Platelet SHARPIN regulates platelet adhesion and inflammatory responses through associations with αIIbß3 and LUBAC.

Platelets form hemostatic plugs to prevent blood loss, and they modulate immunity and inflammation in several ways. A key event during hemostasis is activation of integrin αIIbß3 through direct interactions of the ß3 cytoplasmic tail with talin and kindlin-3. Recently, we showed that human platelets express the adapter molecule Shank-associated RH domain interacting protein (SHARPIN), which can associate directly with the αIIb cytoplasmic tail and separately promote NF-κB pathway activation as a member of the Met-1 linear ubiquitination activation complex (LUBAC). Here we investigated the role of SHARPIN in platelets after crossing Sharpin flox/flox (fl/fl) mice with PF4-Cre or GPIbα-Cre mice to selectively delete SHARPIN in platelets. SHARPIN-null platelets adhered to immobilized fibrinogen through αIIbß3, and they spread more extensively than littermate control platelets in a manner dependent on feedback stimulation by platelet adenosine diphosphate (ADP) (P < .01). SHARPIN-null platelets showed increased colocalization of αIIbß3 with talin as assessed by super-resolution microscopy and increased binding of soluble fibrinogen in response to submaximal concentrations of ADP (P < .05). However, mice with SHARPIN-null platelets showed compromised thrombus growth on collagen and slightly prolonged tail bleeding times. Platelets lacking SHARPIN also showed reduced NF-κB activation and linear ubiquitination of protein substrates upon challenge with classic platelet agonists. Furthermore, the loss of platelet SHARPIN resulted in significant reduction in inflammation in murine models of colitis and peritonitis (P < .01). Thus, SHARPIN plays differential and context-dependent roles in platelets to regulate important inflammatory and integrin adhesive functions of these anucleate cells.

Optogenetics-based localization of talin to the plasma membrane promotes activation of ß3 integrins.

Interaction of talin with the cytoplasmic tails of integrin ß triggers integrin activation, leading to an increase of integrin affinity/avidity for extracellular ligands. In talin KO mice, loss of talin interaction with platelet integrin αIIbß3 causes a severe hemostatic defect, and loss of talin interaction with endothelial cell integrin αVß3 affects angiogenesis. In normal cells, talin is autoinhibited and localized in the cytoplasm. Here, we used an optogenetic platform to assess whether recruitment of full-length talin to the plasma membrane was sufficient to induce integrin activation. A dimerization module (Arabidopsis cryptochrome 2 fused to the N terminus of talin; N-terminal of cryptochrome-interacting basic helix-loop-helix domain ended with a CAAX box protein [C: cysteine; A: aliphatic amino acid; X: any C-terminal amino acid]) responsive to 450 nm (blue) light was inserted into Chinese hamster ovary cells and endothelial cells also expressing αIIbß3 or αVß3, respectively. Thus, exposure of the cells to blue light caused a rapid and reversible recruitment of Arabidopsis cryptochrome 2-talin to the N-terminal of cryptochrome-interacting basic helix-loop-helix domain ended with a CAAX box protein [C: cysteine; A: aliphatic amino acid; X: any C-terminal amino acid]-decorated plasma membrane. This resulted in ß3 integrin activation in both cell types, as well as increasing migration of the endothelial cells. However, membrane recruitment of talin was not sufficient for integrin activation, as membrane-associated Ras-related protein 1 (Rap1)-GTP was also required. Moreover, talin mutations that interfered with its direct binding to Rap1 abrogated ß3 integrin activation. Altogether, these results define a role for the plasma membrane recruitment of talin in ß3 integrin activation, and they suggest a nuanced sequence of events thereafter involving Rap1-GTP.

Genetic Instruction of Megakaryocytes and Platelets Derived from Human Induced Pluripotent Stem Cells for Studies of Integrin Regulation.

