Systemically administered wound-homing peptide accelerates wound healing by modulating syndecan-4 function.

CAR (CARSKNKDC) is a wound-homing peptide that recognises angiogenic neovessels. Here we discover that systemically administered CAR peptide has inherent ability to promote wound healing: wounds close and re-epithelialise faster in CAR-treated male mice. CAR promotes keratinocyte migration in vitro. The heparan sulfate proteoglycan syndecan-4 regulates cell migration and is crucial for wound healing. We report that syndecan-4 expression is restricted to epidermis and blood vessels in mice skin wounds. Syndecan-4 regulates binding and internalisation of CAR peptide and CAR-mediated cytoskeletal remodelling. CAR induces syndecan-4-dependent activation of the small GTPase ARF6, via the guanine nucleotide exchange factor cytohesin-2, and promotes syndecan-4-, ARF6- and Cytohesin-2-mediated keratinocyte migration. Finally, we show that genetic ablation of syndecan-4 in male mice eliminates CAR-induced wound re-epithelialisation following systemic administration. We propose that CAR peptide activates syndecan-4 functions to selectively promote re-epithelialisation. Thus, CAR peptide provides a therapeutic approach to enhance wound healing in mice; systemic, yet target organ- and cell-specific.

Lineage Plasticity in SCLC Generates Non-Neuroendocrine Cells Primed for Vasculogenic Mimicry.

INTRODUCTION: Vasculogenic mimicry (VM), the process of tumor cell transdifferentiation to endow endothelial-like characteristics supporting de novo vessel formation, is associated with poor prognosis in several tumor types, including SCLC. In genetically engineered mouse models (GEMMs) of SCLC, NOTCH, and MYC co-operate to drive a neuroendocrine (NE) to non-NE phenotypic switch, and co-operation between NE and non-NE cells is required for metastasis. Here, we define the phenotype of VM-competent cells and molecular mechanisms underpinning SCLC VM using circulating tumor cell-derived explant (CDX) models and GEMMs. METHODS: We analyzed perfusion within VM vessels and their association with NE and non-NE phenotypes using multiplex immunohistochemistry in CDX, GEMMs, and patient biopsies. We evaluated their three-dimensional structure and defined collagen-integrin interactions. RESULTS: We found that VM vessels are present in 23/25 CDX models, 2 GEMMs, and in 20 patient biopsies of SCLC. Perfused VM vessels support tumor growth and only NOTCH-active non-NE cells are VM-competent in vivo and ex vivo, expressing pseudohypoxia, blood vessel development, and extracellular matrix organization signatures. On Matrigel, VM-primed non-NE cells remodel extracellular matrix into hollow tubules in an integrin ß1-dependent process. CONCLUSIONS: We identified VM as an exemplar of functional heterogeneity and plasticity in SCLC and these findings take considerable steps toward understanding the molecular events that enable VM. These results support therapeutic co-targeting of both NE and non-NE cells to curtail SCLC progression and to improve the outcomes of patients with SCLC in the future.

Talin2 and KANK2 functionally interact to regulate microtubule dynamics, paclitaxel sensitivity and cell migration in the MDA-MB-435S melanoma cell line.

