Integrin Adhesion Complexes Research Articles

The role and regulation of integrins in cell migration and invasion.

Integrin receptors are the main molecular link between cells and the extracellular matrix (ECM) as well as mediating cell-cell interactions. Integrin-ECM binding triggers the formation of heterogeneous multi-protein assemblies termed integrin adhesion complexes (IACs) that enable integrins to transform extracellular cues into intracellular signals that affect many cellular processes, especially cell motility. Cell migration is essential for diverse physiological and pathological processes and is dysregulated in cancer to favour cell invasion and metastasis. Here, we discuss recent findings on the role of integrins in cell migration with a focus on cancer cell dissemination. We review how integrins regulate the spatial distribution and dynamics of different IACs, covering classical focal adhesions, emerging adhesion types and adhesion regulation. We discuss the diverse roles integrins have during cancer progression from cell migration across varied ECM landscapes to breaching barriers such as the basement membrane, and eventual colonization of distant organs.

Extracellular vesicles regulate metastable phenotypes of lymphangioleiomyomatosis cells via shuttling ATP synthesis to pseudopodia and activation of integrin adhesion complexes.

Pulmonary lymphangioleiomyomatosis (LAM) is metastatic sarcoma but mechanisms regulating LAM metastasis are unknown. Extracellular vesicle (EV) regulate cancer metastasis but their roles in LAM have not yet been investigated. Here, we report that EV biogenesis is increased in LAM and LAM EV cargo is enriched with lung tropic integrins, metalloproteinases, and cancer stem cell markers. LAM-EV increase LAM cell migration and invasion via the ITGα6/β1-c-Src-FAK-AKT axis. Metastable (hybrid) phenotypes of LAM metastasizing cells, pivotal for metastasis, are regulated by EV from primary tumor or metastasizing LAM cells via shuttling ATP synthesis to cell pseudopodia or activation of integrin adhesion complex, respectively. In mouse models of LAM, LAM-EV increase lung metastatic burden through mechanisms involving lung extracellular matrix remodeling. Collectively, these data provide evidence for the role of EV in promoting LAM lung metastasis and identify novel EV-dependent mechanisms regulating metastable phenotypes of tumor cells. Clinical impact of research is that it establishes LAM pathway as novel target for LAM therapy.

A multiscale dynamic model of cell–substrate interfaces

Cell–extracellular matrix (ECM) interactions play a pivotal role in many functions of cells, for example, sensing, signaling, migration, and gene expression. The spatial–temporal dynamic evolution of cell–substrate adhesions involves complicated mechano-bio-chemical coupling mechanisms of integrin, adaptor and signaling proteins, as well as the interplay between the cytoskeleton and ECM. In this paper, we establish a multiscale dynamic model of cell–substrate interfaces considering intermolecular force transmission pathways, i.e., intra- and extra-cellular bond dynamics, and mechanochemical coupling regulations. To illustrate its applications, this model is used to reproduce several adhesion-related experimental phenomena of cells, including substrate rigidity sensing, actin-assisted focal adhesion formation, and biphasic interface stress–actin speed relation. The results suggest that the cluster growth patterns are mediated by the intermolecular force transmission pathway and the actin flow speed, and that the integrin–ligand (IL) bond strengthening tends to inhibit the motion of clusters. We reveal that the monotonic-to-biphasic transition of force transmission and adhesion assembly is regulated by the bond rupture mechanosensitivity of integrin adhesion complexes (IACs). It is also demonstrated that this multiscale dynamic model can well describe the spatial–temporal evolutions of collective cell–substrate adhesions with the effects of actin flow and substrate deformation, and can also capture the morphological features of adhesion clusters and related interface force transmissions. This work provides a theoretical tool for studying mechano-biochemical mechanisms of cell–substrate interactions and for understanding various subcellular and cellular dynamic processes in, for example, tumors.