Platelets are small, anucleate cells that play oversized roles in hemostasis, immunity, and inflammation. An important mediator of platelet function is integrin αIIbß3, which is required for fibrinogen-dependent platelet aggregation during hemostasis. This platelet response is dependent on conformational changes in the integrin induced by "inside-out" biochemical signals that are triggered by platelet agonists. In turn, fibrinogen binding to αIIbß3 initiates "outside-in" biochemical and mechanical signals that regulate the platelet cytoskeleton and help to promote full platelet aggregation and secretory responses. Without a nucleus, there is a limited range of experimental manipulations that are possible with human platelets to study the molecular basis of integrin signaling in these primary cells. Consequently, many studies of αIIbß3 function use genetic approaches that rely on heterologous expression systems or platelets from gene-targeted mice, sometimes with uncertain applicability to human platelets. This chapter will detail a method for genetic manipulation of megakaryocytes and platelets derived from human induced pluripotent stem cells for molecular studies of αIIbß3 signaling and for modeling of human platelet functions potentially relevant to hemostasis, immunity, and inflammation.

Underlying Immune Disorder May Predispose Some Transthyretin Amyloidosis Subjects to Inotersen-Mediated Thrombocytopenia.

Inotersen, a 2'-O-methoxyethyl (2'-MOE) phosphorothioate antisense oligonucleotide, reduced disease progression and improved quality of life in patients with hereditary transthyretin amyloidosis with polyneuropathy (hATTR-PN) in the NEURO-TTR and NEURO-TTR open-label extension (OLE) trials. However, 300 mg/week inotersen treatment was associated with platelet count reductions in several patients. Mean platelet counts in patients in the NEURO-TTR-inotersen group remained ≥140 × 109/L in 50% and ≥100 × 109/L in 80% of the subjects. However, grade 4 thrombocytopenia (<25 × 109/L) occurred in three subjects in NEURO-TTR trial, and one of these suffered a fatal intracranial hemorrhage. The two others were treated successfully with corticosteroids and discontinuation of inotersen. Investigations in a subset of subjects in NEURO-TTR (n = 17 placebo; n = 31 inotersen) and OLE (n = 33) trials ruled out direct myelotoxicity, consumptive coagulopathy, and heparin-induced thrombocytopenia. Antiplatelet immunoglobulin G (IgG) antibodies were detected at baseline in 5 of 31 (16%) inotersen-treated subjects in NEURO-TTR, 4 of whom eventually developed grade 1 or 2 thrombocytopenia while on the drug. In addition, 24 subjects in the same group developed treatment-emergent antiplatelet IgG antibodies, of which 2 developed grade 2, and 3 developed grade 4 thrombocytopenia. Antiplatelet IgG antibodies in two of the three grade 4 thrombocytopenia subjects targeted GPIIb/IIIa. Plasma cytokines previously implicated in immune dysregulation, such as interleukin (IL)-23 and a proliferation-inducing ligand (APRIL) were often above the normal range at baseline. Collectively, these findings suggest an underlying immunologic dysregulation predisposing some individuals to immune-mediated thrombocytopenia during inotersen treatment.

SHARPIN at the nexus of integrin, immune, and inflammatory signaling in human platelets.

Platelets mediate primary hemostasis, and recent work has emphasized platelet participation in immunity and inflammation. The function of the platelet-specific integrin αIIbß3 as a fibrinogen receptor in hemostasis is well defined, but the roles of αIIbß3 or integrin-associated proteins in nonhemostatic platelet functions are poorly understood. Here we show that human platelets express the integrin-associated protein SHARPIN with functional consequences. In leukocytes, SHARPIN interacts with integrin α cytoplasmic tails, and it is also an obligate member of the linear ubiquitin chain assembly complex (LUBAC), which mediates Met1 linear ubiquitination of proteins leading to canonical NF-κB activation. SHARPIN interacted with αIIb in pull-down and coimmunoprecipitation assays. SHARPIN was partially localized, as was αIIbß3, at platelet edges, and thrombin stimulation induced more central SHARPIN localization. SHARPIN also coimmunoprecipitated from platelets with the two other proteins comprising LUBAC, the E3 ligase HOIP and HOIL-1. Platelet stimulation with thrombin or inflammatory agonists, including lipopolysaccharide or soluble CD40 ligand (sCD40L), induced Met1 linear ubiquitination of the NF-κB pathway protein NEMO and serine-536 phosphorylation of the p65 RelA subunit of NF-κB. In human megakaryocytes and/or platelets derived from induced pluripotent stem (iPS) cells, SHARPIN knockdown caused increased basal and agonist-induced fibrinogen binding to αIIbß3 as well as reduced Met1 ubiquitination and RelA phosphorylation. Moreover, these SHARPIN knockdown cells exhibited increased surface expression of MHC class I molecules and increased release of sCD40L. These results establish that SHARPIN functions in the human megakaryocyte/platelet lineage through protein interactions at the nexus of integrin and immune/inflammatory signaling.

