Assembly Of Adhesion Complexes Research Articles

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.

Leishmania amazonensis infection impairs VLA-4 clustering and adhesion complex assembly at the adhesion site of J774 cells.

Cutaneous leishmaniasis is an infectious disease that may lead to a single or multiple disseminated cutaneous lesions. The mechanisms involved in Leishmania dissemination to different areas of the skin and the internal organs remain poorly understood. Evidence shows that Very Late Antigen-4 (VLA-4)-dependent phagocyte adhesion is impaired by Leishmania infection, which may be related to the mechanisms of parasite dissemination. We investigated factors potentially associated with decreased VLA-4-mediated adhesion in Leishmania-infected macrophages, including lipid raft-mediated VLA-4 mobilization along the cellular membrane, integrin cluster formation at the cell base (adhesion site), and focal adhesion complex assembly. Phagocytes treated with Methyl-β-Cyclodextrin (MβCD) demonstrated reduced adhesion, similarly to Leishmania amazonensis-infected J774 cells. Infected and MβCD-treated macrophages presented decreased VLA-4 mobilization to the adhesion plane, as well as reduced integrin clustering. Leishmania amazonensis-infected cells exhibited talin depletion, as well as a decreased mobilization of adhesion complex proteins, such as talin and viculin, which were associated with lower VLA-4 concentrations at the adhesion site and limited cell-spreading. Our results suggest that Leishmania infection may modulate the firm adhesion phase of the cell-spreading process, which could contribute to the bloodstream dissemination of infected cells.

Integrin Adhesion Complex Organization in Sheep Myometrium Reflects Changing Mechanical Forces during Pregnancy and Postpartum.

Simple SummaryIACs assemble within the sheep myometrium during early-to-mid gestation in response to increased stretch of the uterine wall and continue to increase as pregnancy progresses. Fibronectin (FN1) is important in its ability to attach to IACs in myometrial cells to generate force to sustain powerful contractions during labor. After parturition, IACs are disassembled but the integrin subunits ITGA5 and ITGB1 remain expressed at the protein level at least two weeks postpartum.Cells respond to extracellular mechanical forces through the assembly of integrin adhesion complexes (IACs) that provide a scaffold through which cells sense and transduce responses to those forces. IACs are composed of transmembrane integrin receptors that bind to extracellular matrix (ECM) proteins externally and connect with the actomyosin cytoskeleton internally. Myometrial smooth muscle cells respond to forces that arise due to increases in fetal growth/weight, placental fluid volumes, and blood flow. As a result, the uterus transforms into an organ that can forcefully expel the fetus and placental membranes during parturition. While earlier studies focused on IAC expression in the myometrial compartment of rodents and humans to explore pregnancy-associated responses, the present study examines IAC assembly in ovine myometrium where mechanical forces are expected to be amplified in a manner similar to humans. Results indicate that the ITGA5 and ITGB1 heterodimers associate with the ECM protein FN1 externally, and with VCL and TLN1 internally, to form IACs in myometrial cells during the first trimester of pregnancy. These IACs become increasingly ordered until parturition. This ordered structure is lost by one day postpartum; however, the abundance of the integrin proteins remains elevated for at least two weeks postpartum. Implications of the present study are that sheep are similar to humans regarding the assembly of IACs in the pregnant myometrium and suggest that IACs may form much earlier in human gestation than was previously implied by the rat model. Results highlight the continued value of the sheep model as a flagship gynecological model for understanding parturition in humans.

Network Analysis of Integrin Adhesion Complexes.

Cell-surface adhesion receptors mediate interactions with the extracellular matrix (ECM) to control many fundamental aspects of cell behavior, including cell migration, survival, and proliferation. Integrin adhesion receptors recruit structural and signaling proteins to form multimolecular adhesion complexes that link the plasma membrane to the actomyosin cytoskeleton. The assembly and turnover of adhesion complexes are tightly regulated, governed in part by the networks of physical protein interactions and functional signaling associations between components of the adhesome. Proteomic profiling of adhesion complexes has begun to reveal their molecular complexity and diversity. To interrogate the composition of cell-ECM adhesions, we detail herein an approach for the network analysis of adhesion complex proteomes. Integration of these proteomic data with adhesome databases in the context of predicted protein interactions enables the mapping of experimentally defined adhesion complex networks. Computational analysis of resultant network models can identify subnetworks of putative functionally linked adhesion protein communities. This approach provides a framework to predict functional adhesion protein relationships and generate new mechanistic hypotheses for further experimental testing.