BACKGROUND: Focal adhesions (FAs) are integrin-containing, multi-protein structures that link intracellular actin to the extracellular matrix and trigger multiple signaling pathways that control cell proliferation, differentiation, survival and motility. Microtubules (MTs) are stabilized in the vicinity of FAs through interaction with the components of the cortical microtubule stabilizing complex (CMSC). KANK (KN motif and ankyrin repeat domains) family proteins within the CMSC, KANK1 or KANK2, bind talin within FAs and thus mediate actin-MT crosstalk. We previously identified in MDA-MB-435S cells, which preferentially use integrin αVß5 for adhesion, KANK2 as a key molecule enabling the actin-MT crosstalk. KANK2 knockdown also resulted in increased sensitivity to MT poisons, paclitaxel (PTX) and vincristine and reduced migration. Here, we aimed to analyze whether KANK1 has a similar role and to distinguish which talin isoform binds KANK2. METHODS: The cell model consisted of human melanoma cell line MDA-MB-435S and stably transfected clone with decreased expression of integrin αV (3αV). For transient knockdown of talin1, talin2, KANK1 or KANK2 we used gene-specific siRNAs transfection. Using previously standardized protocol we isolated integrin adhesion complexes. SDS-PAGE and Western blot was used for protein expression analysis. The immunofluorescence analysis and live cell imaging was done using confocal microscopy. Cell migration was analyzed with Transwell Cell Culture Inserts. Statistical analysis using GraphPad Software consisted of either one-way analysis of variance (ANOVA), unpaired Student's t-test or two-way ANOVA analysis. RESULTS: We show that KANK1 is not a part of the CMSC associated with integrin αVß5 FAs and its knockdown did not affect the velocity of MT growth or cell sensitivity to PTX. The talin2 knockdown mimicked KANK2 knockdown i.e. led to the perturbation of actin-MT crosstalk, which is indicated by the increased velocity of MT growth and increased sensitivity to PTX and also reduced migration. CONCLUSION: We conclude that KANK2 functionally interacts with talin2 and that the mechanism of increased sensitivity to PTX involves changes in microtubule dynamics. These data elucidate a cell-type-specific role of talin2 and KANK2 isoforms and we propose that talin2 and KANK2 are therefore potential therapeutic targets for improved cancer therapy.

Connexin 37 sequestering of activated-ERK in the cytoplasm promotes p27-mediated endothelial cell cycle arrest.

Connexin37-mediated regulation of cell cycle modulators and, consequently, growth arrest lack mechanistic understanding. We previously showed that arterial shear stress up-regulates Cx37 in endothelial cells and activates a Notch/Cx37/p27 signaling axis to promote G1 cell cycle arrest, and this is required to enable arterial gene expression. However, how induced expression of a gap junction protein, Cx37, up-regulates cyclin-dependent kinase inhibitor p27 to enable endothelial growth suppression and arterial specification is unclear. Herein, we fill this knowledge gap by expressing wild-type and regulatory domain mutants of Cx37 in cultured endothelial cells expressing the Fucci cell cycle reporter. We determined that both the channel-forming and cytoplasmic tail domains of Cx37 are required for p27 up-regulation and late G1 arrest. Mechanistically, the cytoplasmic tail domain of Cx37 interacts with, and sequesters, activated ERK in the cytoplasm. This then stabilizes pERK nuclear target Foxo3a, which up-regulates p27 transcription. Consistent with previous studies, we found this Cx37/pERK/Foxo3a/p27 signaling axis functions downstream of arterial shear stress to promote endothelial late G1 state and enable up-regulation of arterial genes.

A Nrf2-OSGIN1&2-HSP70 axis mediates cigarette smoke-induced endothelial detachment: implications for plaque erosion.

AIMS: Endothelial erosion of plaques is responsible for ∼30% of acute coronary syndromes (ACS). Smoking is a risk factor for plaque erosion, which most frequently occurs on the upstream surface of plaques where the endothelium experiences elevated shear stress. We sought to recreate these conditions in vitro to identify potential pathological mechanisms that might be of relevance to plaque erosion. METHODS AND RESULTS: Culturing human coronary artery endothelial cells (HCAECs) under elevated flow (shear stress of 7.5 Pa) and chronically exposing them to cigarette smoke extract (CSE) and tumour necrosis factor-alpha (TNFα) recapitulated a defect in HCAEC adhesion, which corresponded with augmented Nrf2-regulated gene expression. Pharmacological activation or adenoviral overexpression of Nrf2 triggered endothelial detachment, identifying Nrf2 as a mediator of endothelial detachment. Growth/Differentiation Factor-15 (GDF15) expression was elevated in this model, with protein expression elevated in the plasma of patients experiencing plaque erosion compared with plaque rupture. The expression of two Nrf2-regulated genes, OSGIN1 and OSGIN2, was increased by CSE and TNFα under elevated flow and was also elevated in the aortas of mice exposed to cigarette smoke in vivo. Knockdown of OSGIN1&2 inhibited Nrf2-induced cell detachment. Overexpression of OSGIN1&2 induced endothelial detachment and resulted in cell cycle arrest, induction of senescence, loss of focal adhesions and actin stress fibres, and disturbed proteostasis mediated in part by HSP70, restoration of which reduced HCAEC detachment. In ACS patients who smoked, blood concentrations of HSP70 were elevated in plaque erosion compared with plaque rupture. CONCLUSION: We identified a novel Nrf2-OSGIN1&2-HSP70 axis that regulates endothelial adhesion, elevated GDF15 and HSP70 as biomarkers for plaque erosion in patients who smoke, and two therapeutic targets that offer the potential for reducing the risk of plaque erosion.