uPAR isoform 2 forms a dimer and induces severe kidney disease in mice.

Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived circulating signaling molecule that has been implicated in chronic kidney disease, such as focal segmental glomerulosclerosis (FSGS). Typically, native uPAR (isoform 1) translates to a 3-domain protein capable of binding and activating integrins, yet the function of additional isoforms generated by alternative splicing is unknown. Here, we characterized mouse uPAR isoform 2 (msuPAR2), encoding domain I and nearly one-half of domain II, as a dimer in solution, as revealed by 3D electron microscopy structural analysis. In vivo, msuPAR2 transgenic mice exhibited signs of severe renal disease characteristic of FSGS with proteinuria, loss of kidney function, and glomerulosclerosis. Sequencing of the glomerular RNAs from msuPAR2-Tg mice revealed a differentially expressed gene signature that includes upregulation of the suPAR receptor Itgb3, encoding ß3 integrin. Crossing msuPAR2-transgenic mice with 3 different integrin ß3 deficiency models rescued msuPAR2-mediated kidney function. Further analyses indicated a central role for ß3 integrin and c-Src in msuPAR2 signaling and in human FSGS kidney biopsies. Administration of Src inhibitors reduced proteinuria in msuPAR2-transgenic mice. In conclusion, msuPAR2 may play an important role in certain forms of scarring kidney disease.

Rap1 binding to the talin 1 F0 domain makes a minimal contribution to murine platelet GPIIb-IIIa activation.

Activation of platelet glycoprotein IIb-IIIa (GPIIb-IIIa; integrin αIIbß3) leads to high-affinity fibrinogen binding and platelet aggregation during hemostasis. Whereas GTP-bound Rap1 GTPase promotes talin 1 binding to the ß3 cytoplasmic domain to activate platelet GPIIb-IIIa, the Rap1 effector that regulates talin association with ß3 in platelets is unknown. Rap1 binding to the talin 1 F0 subdomain was proposed to forge the talin 1-Rap1 link in platelets. Here, we report a talin 1 point mutant (R35E) that significantly reduces Rap1 affinity without a significant effect on its structure or expression. Talin 1 head domain (THD) (R35E) was of similar potency to wild-type THD in activating αIIbß3 in Chinese hamster ovary cells. Coexpression with activated Rap1b increased activation, and coexpression with Rap1GAP1 reduced activation caused by transfection of wild-type THD or THD(R35E). Furthermore, platelets from Tln1R35E/R35E mice showed similar GPIIb-IIIa activation to those from wild-type littermates in response to multiple agonists. Tln1R35E/R35E platelets exhibited slightly reduced platelet aggregation in response to low doses of agonists; however, there was not a significant hemostatic defect, as judged by tail bleeding times. Thus, the Rap1-talin 1 F0 interaction has little effect on platelet GPIIb-IIIa activation and hemostasis and cannot account for the dramatic effects of loss of Rap1 activity on these platelet functions.

Optogenetic interrogation of integrin αVß3 function in endothelial cells.