#1115 TGFβ induced mechanosignaling controls the transcriptome and microenvironmental composition of proximal tubules in chronic kidney disease

Abstract Background and Aims Interstitial fibrosis of the renal parenchyma and progressive atrophy of proximal tubules (PT) are hallmark features of chronic kidney disease (CKD). The latter is characterized by pronounced thickening and multilamellation of the tubular basement membrane, whereas fibrosis is promoted by accumulation of extracellular matrix (ECM) translating into increased matrix rigidity. The role of mechanotransduction via the integrin adhesion complex (IAC) as well as the corresponding signaling programs activated in this context remain largely elusive. Here, we aimed to elucidate the functional role of the ILK-Pinch-Parvin (IPP)-complex as an essential part of the IAC in response to PT damage and CKD. Method Morphometric assessment of a CKD patient biopsy cohort as well as ischaemia-reperfusion injury (IRI) mouse models was performed. Transcriptome studies of human proximal tubular epithelial cells (hRPTECs) under pro-fibrotic and rigid matrix conditions were analyzed. An in vitro model, mimicking CKD, was established and functionally characterized. Reanalysis of single-cell RNA sequencing (scRNA-seq) from a human CKD cohort was employed for cross-correlation and validation. Knockout (KO) models for the IPP-complex were generated by CRISPR/Cas9 genome editing and combined with functional assays (e.g. ECM deposition and sensing). Results In IRI mice, we observed a strong recruitment of the ILK-Pinch-Parvin (IPP)-complex and related-IAC proteins to the tubular basement membrane of proximal tubules (PTs) showing features of tubular atrophy. These findings were translated to CKD patients further assessing markers of active mechanosignaling (YAP) as well as tubular damage (SOX9). Rigidity sensing assays in a hRPTEC model were employed to define the underlying mechanisms. Remarkably, extensive transcriptome analysis revealed that only under pro-fibrotic signaling conditions, hRPTECs sensed rigidity alterations in the underlying substratum, and translated these changes into regulation of matrisome- and adhesome-related gene signatures (including members of the IPP-complex). Further filtering and cross-correlation with available scRNA-seq data from CKD-patients highlighted differential expression of secreted signaling proteins including the matricellular molecule CCN1. Subsequent functional studies demonstrated the underlying pivotal role of the IPP-complex as a central signaling node required for active mechanosignaling in damaged hRPTECs. Conclusion In CKD, IPP and other IAC proteins are recruited to the massively thickened tubular basement membrane of atrophic tubules, indicating a critical role in disease progression. Our results demonstrate that hRPTECs do not express IAC proteins in healthy conditions. However, when inflammation processes (TGFβ treatment) occur, PTs undergo a phenotypic switch, resulting in altered IAC expression signatures. Our data indicate that IAC proteins are crucially involved in sensing of the microenvironmental composition of PTs and thereby reciprocally fine-tune auto-/paracrine signaling properties of epithelial cells.

Mechanotransduction in stem cells

Nowadays, it is an established concept that the capability to reach a specialised cell identity via differentiation, as in the case of multi- and pluripotent stem cells, is not only determined by biochemical factors, but that also physical aspects of the microenvironment play a key role; interpreted by the cell through a force-based signalling pathway called mechanotransduction. However, the intricate ties between the elements involved in mechanotransduction, such as the extracellular matrix, the glycocalyx, the cell membrane, Integrin adhesion complexes, Cadherin-mediated cell/cell adhesion, the cytoskeleton, and the nucleus, are still far from being understood in detail. Here we report what is currently known about these elements in general and their specific interplay in the context of multi- and pluripotent stem cells. We furthermore merge this overview to a more comprehensive picture, that aims to cover the whole mechanotransductive pathway from the cell/microenvironment interface to the regulation of the chromatin structure in the nucleus. Ultimately, with this review we outline the current picture of the interplay between mechanotransductive cues and epigenetic regulation and how these processes might contribute to stem cell dynamics and fate.

Actin Cytoskeleton Remodeling Accompanied by Redistribution of Adhesion Proteins Drives Migration of Cells in Different EMT States.