The Rho-GEF PIX-1 directs assembly or stability of lateral attachment structures between muscle cells

PIX proteins are guanine nucleotide exchange factors (GEFs) that activate Rac and Cdc42, and are known to have numerous functions in various cell types. Here, we show that a PIX protein has an important function in muscle. From a genetic screen in C. elegans, we found that pix-1 is required for the assembly of integrin adhesion complexes (IACs) at borders between muscle cells, and is required for locomotion of the animal. A pix-1 null mutant has a reduced level of activated Rac in muscle. PIX-1 localizes to IACs at muscle cell boundaries, M-lines and dense bodies. Mutations in genes encoding proteins at known steps of the PIX signaling pathway show defects at muscle cell boundaries. A missense mutation in a highly conserved residue in the RacGEF domain results in normal levels of PIX-1 protein, but a reduced level of activated Rac in muscle, and abnormal IACs at muscle cell boundaries.

S100A4 is activated by RhoA and catalyses the polymerization of non-muscle myosin, adhesion complex assembly and contraction in airway smooth muscle.

S100A4 is expressed in many tissues, including smooth muscle (SM), but its physiologic function is unknown. S100A4 regulates the motility of metastatic cancer cells by binding to non-muscle (NM) myosin II. Contractile stimulation causes the polymerization of NM myosin in airway SM, which is necessary for tension development. NM myosin regulates the assembly of adhesion junction signalling complexes (adhesomes) that catalyse actin polymerization. In airway SM, ACh (acetylcholine) stimulated the binding of S100A4 to the NM myosin heavy chain, which was catalysed by RhoA GTPase via the RhoA-binding protein, rhotekin. The binding of S100A4 to NM myosin was required for NM myosin polymerization, adhesome assembly and actin polymerization. S100A4 plays a critical function in the regulation of airway SM contraction by catalysing NM myosin filament assembly. The interaction of S100A4 with NM myosin may also play an important role in the physiologic function of other tissues. S100A4 binds to the heavy chain of non-muscle (NM) myosin II and can regulate the motility of crawling cells. S100A4 is widely expressed in many tissues including smooth muscle (SM), although its role in the regulation of their physiologic function is not known. We hypothesized that S100A4 contributes to the regulation of contraction in airway SM by regulating a pool of NM myosin II at the cell cortex. NM myosin II undergoes polymerization in airway SM and regulates contraction by catalysing the assembly of integrin-associated adhesome complexes that activate pathways that catalyse actin polymerization. ACh stimulated the interaction of S100A4 with NM myosin II in airway SM at the cell cortex and catalysed NM myosin filament assembly. RhoA GTPase regulated the activation of S100A4 via rhotekin, which facilitated the formation of a complex between RhoA, S100A4 and NM myosin II. The depletion of S100A4, RhoA or rhotekin from airway SM tissues using short hairpin RNA or small interfering RNA prevented NM myosin II polymerization as well as the recruitment of vinculin and paxillin to adhesome signalling complexes in response to ACh, and inhibited actin polymerization and tension development. S100A4 depletion did not affect ACh-stimulated SM myosin regulatory light chain phosphorylation. The results show that S100A4 plays a critical role in tension development in airway SM tissue by catalysing NM myosin filament assembly, and that the interaction of S100A4 with NM myosin in response to contractile stimulation is activated by RhoA GTPase. These results may be broadly relevant to the physiologic function of S100A4 in other cell and tissue types.

