix play important roles in supporting and protecting the nervous system, but the interactions between these components have not been well characterized. Our study identified expression of a conserved Ig superfamily protein, Basigin, at the glial membrane of Drosophila where it associates with the integrin-based focal adhesion complexes to ensure proper ensheathment of the CNS and PNS. Loss of Basigin in the glia results in an overall compression of the nervous system due to integrin dysregulation, which causes locomotor defects in the animals. This underlies the importance of glia-matrix communication for structural and functional support of the nervous system.

Inward Tension of Talin and Integrin-related Osmotic Pressure are involved Synergetically in the Invasion and Metastasis of Non-small Cell Lung Cancer.

The integrin receptor protein talin plays vital roles in intracellular chemical and mechanical activities, and it is implicated in the high invasion and poor prognosis of non-small cell lung cancer (NSCLC). To better understand the mechanism underlying the function of talin in NSCLC invasion and metastasis, a few newly designed tension probe based on Förster resonance energy transfer was used for real-time observation of tension changes in A549 cells. High NSCLC cell aggressiveness was found to be accompanied with inward talin and outward glial fibrillary acidic protein (GFAP) tensions, which are closely associated with microfilament (MF) force and intracellular osmotic potential. The increased osmotic pressure resulted from the production of intracellular protein nanoparticles and the related ion influx. Furthermore, integrin activation was found to adjust the talin and GFAP tensions. Disruption of the interaction between talin and MFs blocked the mechanical source of talin, reducing both talin tension and osmotic pressure and thus inhibiting NSCLC cell invasion and migration. Consequently, our study demonstrates that talin is involved in NSCLC invasion and migration via its inward tension and that the integrin pathway is correlated closely with protein-nanoparticle-induced outward osmotic pressure.

Inhibition of Smooth Muscle Cell Proliferation and Migration by a Talin Modulator Attenuates Neointimal Formation after Femoral Arterial Injury.

Background and ObjectivesVascular smooth muscle cell (SMC) proliferation and migration play a critical role in neointimal formation. Focal adhesion is involved in cell proliferation and migration, and talin is known to be a key regulator of these processes. We synthesized a new talin modulator that binds to the talin protein, and investigated its effects on SMCs and neointimal formation after vascular injury.MethodsHuman aortic SMCs (HAoSMCs) were treated with a newly synthesized talin modulator. Apolipoprotein E knockout (ApoE KO) mice were subjected to left femoral arterial injury and orally administered with the talin modulator daily. Laser Doppler imager was used to compare the blood flow, and injured femoral arteries and blood serum were analyzed after 28 days.ResultsThe talin modulator significantly inhibited cell proliferation in a concentration-dependent manner and suppressed the migration of HAoSMCs. Treatment with a talin modulator resulted in a significant reduction in the phosphorylation of focal adhesion molecules and downstream signaling molecules related to cell proliferation and migration. The effects of the talin modulator in HAoSMCs were found to be reversible, as evidenced by the reactivation of signaling pathways upon its removal. After 28 days of administration of the talin modulator, an improvement in the blood flow and reduction in neointimal formation in the injured femoral arteries were observed.ConclusionsWe demonstrated the inhibitory effects of a talin modulator on SMC proliferation and migration, and that were associated with downregulation of signaling pathways, resulting in the attenuation of neointimal formation in ApoE KO mice.

Fibronectin is up‐regulated in podocytes by mechanical stress

Hypertension is one of the central causes of kidney damage. In the past it was shown that glomerular hypertension leads to morphologic changes of podocytes and effacement and is responsible for detachment of these postmitotic cells. Because we have shown that podocytes are mechanosensitive and respond to mechanical stress by reorganization of the actin cytoskeleton in vitro, we look for mechanotransducers in podocytes. In this study, we demonstrate that the extracellular matrix protein fibronectin (Fn1) might be a potential candidate. The present study shows that Fn1 is essential for the attachment of podocytes during mechanical stress. By real-time quantitative PCR as well as by liquid chromatography-mass spectrometry, we found a significant up-regulation of Fn1 caused by mechanical stretch (3 d, 0.5 Hz, and 5% extension). To study the role of Fn1 in cultured podocytes under mechanical stress, Fn1 was knocked down (Fn1 KD) by a specific small interfering RNA. Additionally, we established a Fn1 knockout (KO) podocyte cell line (Fn1 KO) by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). During mechanical stress, a significant loss of podocytes (>80%) was observed in Fn1 KD as well as Fn1 KO podocytes compared with control cells. Furthermore, Fn1 KO podocytes showed a significant down-regulation of the focal adhesion proteins talin, vinculin, and paxillin and a reduced cell spreading, indicating an important role of Fn1 in adhesion. Analyses of kidney sections from patients with diabetic nephropathy have shown a significant up-regulation of FN1 in contrast to control biopsies. In summary, we show that Fn1 plays an important role in the adaptation of podocytes to mechanical stress.-Kliewe, F., Kaling, S., Lötzsch, H., Artelt, N., Schindler, M., Rogge, H., Schröder, S., Scharf, C., Amann, K., Daniel, C., Lindenmeyer, M. T., Cohen, C. D., Endlich, K., Endlich, N. Fibronectin is up-regulated in podocytes by mechanical stress.