Epithelial-mesenchymal transition (EMT) is a process during which epithelial cells lose epithelial characteristics and gain mesenchymal features. Here, we used several cell models to study migratory activity and redistribution of cell-cell adhesion proteins in cells in different EMT states: EGF-induced EMT of epithelial IAR-20 cells; IAR-6-1 cells with a hybrid epithelial-mesenchymal phenotype; and their more mesenchymal derivatives, IAR-6-1-DNE cells lacking adherens junctions. In migrating cells, the cell-cell adhesion protein α-catenin accumulated at the leading edges along with ArpC2/p34 and α-actinin. Suppression of α-catenin shifted cell morphology from fibroblast-like to discoid and attenuated cell migration. Expression of exogenous α-catenin in MDA-MB-468 cells devoid of α-catenin drastically increased their migratory capabilities. The Y654 phosphorylated form of β-catenin was detected at integrin adhesion complexes (IACs). Co-immunoprecipitation studies indicated that α-catenin and pY654-β-catenin were associated with IAC proteins: vinculin, zyxin, and α-actinin. Taken together, these data suggest that in cells undergoing EMT, catenins not participating in assembly of adherens junctions may affect cell migration.

The RhoGAP RRC-1 is required for the assembly or stability of integrin adhesion complexes and is a member of the PIX pathway in muscle.

GTPases cycle between active GTP bound and inactive GDP bound forms. Exchange of GDP for GTP is catalyzed by guanine nucleotide exchange factors (GEFs). GTPase activating proteins (GAPs) accelerate GTP hydrolysis, to promote the GDP bound form. We reported that the RacGEF, PIX-1, is required for assembly of integrin adhesion complexes (IAC) in striated muscle of Caenorhabditis elegans. In C. elegans, IACs are found at the muscle cell boundaries (MCBs), and bases of sarcomeric M-lines and dense bodies (Z-disks). Screening C. elegans mutants in proteins containing RhoGAP domains revealed that loss of function of rrc-1 results in loss of IAC components at MCBs, disorganization of M-lines and dense bodies, and reduced whole animal locomotion. RRC-1 localizes to MCBs, like PIX-1. The localization of RRC-1 at MCBs requires PIX-1, and the localization of PIX-1 requires RRC-1. Loss of function of CED-10 (Rac) shows lack of PIX-1 and RRC-1 at MCBs. RRC-1 exists in a complex with PIX-1. Transgenic rescue of rrc-1 was achieved with wild type RRC-1 but not RRC-1 with a missense mutation in a highly conserved residue of the RhoGAP domain. Our results are consistent with RRC-1 being a RhoGAP for the PIX pathway in muscle.

Global research trends and emerging opportunities for integrin adhesion complexes in cardiac repair: a scientometric analysis.

Cardiac regenerative medicine has gained significant attention in recent years, and integrins are known to play a critical role in mediating cardiac development and repair, especially after an injury from the myocardial infarction (MI). Given the extensive research history and interdisciplinary nature of this field, a quantitative retrospective analysis and visualization of related topics is necessary. We performed a scientometric analysis of published papers on cardiac integrin adhesion complexes (IACs), including analysis of annual publications, disciplinary evolution, keyword co-occurrence, and literature co-citation. A total of 2,664 publications were finally included in the past 20 years. The United States is the largest contributor to the study and is leading this area of research globally. The journal Circulation Research attracts the largest number of high-quality publications. The study of IACs in cardiac repair/regenerative therapies involves multiple disciplines, particularly in materials science and developmental biology. Keywords of research frontiers were represented by Tenasin-C (2019-2023) and inflammation (2020-2023). Integrins are topics with ongoing enthusiasm in biological development and tissue regeneration. The rapidly emerging role of matricellular proteins and non-protein components of the extracellular matrix (ECM) in regulating matrix structure and function may be a further breakthrough point in the future; the emerging role of IACs and their downstream molecular signaling in cardiac repair are also of great interest, such as induction of cardiac proliferation, differentiation, maturation, and metabolism, fibroblast activation, and inflammatory modulation.