Clustering of integrin \u03b2 cytoplasmic domains triggers nascent adhesion formation and reveals a protozoan origin of the integrin-talin interaction

Integrins and integrin-dependent cell-matrix adhesions are essential for a number of physiological processes. Integrin function is tightly regulated via binding of cytoplasmic proteins to integrin intracellular domains. Yet, the complexity of cell-matrix adhesions in mammals, with more than 150 core adhesome proteins, complicates the analysis of integrin-associated protein complexes. Interestingly, the evolutionary origin of integrins dates back before the transition from unicellular life to complex multicellular animals. Though unicellular relatives of metazoa have a less complex adhesome, nothing is known about the initial steps of integrin activation and adhesion complex assembly in protozoa. Therefore, we developed a minimal, microscope-based system using chimeric integrins to investigate receptor-proximal events during focal adhesion assembly. Clustering of the human integrin β1 tail led to recruitment of talin, kindlin, and paxillin and mutation of the known talin binding site abolished recruitment of this protein. Proteins indirectly linked to integrins, such as vinculin, migfilin, p130CAS, or zyxin were not enriched around the integrin β1 tail. With the exception of integrin β4 and integrin β8, the cytoplasmic domains of all human integrin β subunits supported talin binding. Likewise, the cytoplasmic domains of integrin β subunits expressed by the protozoan Capsaspora owczarzaki readily recruited talin and this interaction was based on an evolutionary conserved NPXY/F amino acid motif. The results we present here validate the use of our novel microscopic assay to uncover details of integrin-based protein-protein interactions in a cellular context and suggest that talin binding to integrin β cytoplasmic tails is an ancient feature of integrin regulation.

Universal Kinetics of the Onset of Cell Spreading on Substrates of Different Stiffness

When plated onto substrates, cell morphology and even stem-cell differentiation are influenced by the stiffness of their environment. Stiffer substrates give strongly spread (eventually polarized) cells with strong focal adhesions and stress fibers; very soft substrates give a less developed cytoskeleton and much lower cell spreading. The kinetics of this process of cell spreading is studied extensively, and important universal relationships are established on how the cell area grows with time. Here, we study the population dynamics of spreading cells, investigating the characteristic processes involved in the cell response to the substrate. We show that unlike the individual cell morphology, this population dynamics does not depend on the substrate stiffness. Instead, a strong activation temperature dependence is observed. Different cell lines on different substrates all have long-time statistics controlled by the thermal activation over a single energy barrier ΔG ≈ 18 kcal/mol, whereas the early-time kinetics follows a power law ∼t5. This implies that the rate of spreading depends on an internal process of adhesion complex assembly and activation; the operational complex must have five component proteins, and the last process in the sequence (which we believe is the activation of focal adhesion kinase) is controlled by the binding energy ΔG.

Scribble: A master scaffold in polarity, adhesion, synaptogenesis, and proliferation.

Key events ranging from cell polarity to proliferation regulation to neuronal signaling rely on the assembly of multiprotein adhesion or signaling complexes at particular subcellular sites. Multidomain scaffolding proteins nucleate assembly and direct localization of these complexes, and the protein Scribble and its relatives in the LAP protein family provide a paradigm for this. Scribble was originally identified because of its role in apical-basal polarity and epithelial integrity in Drosophila melanogaster It is now clear that Scribble acts to assemble and position diverse multiprotein complexes in processes ranging from planar polarity to adhesion to oriented cell division to synaptogenesis. Here, we explore what we have learned about the mechanisms of action of Scribble in the context of its multiple known interacting partners and discuss how this knowledge opens new questions about the full range of Scribble protein partners and their structural and signaling roles.

Adiponectin is an endogenous anti-fibrotic mediator and therapeutic target

Skin fibrosis in systemic sclerosis (SSc) is accompanied by attrition of dermal white adipose tissue (dWAT) and reduced levels of circulating adiponectin. Since adiponectin has potent regulatory effects on fibroblasts, we sought to assess adiponectin signaling in SSc skin biopsies, and evaluate fibrosis in mice with adiponectin gain- and loss-of-function mutations. Furthermore, we investigated the effects and mechanism of action of agonist peptides targeting adiponectin receptors in vitro and in vivo. We found that adiponectin pathway activity was significantly reduced in a subset of SSc skin biopsies. Mice lacking adiponectin mounted an exaggerated dermal fibrotic response, while transgenic mice with constitutively elevated adiponectin showed selective dWAT expansion and protection from skin and peritoneal fibrosis. Adiponectin receptor agonists abrogated ex vivo fibrotic responses in explanted normal and SSc fibroblasts and in 3D human skin equivalents, in part by attenuating focal adhesion complex assembly, and prevented and reversed experimentally-induced organ fibrosis in mice. These results implicate aberrant adiponectin pathway activity in skin fibrosis, identifying a novel function for this pleiotropic adipokine in regulation of tissue remodeling. Restoring adiponectin signaling in SSc patients therefore might represent an innovative pharmacological strategy for intractable organ fibrosis.