The ßI domain promotes active ß1 integrin clustering into mature adhesion sites.

Modulation of integrin function is required in many physiological and pathological settings, such as angiogenesis and cancer. Integrin allosteric changes, clustering, and trafficking cooperate to regulate cell adhesion and motility on extracellular matrix proteins via mechanisms that are partly defined. By exploiting four monoclonal antibodies recognizing distinct conformational epitopes, we show that in endothelial cells (ECs), the extracellular ßI domain, but not the hybrid or I-EGF2 domain of active ß1 integrins, promotes their FAK-regulated clustering into tensin 1-containing fibrillar adhesions and impairs their endocytosis. In this regard, the ßI domain-dependent clustering of active ß1 integrins is necessary to favor fibronectin-elicited directional EC motility, which cannot be effectively promoted by ß1 integrin conformational activation alone.

Pancreatic ductal adenocarcinoma cells employ integrin α6ß4 to form hemidesmosomes and regulate cell proliferation.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis due to its aggressive progression, late detection and lack of druggable driver mutations, which often combine to result in unsuitability for surgical intervention. Together with activating mutations of the small GTPase KRas, which are found in over 90% of PDAC tumours, a contributory factor for PDAC tumour progression is formation of a rigid extracellular matrix (ECM) and associated desmoplasia. This response leads to aberrant integrin signalling, and accelerated proliferation and invasion. To identify the integrin adhesion systems that operate in PDAC, we analysed a range of pancreatic ductal epithelial cell models using 2D, 3D and organoid culture systems. Proteomic analysis of isolated integrin receptor complexes from human pancreatic ductal epithelial (HPDE) cells predominantly identified integrin α6ß4 and hemidesmosome components, rather than classical focal adhesion components. Electron microscopy, together with immunofluorescence, confirmed the formation of hemidesmosomes by HPDE cells, both in 2D and 3D culture systems. Similar results were obtained for the human PDAC cell line, SUIT-2. Analysis of HPDE cell secreted proteins and cell-derived matrices (CDM) demonstrated that HPDE cells secrete a range of laminin subunits and form a hemidesmosome-specific, laminin 332-enriched ECM. Expression of mutant KRas (G12V) did not affect hemidesmosome composition or formation by HPDE cells. Cell-ECM contacts formed by mouse and human PDAC organoids were also assessed by electron microscopy. Organoids generated from both the PDAC KPC mouse model and human patient-derived PDAC tissue displayed features of acinar-ductal cell polarity, and hemidesmosomes were visible proximal to prominent basement membranes. Furthermore, electron microscopy identified hemidesmosomes in normal human pancreas. Depletion of integrin ß4 reduced cell proliferation in both SUIT-2 and HPDE cells, reduced the number of SUIT-2 cells in S-phase, and induced G1 cell cycle arrest, suggesting a requirement for α6ß4-mediated adhesion for cell cycle progression and growth. Taken together, these data suggest that laminin-binding adhesion mechanisms in general, and hemidesmosome-mediated adhesion in particular, may be under-appreciated in the context of PDAC. Proteomic data are available via ProteomeXchange with the identifiers PXD027803, PXD027823 and PXD027827.