The Potential of Extracellular Matrix- and Integrin Adhesion Complex-Related Molecules for Prostate Cancer Biomarker Discovery.

Prostate cancer is among the top five cancer types according to incidence and mortality. One of the main obstacles in prostate cancer management is the inability to foresee its course, which ranges from slow growth throughout years that requires minimum or no intervention to highly aggressive disease that spreads quickly and resists treatment. Therefore, it is not surprising that numerous studies have attempted to find biomarkers of prostate cancer occurrence, risk stratification, therapy response, and patient outcome. However, only a few prostate cancer biomarkers are used in clinics, which shows how difficult it is to find a novel biomarker. Cell adhesion to the extracellular matrix (ECM) through integrins is among the essential processes that govern its fate. Upon activation and ligation, integrins form multi-protein intracellular structures called integrin adhesion complexes (IACs). In this review article, the focus is put on the biomarker potential of the ECM- and IAC-related molecules stemming from both body fluids and prostate cancer tissue. The processes that they are involved in, such as tumor stiffening, bone turnover, and communication via exosomes, and their biomarker potential are also reviewed.

Thy-1 (CD90)-regulated cell adhesion and migration of mesenchymal cells: insights into adhesomes, mechanical forces, and signaling pathways

Cell adhesion and migration depend on the assembly and disassembly of adhesive structures known as focal adhesions. Cells adhere to the extracellular matrix (ECM) and form these structures via receptors, such as integrins and syndecans, which initiate signal transduction pathways that bridge the ECM to the cytoskeleton, thus governing adhesion and migration processes. Integrins bind to the ECM and soluble or cell surface ligands to form integrin adhesion complexes (IAC), whose composition depends on the cellular context and cell type. Proteomic analyses of these IACs led to the curation of the term adhesome, which is a complex molecular network containing hundreds of proteins involved in signaling, adhesion, and cell movement. One of the hallmarks of these IACs is to sense mechanical cues that arise due to ECM rigidity, as well as the tension exerted by cell-cell interactions, and transduce this force by modifying the actin cytoskeleton to regulate cell migration. Among the integrin/syndecan cell surface ligands, we have described Thy-1 (CD90), a GPI-anchored protein that possesses binding domains for each of these receptors and, upon engaging them, stimulates cell adhesion and migration. In this review, we examine what is currently known about adhesomes, revise how mechanical forces have changed our view on the regulation of cell migration, and, in this context, discuss how we have contributed to the understanding of signaling mechanisms that control cell adhesion and migration.

Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly.

Endothelial cell interactions with their extracellular matrix are essential for vascular homeostasis and expansion. Large-scale proteomic analyses aimed at identifying components of integrin adhesion complexes have revealed the presence of several RNA binding proteins (RBPs) of which the functions at these sites remain poorly understood. Here, we explored the role of the RBP SAM68 (Src associated in mitosis, of 68 kDa) in endothelial cells. We found that SAM68 is transiently localized at the edge of spreading cells where it participates in membrane protrusive activity and the conversion of nascent adhesions to mechanically loaded focal adhesions by modulation of integrin signaling and local delivery of β-actin mRNA. Furthermore, SAM68 depletion impacts cell-matrix interactions and motility through induction of key matrix genes involved in vascular matrix assembly. In a 3D environment SAM68-dependent functions in both tip and stalk cells contribute to the process of sprouting angiogenesis. Altogether, our results identify the RBP SAM68 as a novel actor in the dynamic regulation of blood vessel networks.