Force-Dependent Regulation of Talin-KANK1 Complex at Focal Adhesions.

KANK proteins mediate cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix. KANKs interact with the integrin/actin-binding protein talin and with several components of microtubule-stabilizing cortical complexes. Because of actomyosin contractility, the talin-KANK complex is likely under mechanical force, and its mechanical stability is expected to be a critical determinant of KANK recruitment to focal adhesions. Here, we quantified the lifetime of the complex of the talin rod domain R7 and the KN domain of KANK1 under shear-force geometry and found that it can withstand forces for seconds to minutes over a physiological force range up to 10 pN. Complex stability measurements combined with cell biological experiments suggest that shear-force stretching promotes KANK1 localization to the periphery of focal adhesions. These results indicate that the talin-KANK1 complex is mechanically strong, enabling it to support the cross-talk between microtubule and actin cytoskeleton at focal adhesions.

A mechano-signalling network linking microtubules, myosin IIA filaments and integrin-based adhesions.

The interrelationship between microtubules and the actin cytoskeleton in mechanoregulation of integrin-mediated adhesions is poorly understood. Here, we show that the effects of microtubules on two major types of cell-matrix adhesion, focal adhesions and podosomes, are mediated by KANK family proteins connecting the adhesion protein talin with microtubule tips. Both total microtubule disruption and microtubule uncoupling from adhesions by manipulations with KANKs trigger a massive assembly of myosin IIA filaments, augmenting focal adhesions and disrupting podosomes. Myosin IIA filaments are indispensable effectors in the microtubule-driven regulation of integrin-mediated adhesions. Myosin IIA filament assembly depends on Rho activation by the RhoGEF GEF-H1, which is trapped by microtubules when they are connected with integrin-mediated adhesions via KANK proteins but released after their disconnection. Thus, microtubule capture by integrin-mediated adhesions modulates the GEF-H1-dependent effect of microtubules on the assembly of myosin IIA filaments. Subsequent actomyosin reorganization then remodels the focal adhesions and podosomes, closing the regulatory loop.

Nephrin Signaling Results in Integrin β1 Activation.

Patients with certain mutations in the gene encoding the slit diaphragm protein Nephrin fail to develop functional slit diaphragms and display severe proteinuria. Many adult-onset glomerulopathies also feature alterations in Nephrin expression and function. Nephrin signals from the podocyte slit diaphragm to the Actin cytoskeleton by recruiting proteins that can interact with C3G, a guanine nucleotide exchange factor of the small GTPase Rap1. Because Rap activity affects formation of focal adhesions, we hypothesized that Nephrin transmits signals to the Integrin receptor complex, which mediates podocyte adhesion to the extracellular matrix. To investigate Nephrin's role in transmitting signals to the Integrin receptor complex, we conducted genetic studies in Drosophila nephrocytes and validated findings from Drosophila in a cultured human podocyte model. Drosophila nephrocytes form a slit diaphragm-like filtration barrier and express the Nephrin ortholog Sticks and stones (Sns). A genetic screen identified c3g as necessary for nephrocyte function. In vivo, nephrocyte-specific gene silencing of sns or c3g compromised nephrocyte filtration and caused nephrocyte diaphragm defects. Nephrocytes with impaired Sns or C3G expression displayed an altered localization of Integrin and the Integrin-associated protein Talin. Furthermore, gene silencing of c3g partly rescued nephrocyte diaphragm defects of an sns overexpression phenotype, pointing to genetic interaction of sns and c3g in nephrocytes. We also found that activated Nephrin recruited phosphorylated C3G and resulted in activation of Integrin β1 in cultured podocytes. Our findings suggest that Nephrin can mediate a signaling pathway that results in activation of Integrin β1 at focal adhesions, which may affect podocyte attachment to the extracellular matrix.