The integrin αVß3 is reported to promote angiogenesis in some model systems but not in others. Here, we used optogenetics to study the effects of αVß3 interaction with the intracellular adapter kindlin-2 (Fermt2) on endothelial cell functions potentially relevant to angiogenesis. Because interaction of kindlin-2 with αVß3 requires the C-terminal three residues of the ß3 cytoplasmic tail (Arg-Gly-Thr; RGT), optogenetic probes LOVpep and ePDZ1 were fused to ß3ΔRGT-GFP and mCherry-kindlin-2, respectively, and expressed in ß3 integrin-null microvascular endothelial cells. Exposure of the cells to 450 nm (blue) light caused rapid and specific interaction of kindlin-2 with αVß3 as assessed by immunofluorescence and total internal reflection fluorescence (TIRF) microscopy, and it led to increased endothelial cell migration, podosome formation and angiogenic sprouting. Analyses of kindlin-2 mutants indicated that interaction of kindlin-2 with other kindlin-2 binding partners, including c-Src, actin, integrin-linked kinase and phosphoinositides, were also likely necessary for these endothelial cell responses. Thus, kindlin-2 promotes αVß3-dependent angiogenic functions of endothelial cells through its simultaneous interactions with ß3 integrin and several other binding partners. Optogenetic approaches should find further use in clarifying spatiotemporal aspects of vascular cell biology.

Interaction of kindlin-2 with integrin ß3 promotes outside-in signaling responses by the αVß3 vitronectin receptor.

The bidirectional signaling and hemostatic functions of platelet αIIbß3 are regulated by kindlin-3 through interactions with the ß3 cytoplasmic tail. Little is known about kindlin regulation of the related "vitronectin receptor," αVß3. These relationships were investigated in endothelial cells, which express αVß3 and kindlin-2 endogenously. "ß3ΔRGT" knock-in mice lack the 3 C-terminal ß3 tail residues, whereas in "ß3/ß1(EGK)" mice, RGT is replaced by the corresponding residues of ß1. The wild-type ß3 tail pulled down kindlin-2 and c-Src in vitro, whereas ß3ΔRGT bound neither protein and ß3/ß1(EGK) bound kindlin-2, but not c-Src. ß3ΔRGT endothelial cells, but not ß3/ß1(EGK) endothelial cells, exhibited migration and spreading defects on vitronectin and reduced sprouting in 3-dimensional fibrin. Short hairpin RNA silencing of kindlin-2, but not c-Src, blocked sprouting by ß3 wild-type endothelial cells. Moreover, defective sprouting by ß3ΔRGT endothelial cells could be rescued by conditional, forced interaction of αVß3ΔRGT with kindlin-2. Stimulation of ß3ΔRGT endothelial cells led to normal extracellular ligand binding to αVß3, pin-pointing their defect to one of outside-in αVß3 signaling. ß3ΔRGT mice, but not ß3/ß1(EGK) mice, exhibited defects in both developmental and tumor angiogenesis, responses that require endothelial cell function. Thus, the ß3/kindlin-2 interaction promotes outside-in αVß3 signaling selectively, with biological consequences in vivo.

Integrin αvß3 drives slug activation and stemness in the pregnant and neoplastic mammary gland.

Although integrin αvß3 is linked to cancer progression, its role in epithelial development is unclear. Here, we show that αvß3 plays a critical role in adult mammary stem cells (MaSCs) during pregnancy. Whereas αvß3 is a luminal progenitor marker in the virgin gland, we noted increased αvß3 expression in MaSCs at midpregnancy. Accordingly, mice lacking αvß3 or expressing a signaling-deficient receptor showed defective mammary gland morphogenesis during pregnancy. This was associated with decreased MaSC expansion, clonogenicity, and expression of Slug, a master regulator of MaSCs. Surprisingly, αvß3-deficient mice displayed normal development of the virgin gland with no effect on luminal progenitors. Transforming growth factor ß2 (TGF-ß2) induced αvß3 expression, enhancing Slug nuclear accumulation and MaSC clonogenicity. In human breast cancer cells, αvß3 was necessary and sufficient for Slug activation, tumorsphere formation, and tumor initiation. Thus, pregnancy-associated MaSCs require a TGF-ß2/αvß3/Slug pathway, which may contribute to breast cancer progression and stemness.

The classical lancefield antigen of group a Streptococcus is a virulence determinant with implications for vaccine design.