A microenvironment-inspired synthetic three-dimensional model for pancreatic ductal adenocarcinoma organoids.

Experimental in vitro models that capture pathophysiological characteristics of human tumours are essential for basic and translational cancer biology. Here, we describe a fully synthetic hydrogel extracellular matrix designed to elicit key phenotypic traits of the pancreatic environment in culture. To enable the growth of normal and cancerous pancreatic organoids from genetically engineered murine models and human patients, essential adhesive cues were empirically defined and replicated in the hydrogel scaffold, revealing a functional role of laminin-integrin α3/α6 signalling in establishment and survival of pancreatic organoids. Altered tissue stiffness-a hallmark of pancreatic cancer-was recapitulated in culture by adjusting the hydrogel properties to engage mechano-sensing pathways and alter organoid growth. Pancreatic stromal cells were readily incorporated into the hydrogels and replicated phenotypic traits characteristic of the tumour environment in vivo. This model therefore recapitulates a pathologically remodelled tumour microenvironment for studies of normal and pancreatic cancer cells in vitro.

FHL-1 interacts with human RPE cells through the α5ß1 integrin and confers protection against oxidative stress.

Retinal pigment epithelial (RPE) cells that underlie the neurosensory retina are essential for the maintenance of photoreceptor cells and hence vision. Interactions between the RPE and their basement membrane, i.e. the inner layer of Bruch's membrane, are essential for RPE cell health and function, but the signals induced by Bruch's membrane engagement, and their contributions to RPE cell fate determination remain poorly defined. Here, we studied the functional role of the soluble complement regulator and component of Bruch's membrane, Factor H-like protein 1 (FHL-1). Human primary RPE cells adhered to FHL-1 in a manner that was eliminated by either mutagenesis of the integrin-binding RGD motif in FHL-1 or by using competing antibodies directed against the α5 and ß1 integrin subunits. These short-term experiments reveal an immediate protein-integrin interaction that were obtained from primary RPE cells and replicated using the hTERT-RPE1 cell line. Separate, longer term experiments utilising RNAseq analysis of hTERT-RPE1 cells bound to FHL-1, showed an increased expression of the heat-shock protein genes HSPA6, CRYAB, HSPA1A and HSPA1B when compared to cells bound to fibronectin (FN) or laminin (LA). Pathway analysis implicated changes in EIF2 signalling, the unfolded protein response, and mineralocorticoid receptor signalling as putative pathways. Subsequent cell survival assays using H2O2 to induce oxidative stress-induced cell death suggest hTERT-RPE1 cells had significantly greater protection when bound to FHL-1 or LA compared to plastic or FN. These data show a non-canonical role of FHL-1 in protecting RPE cells against oxidative stress and identifies a novel interaction that has implications for ocular diseases such as age-related macular degeneration.

A SNAI2-PEAK1-INHBA stromal axis drives progression and lapatinib resistance in HER2-positive breast cancer by supporting subpopulations of tumor cells positive for antiapoptotic and stress signaling markers.