Surface-induced phase separation of reconstituted nascent integrin clusters on lipid membranes

Integrin adhesion complexes are essential membrane-associated cellular compartments for metazoan life. The formation of initial integrin adhesion complexes is a dynamic process involving focal adhesion proteins assembled at the integrin cytoplasmic tails and the inner leaflet of the plasma membrane. The weak multivalent protein interactions within the complex and with the plasma membrane suggest that liquid-liquid phase separation could play a role in the nascent adhesion assembly. Here, we report that solid-supported lipid membranes supplemented with phosphoinositides induce the phase separation of minimal integrin adhesion condensates composed of integrin β1 tails, kindlin, talin, paxillin, and FAK at physiological ionic strengths and protein concentrations. We show that the presence of phosphoinositides is key to enriching kindlin and talin on the lipid membrane, which is necessary to further induce the phase separation of paxillin and FAK at the membrane. Our data demonstrate that lipid membrane surfaces set the local solvent conditions for steering the membrane-localized phase separation even in a regime where no condensate formation of proteins occurs in bulk solution.

Extracellular Matrix- and Integrin Adhesion Complexes-Related Genes in the Prognosis of Prostate Cancer Patients' Progression-Free Survival.

Prostate cancer is a heterogeneous disease, and one of the main obstacles in its management is the inability to foresee its course. Therefore, novel biomarkers are needed that will guide the treatment options. The extracellular matrix (ECM) is an important part of the tumor microenvironment that largely influences cell behavior. ECM components are ligands for integrin receptors which are involved in every step of tumor progression. An underlying characteristic of integrin activation and ligation is the formation of integrin adhesion complexes (IACs), intracellular structures that carry information conveyed by integrins. By using The Cancer Genome Atlas data, we show that the expression of ECM- and IACs-related genes is changed in prostate cancer. Moreover, machine learning methods revealed that they are a source of biomarkers for progression-free survival of patients that are stratified according to the Gleason score. Namely, low expression of FMOD and high expression of PTPN2 genes are associated with worse survival of patients with a Gleason score lower than 9. The FMOD gene encodes protein that may play a role in the assembly of the ECM and the PTPN2 gene product is a protein tyrosine phosphatase activated by integrins. Our results suggest potential biomarkers of prostate cancer progression.

A luciferase fragment complementation assay to detect focal adhesion kinase (FAK) signaling events

Integrin Adhesion Complexes (IACs) serve as links between the cytoskeleton and extracellular environment, acting as mechanosensing and signaling hubs. As such, IACs participate in many aspects of cellular motility, tissue morphogenesis, anchorage-dependent growth and cell survival. Focal Adhesion Kinase (FAK) has emerged as a critical organizer of IAC signaling events due to its early recruitment and diverse substrates, and thus has become a genetic and therapeutic target. Here we present the design and characterization of simple, reversible, and scalable Bimolecular Complementation sensors to monitor FAK phosphorylation in living cells. These probes provide novel means to quantify IAC signaling, expanding on the currently available toolkit for interrogating FAK phosphorylation during diverse cellular processes.

Mena regulates nesprin-2 to control actin–nuclear lamina associations, trans-nuclear membrane signalling and gene expression

Interactions between cells and the extracellular matrix, mediated by integrin adhesion complexes, play key roles in fundamental cellular processes, including the sensing and transduction of mechanical cues. Here, we investigate systems-level changes in the integrin adhesome in patient-derived cutaneous squamous cell carcinoma cells and identify the actin regulatory protein Mena as a key node in the adhesion complex network. Mena is connected within a subnetwork of actin-binding proteins to the LINC complex component nesprin-2, with which it interacts and co-localises at the nuclear envelope. Moreover, Mena potentiates the interactions of nesprin-2 with the actin cytoskeleton and the nuclear lamina. CRISPR-mediated Mena depletion causes altered nuclear morphology, reduces tyrosine phosphorylation of the nuclear membrane protein emerin and downregulates expression of the immunomodulatory gene PTX3 via the recruitment of its enhancer to the nuclear periphery. We uncover an unexpected role for Mena at the nuclear membrane, where it controls nuclear architecture, chromatin repositioning and gene expression. Our findings identify an adhesion protein that regulates gene transcription via direct signalling across the nuclear envelope.