Group A Streptococcus (GAS) is a leading cause of infection-related mortality in humans. All GAS serotypes express the Lancefield group A carbohydrate (GAC), comprising a polyrhamnose backbone with an immunodominant N-acetylglucosamine (GlcNAc) side chain, which is the basis of rapid diagnostic tests. No biological function has been attributed to this conserved antigen. Here we identify and characterize the GAC biosynthesis genes, gacA through gacL. An isogenic mutant of the glycosyltransferase gacI, which is defective for GlcNAc side-chain addition, is attenuated for virulence in two infection models, in association with increased sensitivity to neutrophil killing, platelet-derived antimicrobials in serum, and the cathelicidin antimicrobial peptide LL-37. Antibodies to GAC lacking the GlcNAc side chain and containing only polyrhamnose promoted opsonophagocytic killing of multiple GAS serotypes and protected against systemic GAS challenge after passive immunization. Thus, the Lancefield antigen plays a functional role in GAS pathogenesis, and a deeper understanding of this unique polysaccharide has implications for vaccine development.

C-terminal COOH of integrin ß1 is necessary for ß1 association with the kindlin-2 adapter protein.

Protein-protein interactions are driving forces in cellular processes. As a prime example, transmembrane integrins link extracellular matrix and intracellular proteins, resulting in bidirectional signaling that regulates cell migration, proliferation, differentiation, and survival. Here we provide the first evidence that interaction between the integrin ß1 cytoplasmic tail and kindlin-2, a member of a family of adapters implicated in human disease pathogenesis, is mainly governed by the ß1 C-terminal carboxylate moiety and is required for laterality organ development in zebrafish. Affinity measurements indicate that this unusual protein-protein interaction mode is coordinated by a putative carboxylate-binding motif in the kindlin-2 FERM subdomain F3. Contrary to the C terminus of proteins that engage PDZ domains, the C-terminal three residues of ß1, per se, do not contribute to kindlin-2 binding or to laterality organ development. Thus, by employing zebrafish as an in situ physiological tool to correlate protein structure and function, we have discovered an unexpected association chemistry between an integrin and a key adapter involved in integrin signaling.

ADAP interactions with talin and kindlin promote platelet integrin αIIbß3 activation and stable fibrinogen binding.

ADAP is a hematopoietic-restricted adapter protein that promotes integrin activation and is a carrier for other adapter proteins, Src kinase-associated phosphoprotein 1 (SKAP1) and SKAP2. In T lymphocytes, SKAP1 is the ADAP-associated molecule that activates integrins through direct linkages with Rap1 effectors (regulator of cell adhesion and polarization enriched in lymphoid tissues; Rap1-interacting adapter molecule). ADAP also promotes integrin αIIbß3 activation in platelets, which lack SKAP1, suggesting an ADAP integrin-regulatory pathway different from those in lymphocytes. Here we characterized a novel association between ADAP and 2 essential integrin-ß cytoplasmic tail-binding proteins involved in αIIbß3 activation, talin and kindlin-3. Glutathione S-transferase pull-downs identified distinct regions in ADAP necessary for association with kindlin or talin. ADAP was physically proximal to talin and kindlin-3 in human platelets, as assessed biochemically, and by immunofluorescence microscopy and proximity ligation. Relative to wild-type mouse platelets, ADAP-deficient platelets exhibited reduced co-localization of talin with αIIbß3, and reduced irreversible fibrinogen binding in response to a protease activated receptor 4 (PAR4) thrombin receptor agonist. When ADAP was heterologously expressed in Chinese hamster ovary cells co-expressing αIIbß3, talin, PAR1, and kindlin-3, it associated with an αIIbß3/talin complex and enabled kindlin-3 to promote agonist-dependent ligand binding to αIIbß3. Thus, ADAP uniquely promotes activation of and irreversible fibrinogen binding to platelet αIIbß3 through interactions with talin and kindlin-3.

The mechanism of kindlin-mediated activation of integrin αIIbß3.