Intercellular mechanisms by which the stromal microenvironment contributes to solid tumor progression and targeted therapy resistance remain poorly understood, presenting significant clinical hurdles. PEAK1 (Pseudopodium-Enriched Atypical Kinase One) is an actin cytoskeleton- and focal adhesion-associated pseudokinase that promotes cell state plasticity and cancer metastasis by mediating growth factor-integrin signaling crosstalk. Here, we determined that stromal PEAK1 expression predicts poor outcomes in HER2-positive breast cancers high in SNAI2 expression and enriched for MSC content. Specifically, we identified that the fibroblastic stroma in HER2-positive breast cancer patient tissue stains positive for both nuclear SNAI2 and cytoplasmic PEAK1. Furthermore, mesenchymal stem cells (MSCs) and cancer-associated fibroblasts (CAFs) express high PEAK1 protein levels and potentiate tumorigenesis, lapatinib resistance and metastasis of HER2-positive breast cancer cells in a PEAK1-dependent manner. Analysis of PEAK1-dependent secreted factors from MSCs revealed INHBA/activin-A as a necessary factor in the conditioned media of PEAK1-expressing MSCs that promotes lapatinib resistance. Single-cell CycIF analysis of MSC-breast cancer cell co-cultures identified enrichment of p-Akthigh/p-gH2AXlow, MCL1high/p-gH2AXlow and GRP78high/VIMhigh breast cancer cell subpopulations by the presence of PEAK1-expressing MSCs and lapatinib treatment. Bioinformatic analyses on a PEAK1-centric stroma-tumor cell gene set and follow-up immunostaining of co-cultures predict targeting antiapoptotic and stress pathways as a means to improve targeted therapy responses and patient outcomes in HER2-positive breast cancer and other stroma-rich malignancies. These data provide the first evidence that PEAK1 promotes tumorigenic phenotypes through a previously unrecognized SNAI2-PEAK1-INHBA stromal cell axis.

Talin mechanosensitivity is modulated by a direct interaction with cyclin-dependent kinase-1.

Talin (TLN1) is a mechanosensitive component of adhesion complexes that directly couples integrins to the actin cytoskeleton. In response to force, talin undergoes switch-like behavior of its multiple rod domains that modulate interactions with its binding partners. Cyclin-dependent kinase-1 (CDK1) is a key regulator of the cell cycle, exerting its effects through synchronized phosphorylation of a large number of protein targets. CDK1 activity maintains adhesion during interphase, and its inhibition is a prerequisite for the tightly choreographed changes in cell shape and adhesion that are required for successful mitosis. Using a combination of biochemical, structural, and cell biological approaches, we demonstrate a direct interaction between talin and CDK1 that occurs at sites of integrin-mediated adhesion. Mutagenesis demonstrated that CDK1 contains a functional talin-binding LD motif, and the binding site within talin was pinpointed to helical bundle R8. Talin also contains a consensus CDK1 phosphorylation motif centered on S1589, a site shown to be phosphorylated by CDK1 in vitro. A phosphomimetic mutant of this site within talin lowered the binding affinity of the cytoskeletal adaptor KANK and weakened the response of this region to force as measured by single molecule stretching, potentially altering downstream mechanotransduction pathways. The direct binding of the master cell cycle regulator CDK1 to the primary integrin effector talin represents a coupling of cell proliferation and cell adhesion machineries and thereby indicates a mechanism by which the microenvironment can control cell division in multicellular organisms.

Identification of an Altered Matrix Signature in Kidney Aging and Disease.

BACKGROUND: Accumulation of extracellular matrix in organs and tissues is a feature of both aging and disease. In the kidney, glomerulosclerosis and tubulointerstitial fibrosis accompany the decline in function, which current therapies cannot address, leading to organ failure. Although histologic and ultrastructural patterns of excess matrix form the basis of human disease classifications, a comprehensive molecular resolution of abnormal matrix is lacking. METHODS: Using mass spectrometry-based proteomics, we resolved matrix composition over age in mouse models of kidney disease. We compared the changes in mice with a global characterization of human kidneymatrix during aging and to existing kidney disease datasets to identify common molecular features. RESULTS: Ultrastructural changes in basement membranes are associated with altered cell adhesion and metabolic processes and with distinct matrix proteomes during aging and kidney disease progression in mice. Within the altered matrix, basement membrane components (laminins, type IV collagen, type XVIII collagen) were reduced and interstitial matrix proteins (collagens I, III, VI, and XV; fibrinogens; and nephronectin) were increased, a pattern also seen in human kidney aging. Indeed, this signature of matrix proteins was consistently modulated across all age and disease comparisons, and the increase in interstitial matrix was also observed in human kidney disease datasets. CONCLUSIONS: This study provides deep molecular resolution of matrix accumulation in kidney aging and disease, and identifies a common signature of proteins that provides insight into mechanisms of response to kidney injury and repair.