Profiling of Ubiquitination Modification Sites in Talin in Colorectal Carcinoma by Mass Spectrometry

Talin protein was partially purified from human colorectal carcinoma tissues, which was subject to tryptic digestion. Immunoaffinity precipitation with specific antibodies that recognize diglycyl-lysine(Lys) remnants from tryptic digestion of ubiquitinated peptides was used to enrich ubiquitinated sites in talin. Mass spectrometry coupled with capillary reverse-phase high-performance liquid chromatography was used to analyze the enriched peptides. Specifically, four peptides containing diglycyl-Lys remnants from talin, namely, TAK(ub)VLVEDTK, QQQYK(ub) FLPSELRDEH, K(ub)STVLQQQYNR, and EGILK(ub)TAK can be determined using mass spectrometric data. This study provides an analytical method for further study in the relationship between ubiquitination modification of talin and its biological activity in colorectal cancer tissues with different pathological processes.

Talin-mediated force transmission and talin rod domain unfolding independently regulate adhesion signaling.

Talin protein is one of the key components in integrin-mediated adhesion complexes. Talins transmit mechanical forces between β-integrin and actin, and regulate adhesion complex composition and signaling through the force-regulated unfolding of talin rod domain. Using modified talin proteins, we demonstrate that these functions contribute to different cellular processes and can be dissected. The transmission of mechanical forces regulates adhesion complex composition and phosphotyrosine signaling even in the absence of the mechanically regulated talin rod subdomains. However, the presence of the rod subdomains and their mechanical activation are required for the reinforcement of the adhesion complex, cell polarization and migration. Talin rod domain unfolding was also found to be essential for the generation of cellular signaling anisotropy, since both insufficient and excess activity of the rod domain severely inhibited cell polarization. Utilizing proteomics tools, we identified adhesome components that are recruited and activated either in a talin rod-dependent manner or independently of the rod subdomains. This study clarifies the division of roles between the force-regulated unfolding of a talin protein (talin 1) and its function as a physical linker between integrins and the cytoskeleton.

A small proportion of Talin molecules transmit forces at developing muscle attachments in vivo.

Cells in developing organisms are subjected to particular mechanical forces that shape tissues and instruct cell fate decisions. How these forces are sensed and transmitted at the molecular level is therefore an important question, one that has mainly been investigated in cultured cells in vitro. Here, we elucidate how mechanical forces are transmitted in an intact organism. We studied Drosophila muscle attachment sites, which experience high mechanical forces during development and require integrin-mediated adhesion for stable attachment to tendons. Therefore, we quantified molecular forces across the essential integrin-binding protein Talin, which links integrin to the actin cytoskeleton. Generating flies expressing 3 Förster resonance energy transfer (FRET)-based Talin tension sensors reporting different force levels between 1 and 11 piconewton (pN) enabled us to quantify physiologically relevant molecular forces. By measuring primary Drosophila muscle cells, we demonstrate that Drosophila Talin experiences mechanical forces in cell culture that are similar to those previously reported for Talin in mammalian cell lines. However, in vivo force measurements at developing flight muscle attachment sites revealed that average forces across Talin are comparatively low and decrease even further while attachments mature and tissue-level tension remains high. Concomitantly, the Talin concentration at attachment sites increases 5-fold as quantified by fluorescence correlation spectroscopy (FCS), suggesting that only a small proportion of Talin molecules are mechanically engaged at any given time. Reducing Talin levels at late stages of muscle development results in muscle–tendon rupture in the adult fly, likely as a result of active muscle contractions. We therefore propose that a large pool of adhesion molecules is required to share high tissue forces. As a result, less than 15% of the molecules experience detectable forces at developing muscle attachment sites at the same time. Our findings define an important new concept of how cells can adapt to changes in tissue mechanics to prevent mechanical failure in vivo.

Talin2 regulates invasion of human breast cancer MDA-MB-231 cells via alteration of the tumor microenvironment.

The talin proteins are a key component of the extracellular matrix-integrin-cytoskeleton system, and our previous study suggested that talin2 contributes to the tumor invasion and metastasis processes regulated by the tumor microenvironment. In the present study, the specific effects of talin2 on the invasive ability of breast cancer cells, as well as the underlying mechanism, were investigated by creating two MDA-MB-231 cell lines with stable talin2 knockdown by specific RNA interference. Initially, it was confirmed that the expression levels of talin2 in human breast cancer tissues were upregulated compared with in normal adjacent tissues. Subsequently, invasion and wound healing assays revealed that depletion of talin2 in MDA-MB-231 cells decreased their migratory and invasive abilities. Western blot analysis demonstrated that knockdown of talin2 in MDA-MB-231 cells caused marked downregulation of the tumor microenvironment markers hypoxia-inducible factor 1α, phosphorylated ribosomal protein S6 kinase, phosphorylated protein kinase B and phosphorylated mechanistic target of rapamycin. Furthermore, knockdown of talin2 decreased the basal contents of glucose and lactic acid in the breast cancer cell line. In conclusion, the findings of the present study demonstrated that talin2 knockdown may inhibit the invasive ability of human breast cancer MDA-MB-23l cells via alterations in the tumor microenvironment.