Increased ligand binding to cellular integrins ("activation") plays important roles in processes such as development, cell migration, extracellular matrix assembly, tumor metastasis, hemostasis, and thrombosis. Integrin activation encompasses both increased integrin monomer affinity and increased receptor clustering and depends on integrin-talin interactions. Loss of kindlins results in reduced activation of integrins. Kindlins might promote talin binding to integrins through a cooperative mechanism; however, kindlins do not increase talin association with integrins. Here, we report that, unlike talin head domain (THD), kindlin-3 has little effect on the affinity of purified monomeric αIIbß3, and it does not enhance activation by THD. Furthermore, studies with ligands of varying valency show that kindlins primarily increase cellular αIIbß3 avidity rather than monomer affinity. In platelets or nucleated cells, loss of kindlins markedly reduces αIIbß3 binding to multivalent but not monovalent ligands. Finally, silencing of kindlins reduces the clustering of ligand-occupied αIIbß3 as revealed by total internal reflection fluorescence and electron microscopy. Thus, in contrast to talins, kindlins have little primary effect on integrin αIIbß3 affinity for monovalent ligands and increase multivalent ligand binding by promoting the clustering of talin-activated integrins.

The primacy of ß1 integrin activation in the metastatic cascade.

After neoplastic cells leave the primary tumor and circulate, they may extravasate from the vasculature and colonize tissues to form metastases. ß1 integrins play diverse roles in tumorigenesis and tumor progression, including extravasation. In blood cells, activation of ß1 integrins can be regulated by "inside-out" signals leading to extravasation from the circulation into tissues. However, a role for inside-out ß1 activation in tumor cell metastasis is uncertain. Here we show that ß1 integrin activation promotes tumor metastasis and that activated ß1 integrin may serve as a biomarker of metastatic human melanoma. To determine whether ß1 integrin activation can influence tumor cell metastasis, the ß1 integrin subunit in melanoma and breast cancer cell lines was stably knocked down with shRNA and replaced with wild-type or constitutively-active ß1. When tumor cells expressing constitutively-active ß1 integrins were injected intravenously into chick embryos or mice, they demonstrated increased colonization of the liver when compared to cells expressing wild-type ß1 integrins. Rescue expression with mutant ß1 integrins revealed that tumor cell extravasation and hepatic colonization required extracellular ligand binding to ß1 as well as ß1 interaction with talin, an intracellular mediator of integrin activation by the Rap1 GTPase. Furthermore, shRNA-mediated knock down of talin reduced hepatic colonization by tumor cells expressing wild-type ß1, but not constitutively-active ß1. Overexpression in tumor cells of the tumor suppressor, Rap1GAP, inhibited Rap1 and ß1 integrin activation as well as hepatic colonization. Using an antibody that detects activated ß1 integrin, we found higher levels of activated ß1 integrins in human metastatic melanomas compared to primary melanomas, suggesting that activated ß1 integrin may serve as a biomarker of invasive tumor cells. Altogether, these studies establish that inside-out activation of ß1 integrins promotes tumor cell extravasation and colonization, suggesting diagnostic and therapeutic approaches for targeting of ß1 integrin signaling in neoplasia.

Kindlins, integrin activation and the regulation of talin recruitment to αIIbß3.

Talins and kindlins bind to the integrin ß3 cytoplasmic tail and both are required for effective activation of integrin αIIbß3 and resulting high-affinity ligand binding in platelets. However, binding of the talin head domain alone to ß3 is sufficient to activate purified integrin αIIbß3 in vitro. Since talin is localized to the cytoplasm of unstimulated platelets, its re-localization to the plasma membrane and to the integrin is required for activation. Here we explored the mechanism whereby kindlins function as integrin co-activators. To test whether kindlins regulate talin recruitment to plasma membranes and to αIIbß3, full-length talin and kindlin recruitment to ß3 was studied using a reconstructed CHO cell model system that recapitulates agonist-induced αIIbß3 activation. Over-expression of kindlin-2, the endogenous kindlin isoform in CHO cells, promoted PAR1-mediated and talin-dependent ligand binding. In contrast, shRNA knockdown of kindlin-2 inhibited ligand binding. However, depletion of kindlin-2 by shRNA did not affect talin recruitment to the plasma membrane, as assessed by sub-cellular fractionation, and neither over-expression of kindlins nor depletion of kindlin-2 affected talin interaction with αIIbß3 in living cells, as monitored by bimolecular fluorescence complementation. Furthermore, talin failed to promote kindlin-2 association with αIIbß3 in CHO cells. In addition, purified talin and kindlin-3, the kindlin isoform expressed in platelets, failed to promote each other's binding to the ß3 cytoplasmic tail in vitro. Thus, kindlins do not promote initial talin recruitment to αIIbß3, suggesting that they co-activate integrin through a mechanism independent of recruitment.