The Tongue Squamous Carcinoma Cell Line Cal27 Primarily Employs Integrin α6ß4-Containing Type II Hemidesmosomes for Adhesion Which Contribute to Anticancer Drug Sensitivity.

Integrins are heterodimeric cell surface glycoproteins used by cells to bind to the extracellular matrix (ECM) and regulate tumor cell proliferation, migration and survival. A causative relationship between integrin expression and resistance to anticancer drugs has been demonstrated in different tumors, including head and neck squamous cell carcinoma. Using a Cal27 tongue squamous cell carcinoma model, we have previously demonstrated that de novo expression of integrin αVß3 confers resistance to several anticancer drugs (cisplatin, mitomycin C and doxorubicin) through a mechanism involving downregulation of active Src, increased cell migration and invasion. In the integrin αVß3 expressing Cal27-derived cell clone 2B1, αVß5 expression was also increased, but unrelated to drug resistance. To identify the integrin adhesion complex (IAC) components that contribute to the changes in Cal27 and 2B1 cell adhesion and anticancer drug resistance, we isolated IACs from both cell lines. Mass spectrometry (MS)-based proteomics analysis indicated that both cell lines preferentially, but not exclusively, use integrin α6ß4, which is classically found in hemidesmosomes. The anticancer drug resistant cell clone 2B1 demonstrated an increased level of α6ß4 accompanied with increased deposition of a laminin-332-containing ECM. Immunofluorescence and electron microscopy demonstrated the formation of type II hemidesmosomes by both cell types. Furthermore, suppression of α6ß4 expression in both lines conferred resistance to anticancer drugs through a mechanism independent of αVß3, which implies that the cell clone 2B1 would have been even more resistant had the upregulation of α6ß4 not occurred. Taken together, our results identify a key role for α6ß4-containing type II hemidesmosomes in regulating anticancer drug sensitivity.

KANK family proteins in cancer.

The Kank (kidney or KN motif and ankyrin repeat domain-containing) family of proteins has been described as essential for crosstalk between actin and microtubules. Kank1, 2, 3 and 4 arose by gene duplication and diversification and share conserved structural domains. KANK proteins are localised mainly to the plasma membrane in focal adhesions, indirectly affecting RhoA and Rac1 thus regulating actin cytoskeleton. In addition, Kank proteins are part of the cortical microtubule stabilisation complex regulating microtubules. Most of the data have been collected for Kank1 protein whose expression promotes apoptosis and cell-cycle arrest while Kank3 was identified as hypoxia-inducible proapoptotic target of p53. A discrepancy in Kanks role in regulation of cell migration and sensitivity to antitumour drugs has been observed in different cell models. Since expression of Kank1 and 3 correlate positively with tumour progression and patient outcome, at least in some tumour types, they are candidates for tumour suppressors.

Multiplexed Proximity Biotinylation Coupled to Mass Spectrometry for Defining Integrin Adhesion Complexes.

BioID, a proximity biotinylation technique, offers a valuable approach to examine the interactions occurring within protein complexes that complements traditional protein biochemical methods. BioID has various advantages that are beneficial to the study of complexes, including an ability to detect insoluble and transient proteins. We have applied BioID to the study of integrin adhesion complexes (IACs), which are located at the junction between the plasma membrane and actin cytoskeleton. The use of multiple BioID baits enables a complex-wide, spatial annotation of IACs, which in turn facilitates the detection of novel proximal interactors and provides insights into IAC architecture. This article describes the labeling and affinity purification of IAC-proximal proteins and their analysis by label-free quantitative mass spectrometry. The article also outlines steps to identify high-confidence proximity interactors, and to interrogate the topology and functional relevance of proximity interaction networks through bioinformatic analyses. © 2020 The Authors. Basic Protocol 1: Proximity biotinylation of integrin adhesion complex components Basic Protocol 2: Mass spectrometry data processing by MaxQuant and detection of high-confidence proximal interactors Basic Protocol 3: Bioinformatic analysis and data visualization.