Multi-omics analysis of sarcospan overexpression in mdx skeletal muscle reveals compensatory remodeling of cytoskeleton-matrix interactions that promote mechanotransduction pathways

BackgroundThe dystrophin-glycoprotein complex (DGC) is a critical adhesion complex of the muscle cell membrane, providing a mechanical link between the extracellular matrix (ECM) and the cortical cytoskeleton that stabilizes the sarcolemma during repeated muscle contractions. One integral component of the DGC is the transmembrane protein, sarcospan (SSPN). Overexpression of SSPN in the skeletal muscle of mdx mice (murine model of DMD) restores muscle fiber attachment to the ECM in part through an associated increase in utrophin and integrin adhesion complexes at the cell membrane, protecting the muscle from contraction-induced injury. In this study, we utilized transcriptomic and ECM protein-optimized proteomics data sets from wild-type, mdx, and mdx transgenic (mdxTG) skeletal muscle tissues to identify pathways and proteins driving the compensatory action of SSPN overexpression.MethodsThe tibialis anterior and quadriceps muscles were isolated from wild-type, mdx, and mdxTG mice and subjected to bulk RNA-Seq and global proteomics analysis using methods to enhance capture of ECM proteins. Data sets were further analyzed through the ingenuity pathway analysis (QIAGEN) and integrative gene set enrichment to identify candidate networks, signaling pathways, and upstream regulators.ResultsThrough our multi-omics approach, we identified 3 classes of differentially expressed genes and proteins in mdxTG muscle, including those that were (1) unrestored (significantly different from wild type, but not from mdx), (2) restored (significantly different from mdx, but not from wild type), and (3) compensatory (significantly different from both wild type and mdx). We identified signaling pathways that may contribute to the rescue phenotype, most notably cytoskeleton and ECM organization pathways. ECM-optimized proteomics revealed an increased abundance of collagens II, V, and XI, along with β-spectrin in mdxTG samples. Using ingenuity pathway analysis, we identified upstream regulators that are computationally predicted to drive compensatory changes, revealing a possible mechanism of SSPN rescue through a rewiring of cell-ECM bidirectional communication. We found that SSPN overexpression results in upregulation of key signaling molecules associated with regulation of cytoskeleton organization and mechanotransduction, including Yap1, Sox9, Rho, RAC, and Wnt.ConclusionsOur findings indicate that SSPN overexpression rescues dystrophin deficiency partially through mechanotransduction signaling cascades mediated through components of the ECM and the cortical cytoskeleton.

Local pH mapping in the cell adhesion nano-interfaces on a pH-responsive fluorescence-dye-immobilized substrate.

Cell-substrate adhesion nano-interfaces can, in principle, exhibit a spatial distribution of local pH values under the influence of the weakly acidic microenvironment of glycocalyx grafted on lipid bilayer cell membrane which is compressed and closely attached to culture substrate in the vicinity of integrin-adhesion complexes. However, a simple local pH distribution imaging methodology has not been developed. In this study, to visualize the local pH distribution at the cell adhesion interface, we prepared glass substrates chemically modified with a pH-responsive fluorescent dye fluorescein isothiocyanate (FITC), observed the distribution of FITC fluorescence intensity at the adhesion interface of fibroblast (NIH/3T3) and cancer cells (HeLa), and compared the FITC images with the observed distribution of focal adhesions. FITC images were converted to pH mapping based on the pH-fluorescence calibration data of surface-immobilized FITC pre-measured in different pH media, which showed significantly larger regions with lowered pH level (6.8-7.0) from outside the cell (pH 7.4) were observed at the thick inner periphery of HeLa cells while 3T3 cells exhibited smaller lowered pH regions at the thin periphery. The lowered pH regions overlapped with many focal adhesions, and image analysis showed that larger focal adhesions tend to possess more lowered pH sites inside, reflecting enhanced glycocalyx compression due to accumulated integrin-adhesion ligand binding. This tendency was stronger for HeLa than for 3T3 cells. The role of glycocalyx compression and the pH reduction at the cell adhesive interface is discussed.