Kindlin-2 regulates podocyte adhesion and fibronectin matrix deposition through interactions with phosphoinositides and integrins.

Kindlin-2 is a FERM and PH domain-containing integrin-binding protein that is emerging as an important regulator of integrin activation. How kindlin-2 functions in integrin activation, however, is not known. We report here that kindlin-2 interacts with multiple phosphoinositides, preferentially with phosphatidylinositol 3,4,5-trisphosphate. Although integrin-binding is essential for focal adhesion localization of kindlin-2, phosphoinositide-binding is not required for this process. Using biologically and clinically relevant glomerular podocytes as a model system, we show that integrin activation and dependent processes are tightly regulated by kindlin-2: depletion of kindlin-2 reduced integrin activation, matrix adhesion and fibronectin matrix deposition, whereas overexpression of kindlin-2 promoted these processes. Furthermore, we provide evidence showing that kindlin-2 is involved in phosphoinositide-3-kinase-mediated regulation of podocyte-matrix adhesion and fibronectin matrix deposition. Mechanistically, kindlin-2 promotes integrin activation and integrin-dependent processes through interacting with both integrins and phosphoinositides. TGF-ß1, a mediator of progressive glomerular failure, markedly increased the level of kindlin-2 and fibronectin matrix deposition, and the latter process was reversed by depletion of kindlin-2. Our results reveal important functions of kindlin-2 in the regulation of podocyte-matrix adhesion and matrix deposition and shed new light on the mechanism whereby kindlin-2 functions in these processes.

Integrin alphaV is necessary for gastrulation movements that regulate vertebrate body asymmetry.

Integrin αV can form heterodimers with several ß subunits to mediate cell-cell and cell-extracellular matrix interactions. During zebrafish gastrulation, αV is expressed maternally and zygotically. Here, we used a morpholino-mediated αV knockdown strategy to study αV function. Although αV morphants displayed vascular defects, they also exhibited left-right body asymmetry defects affecting multiple visceral organs. This was preceded by mislocalization of dorsal forerunner cells (DFCs) and malformation of the Kupffer's vesicle (KV) laterality organ. These defects were rescued with morpholino-resistant αV mRNA. Like αV, integrin ß1b was expressed in DFCs, and ß1b knockdown largely recapitulated the laterality phenotype of αV morphants. When tracked in real-time, individual DFCs of both morphants showed defects in DFC migration, preventing them from organizing into a KV of normal shape and size. Thus, we propose that αVß1b mediates cellular interactions that are necessary for DFC clustering and movements necessary for Kupffer's vesicle formation, uncovering an early contribution of integrins to the regulation of vertebrate laterality.

The final steps of integrin activation: the end game.

Cell-directed changes in the ligand-binding affinity ('activation') of integrins regulate cell adhesion and migration, extracellular matrix assembly and mechanotransduction, thereby contributing to embryonic development and diseases such as atherothrombosis and cancer. Integrin activation comprises triggering events, intermediate signalling events and, finally, the interaction of integrins with cytoplasmic regulators, which changes an integrin's affinity for its ligands. The first two events involve diverse interacting signalling pathways, whereas the final steps are immediately proximal to integrins, thus enabling integrin-focused therapeutic strategies. Recent progress provides insight into the structure of integrin transmembrane domains, and reveals how the final steps of integrin activation are mediated by integrin-binding proteins such as talins and kindlins.