Talin-Dependent Integrin Activation Regulates VE-Cadherin Localization and Endothelial Cell Barrier Function.

Endothelial barrier function depends on the proper localization and function of the adherens junction protein VE (vascular endothelial)-cadherin. Previous studies have suggested a functional relationship between integrin-mediated adhesion complexes and VE-cadherin yet the underlying molecular links are unclear. Binding of the cytoskeletal adaptor protein talin to the β-integrin cytoplasmic domain is a key final step in regulating the affinity of integrins for extracellular ligands (activation) but the role of integrin activation in VE-cadherin mediated endothelial barrier function is unknown. To test the requirement of talin-dependent activation of β1 integrin in VE-cadherin organization and endothelial cell (EC) barrier function. EC-specific deletion of talin in adult mice resulted in impaired stability of intestinal microvascular blood vessels, hemorrhage, and death. Talin-deficient endothelium showed altered VE-cadherin organization at EC junctions in vivo. shRNA (short hairpin RNA)-mediated knockdown of talin1 expression in cultured ECs led to increased radial actin stress fibers, increased adherens junction width and increased endothelial monolayer permeability measured by electrical cell-substrate impedance sensing. Restoring β1-integrin activation in talin-deficient cells with a β1-integrin activating antibody normalized both VE-cadherin organization and EC barrier function. In addition, VE-cadherin organization was normalized by reexpression of talin or integrin activating talin head domain but not a talin head domain mutant that is selectively deficient in activating integrins. Talin-dependent activation of EC β1-integrin stabilizes VE-cadherin at endothelial junctions and promotes endothelial barrier function.

Talin-2 regulates integrin functions in exosomes

Integrins on exosomes have been shown to mediate binding to recipient cells, potentially playing important roles in controlling exosomal internalization and organ distributions. Although the ability of cellular integrins to mediate cell adhesion is known to be regulated by the cytoplasmic adaptor protein talin, whether the activity of exosomal integrins is similarly regulated by talin remains to be elucidated. Here we have studied this question in T-cell exosomes that surface express the integrins αLβ2 and α4β7. T-cells and T-cell exosomes engineered to lack talin-2 showed reduced binding to the integrin ligand ICAM-1 and MAdCAM-1 compared with control T-cells and exosomes, despite the fact that those T cells and exosomes express intact levels of the other isoform talin-1. In addition, talin-2-deficient T-cell exosomes were less efficiently internalized by endothelial cells, compared with control exosomes. These results suggest that the mechanisms of talin-mediated integrin regulation operate similarly in cells and exosomes.

The activated β-integrin (CgβV) enhances RGD-binding and phagocytic capabilities of hemocytes in Crassostrea gigas

Integrins are an important family of cell receptors that can bind foreign particles and promote cell phagocytosis after they are activated. In the present study, a novel β integrin was identified from pacific oyster Crassostrea gigas with an extracellular domain, a single transmembrane segment, and a short cytoplasmic domain. It was phylogenetically clustered with phagocytosis-related insecta βV, and designated as CgβV. CgβV shared a conserved NPX[Y/F] motif related to integrin activation with other phagocytosis-related β integrins. The mRNA transcripts of CgβV were widely detected in oyster tissues including hemocytes, gonad, adductor muscle, mantle, gill, and hepatopancreas, and the expression level in hemocytes was significantly up-regulated at 12 h after lipopolysaccharide (LPS) stimulation (p < 0.05), which was 2.29-fold higher than that in the control group. CgβV proteins were mainly observed on the hemocytes surface. The oyster hemocytes were found to bind fluorescein isothiocyanate (FITC)-labeled Arg-Gly-Asp-containing peptides (RGDCPs), and the binding capability was significantly up-regulated with the peak percentage of 37.6% at 12 h post LPS stimulation, which was higher than that in the control group (8.8%, p < 0.05), suggesting the activation of RGD-binding integrins on oyster hemocytes surface. The label-free RGDCPs and anti-CgβV antibody inhibited the binding capability of hemocytes towards FITC-labeled RGDCPs, which were significant lower in RGD blocking group (7.4%, p < 0.05) and anti-CgβV blocking group (22.1%, p < 0.05) than that in the control group (37.6%), indicating that CgβV could be a RGD-binding integrin. Phagocytosis assay demonstrated that LPS could enhance the phagocytosis of hemocytes towards Escherichia coli and fluorescent beads with the phagocytic rate (PR) of 18.3% and 17.4%, and phagocytic index (PI) of 5.29 and 37.71, respectively, which were significant higher than that in the control group (11.0% and 3.65 for E. coli, 9.8% and 29.26 for fluorescent beads, respectively, p < 0.05). In addition, both the label-free RGDCPs and anti-CgβV antibody significantly hindered the phagocytosis of hemocytes towards E. coli and fluorescent beads. After the E. coli and fluorescent beads were opsonized by oyster serum, the label-free RGDCPs still inhibited the phagocytosis of hemocytes towards them, while the anti-CgβV antibody could only inhibit the phagocytosis of hemocytes towards E. coli, suggesting that only the activated CgβV was involved in the enhancing phagocytosis for bacteria in oyster. Moreover, the key components of conserved integrin-mediated phagocytosis pathway including GTPases, talin proteins, Ca2+ and cAMP were all induced by LPS in hemocytes of oyster. All these results suggested that the activated CgβV enhanced RGD-binding and phagocytic capabilities of hemocytes, shedding lights on the mechanisms of integrin-mediated phagocytosis in mollusks.