Vinculin recruitment to α-catenin halts the differentiation and maturation of enterocyte progenitors to maintain homeostasis of the Drosophila intestine.

Mechanisms communicating changes in tissue stiffness and size are particularly relevant in the intestine because it is subject to constant mechanical stresses caused by peristalsis of its variable content. Using the Drosophila intestinal epithelium, we investigate the role of vinculin, one of the best characterised mechanoeffectors, which functions in both cadherin and integrin adhesion complexes. We discovered that vinculin regulates cell fate decisions, by preventing precocious activation and differentiation of intestinal progenitors into absorptive cells. It achieves this in concert with α-catenin at sites of cadherin adhesion, rather than as part of integrin function. Following asymmetric division of the stem cell into a stem cell and an enteroblast (EB), the two cells initially remain connected by adherens junctions, where vinculin is required, only on the EB side, to maintain the EB in a quiescent state and inhibit further divisions of the stem cell. By manipulating cell tension, we show that vinculin recruitment to adherens junction regulates EB activation and numbers. Consequently, removing vinculin results in an enlarged gut with improved resistance to starvation. Thus, mechanical regulation at the contact between stem cells and their progeny is used to control tissue cell number.

Cytoophidia coupling adipose architecture and metabolism.

Tissue architecture determines its unique physiology and function. How these properties are intertwined has remained unclear. Here we show that the metabolic enzyme CTP synthase (CTPS) form filamentous structures termed cytoophidia along the adipocyte cortex in Drosophila adipose tissue. Loss of cytoophidia, whether due to reduced CTPS expression or a point mutation that specifically abrogates its polymerization ability, causes impaired adipocyte adhesion and defective adipose tissue architecture. Moreover, CTPS influences integrin distribution and dot-like deposition of type IVcollagen(Col IV). Col IV-integrin signaling reciprocally regulates the assembly of cytoophidia in adipocytes. Our results demonstrate that a positive feedback signaling loop containing both cytoophidia and integrin adhesion complex couple tissue architecture and metabolism in Drosophila adipose tissue.

Genetic analysis suggests a surface of PAT-4 (ILK) that interacts with UNC-112 (kindlin).

Integrin plays a crucial role in the attachment of cells to the extracellular matrix. Integrin recruits many proteins intracellularly, including a 4-protein complex (kindlin, ILK, PINCH, and parvin). Caenorhabditis elegans muscle provides an excellent model to study integrin adhesion complexes. In Caenorhabditis elegans, UNC-112 (kindlin) binds to the cytoplasmic tail of PAT-3 (β-integrin) and to PAT-4 (ILK). We previously reported that PAT-4 binding to UNC-112 is essential for the binding of UNC-112 to PAT-3. Although there are crystal structures for ILK and a kindlin, there is no co-crystal structure available. To understand the molecular interaction between PAT-4 and UNC-112, we took a genetic approach. First, using a yeast 2-hybrid method, we isolated mutant PAT-4 proteins that cannot bind to UNC-112 and then isolated suppressor mutant UNC-112 proteins that restore interaction with mutant PAT-4 proteins. Second, we demonstrated that these mutant PAT-4 proteins cannot localize to attachment structures in nematode muscle, but upon co-expression of an UNC-112 suppressor mutant protein, mutant PAT-4 proteins could localize to attachment structures. Third, overexpression of a PAT-4 mutant results in the disorganization of adhesion plaques at muscle cell boundaries and co-expression of the UNC-112 suppressor mutant protein alleviates this defect. Thus, we demonstrate that UNC-112 binding to PAT-4 is required for the localization and function of PAT-4 in integrin adhesion complexes in vivo. The missense mutations were mapped onto homology models of PAT-4 and UNC-112, and taking into account previously isolated mutations, we suggest a surface of PAT-4 that binds to UNC-112.