Integrin Crosstalk Contributes to the Complexity of Signalling and Unpredictable Cancer Cell Fates.

Integrins are heterodimeric cell surface receptors composed of α and ß subunits that control adhesion, proliferation and gene expression. The integrin heterodimer binding to ligand reorganises the cytoskeletal networks and triggers multiple signalling pathways that can cause changes in cell cycle, proliferation, differentiation, survival and motility. In addition, integrins have been identified as targets for many different diseases, including cancer. Integrin crosstalk is a mechanism by which a change in the expression of a certain integrin subunit or the activation of an integrin heterodimer may interfere with the expression and/or activation of other integrin subunit(s) in the very same cell. Here, we review the evidence for integrin crosstalk in a range of cellular systems, with a particular emphasis on cancer. We describe the molecular mechanisms of integrin crosstalk, the effects of cell fate determination, and the contribution of crosstalk to therapeutic outcomes. Our intention is to raise awareness of integrin crosstalk events such that the contribution of the phenomenon can be taken into account when researching the biological or pathophysiological roles of integrins.

Topological features of integrin adhesion complexes revealed by multiplexed proximity biotinylation.

Integrin adhesion complexes (IACs) bridge the extracellular matrix to the actin cytoskeleton and transduce signals in response to both chemical and mechanical cues. The composition, interactions, stoichiometry, and topological organization of proteins within IACs are not fully understood. To address this gap, we used multiplexed proximity biotinylation (BioID) to generate an in situ, proximity-dependent adhesome in mouse pancreatic fibroblasts. Integration of the interactomes of 16 IAC-associated baits revealed a network of 147 proteins with 361 proximity interactions. Candidates with underappreciated roles in adhesion were identified, in addition to established IAC components. Bioinformatic analysis revealed five clusters of IAC baits that link to common groups of prey, and which therefore may represent functional modules. The five clusters, and their spatial associations, are consistent with current models of IAC interaction networks and stratification. This study provides a resource to examine proximal relationships within IACs at a global level.

KANK2 Links αVß5 Focal Adhesions to Microtubules and Regulates Sensitivity to Microtubule Poisons and Cell Migration.

Integrins are heterodimeric glycoproteins that bind cells to extracellular matrix. Upon integrin clustering, multimolecular integrin adhesion complexes (IACs) are formed, creating links to the cell cytoskeleton. We have previously observed decreased cell migration and increased sensitivity to microtubule (MT) poisons, paclitaxel and vincristine, in the melanoma cell line MDA-MB-435S upon transfection with integrin αV-specific siRNA, suggesting a link between adhesion and drug sensitivity. To elucidate the underlying mechanism, we determined αV-dependent changes in IAC composition. Using mass spectrometry (MS)-based proteomics, we analyzed the components of isolated IACs of MDA-MB-435S cells and two MDA-MB-435S-derived integrin αV-specific shRNA-expressing cell clones with decreased expression of integrin αV. MS analysis showed that cells preferentially use integrin αVß5 for the formation of IACs. The differential analysis between MDA-MB-435S cells and clones with decreased expression of integrin αV identified key components of integrin αVß5 adhesion complexes as talins 1 and 2, α-actinins 1 and 4, filamins A and B, plectin and vinculin. The data also revealed decreased levels of several components of the cortical microtubule stabilization complex, which recruits MTs to adhesion sites (notably liprins α and ß, ELKS, LL5ß, MACF1, KANK1, and KANK2), following αV knockdown. KANK2 knockdown in MDA-MB-435S cells mimicked the effect of integrin αV knockdown and resulted in increased sensitivity to MT poisons and decreased migration. Taken together, we conclude that KANK2 is a key molecule linking integrin αVß5 IACs to MTs, and enabling the actin-MT crosstalk that is important for both sensitivity to MT poisons and cell migration.