Visualizing the 'backbone' of focal adhesions.

To understand how complex machines perform their functions, it is essential to map out the 'blueprints' of how their internal components are organized. Focal adhesions (FAs) are complex mechanobiological structures involved in a plethora of cell biological processes. The application of super-resolution microscopy in concert with protein engineering offers one approach to unravel the complexity of how individual proteins are organized within FAs. In our recent application, the FA protein talin was found to form a direct structural and physical link between integrin and actin. Interestingly, engineered talin constructs with alternate lengths rescaled the FA nanostructure accordingly. This helped establish that talin could be analogous to the backbone of FAs, serving as the mechanosensitive master coordinator of FA structural organization.

Direct binding of Talin to Rap1 is required for cell-ECM adhesion in Drosophila.

Attachment of cells to the extracellular matrix (ECM) via integrins is essential for animal development and tissue maintenance. The cytoplasmic protein Talin (encoded by rhea in flies) is necessary for linking integrins to the cytoskeleton, and its recruitment is a key step in the assembly of the adhesion complex. However, the mechanisms that regulate Talin recruitment to sites of adhesion in vivo are still not well understood. Here, we show that Talin recruitment to, and maintenance at, sites of integrin-mediated adhesion requires a direct interaction between Talin and the GTPase Rap1. A mutation that blocks the direct binding of Talin to Rap1 abolished Talin recruitment to sites of adhesion and the resulting phenotype phenocopies that seen with null alleles of Talin. Moreover, we show that Rap1 activity modulates Talin recruitment to sites of adhesion via its direct binding to Talin. These results identify the direct Talin-Rap1 interaction as a key in vivo mechanism for controlling integrin-mediated cell-ECM adhesion.

Inhibition of calpain delays early muscle atrophy after rotator cuff tendon release in sheep.

Chronic rotator cuff (RC) tears are characterized by retraction, fat accumulation, and atrophy of the affected muscle. These features pose an intractable problem for surgical repair and subsequent recovery, and their prevention may be easier than reversal. Using an established ovine model, we tested the hypothesis that inhibition of the protease calpain mitigates m. infraspinatus atrophy by preservation of the myofibers’ structural anchors in the sarcolemma (the costameres). Already 2 weeks of distal tendon release led to a reduction in muscle volume (−11.6 ± 9.1 cm3, P = 0.038) and a 8.3% slow‐to‐fast shift of the fiber area (P = 0.046), which were both entirely abolished by chronic local administration of the calpain inhibitor calpeptin alone, and in combination with sildenafil. Calpain inhibition blunted the retraction of the muscle‐tendon unit by 0.8–1.0 cm (P = 0.020) compared with the control group, and prevented cleavage of the costameric protein talin. Calpain 1 and 2 protein levels increased in the medicated groups after 4 weeks, counteracting the efficacy of calpeptin. Hence atrophic changes emerged after 4 weeks despite ongoing treatment. These findings suggest that the early muscular adaptations in the specific case of RC tear in the ovine model are indistinguishable from the atrophy and slow‐to‐fast fiber transformation observed with conventional unloading and can be prevented for 2 weeks. Concluding, calpain is a potential target to extend the temporal window for reconstruction of the ruptured RC tendon before recovery turns impossible.