Receptor Nucleation and Clustering in Cellular Adhesion and Mechanical Signal Transduction

E-cadherin-based cell-cell adhesions are key to development and maintenance of the epithelial tissue, and a loss of these adhesions may contribute to cancer development. These are mechanosensitive structures in that they are strengthened under tension. Mechanotransduction in these adhesions has been postulated to be mediated, in part, by a force-dependent conformational activation of α-catenin, which allows it to interact with vinculin, in addition to F-actin, resulting in strengthening of junctions. Here, using E-cadherin adhesions reconstituted on synthetic, nanopatterned membranes, we show that activation of α-catenin is dependent on E-cadherin clustering, and is sustained in the absence of mechanical force or association with F-actin or vinculin. Adhesions are formed by filopodia-mediated nucleation and micron-scale assembly of E-cadherin clusters, which could be distinguished as either peripheral or central depending on their relative location at the cell-bilayer adhesion. While F-actin, vinculin and phosphorylated myosin light chain associate only with the peripheral assemblies, activated α-catenin is present in both peripheral and central assemblies, and persisted in the central assemblies in the absence of actomyosin tension. Impeding filopodia-mediated nucleation and micron-scale assembly of E-cadherin adhesion complexes, by confining bilayer bound E-cadherin extracellular domain movement on nanopatterned substrates, reduced levels of activated α-catenin. Taken together, although the initial activation of α-catenin requires micron-scale clustering that may allow development of mechanical forces, sustained force is not required for maintaining α-catenin in the active state.

Sustained α-catenin Activation at E-cadherin Junctions in the Absence of Mechanical Force

Mechanotransduction at E-cadherin junctions has been postulated to be mediated in part by a force-dependent conformational activation of α-catenin. Activation of α-catenin allows it to interact with vinculin in addition to F-actin, resulting in a strengthening of junctions. Here, using E-cadherin adhesions reconstituted on synthetic, nanopatterned membranes, we show that activation of α-catenin is dependent on E-cadherin clustering, and is sustained in the absence of mechanical force or association with F-actin or vinculin. Adhesions were formed by filopodia-mediated nucleation and micron-scale assembly of E-cadherin clusters, which could be distinguished as either peripheral or central assemblies depending on their relative location at the cell-bilayer adhesion. Whereas F-actin, vinculin, and phosphorylated myosin light chain associated only with the peripheral assemblies, activated α-catenin was present in both peripheral and central assemblies, and persisted in the central assemblies in the absence of actomyosin tension. Impeding filopodia-mediated nucleation and micron-scale assembly of E-cadherin adhesion complexes by confining the movement of bilayer-bound E-cadherin on nanopatterned substrates reduced the levels of activated α-catenin. Taken together, these results indicate that although the initial activation of α-catenin requires micron-scale clustering that may allow the development of mechanical forces, sustained force is not required for maintaining α-catenin in the active state.

Vinculin controls talin engagement with the actomyosin machinery.

The link between extracellular-matrix-bound integrins and intracellular F-actin is essential for cell spreading and migration. Here, we demonstrate how the actin-binding proteins talin and vinculin cooperate to provide this link. By expressing structure-based talin mutants in talin null cells, we show that while the C-terminal actin-binding site (ABS3) in talin is required for adhesion complex assembly, the central ABS2 is essential for focal adhesion (FA) maturation. Thus, although ABS2 mutants support cell spreading, the cells lack FAs, fail to polarize and exert reduced force on the surrounding matrix. ABS2 is inhibited by the preceding mechanosensitive vinculin-binding R3 domain, and deletion of R2R3 or expression of constitutively active vinculin generates stable force-independent FAs, although cell polarity is compromised. Our data suggest a model whereby force acting on integrin-talin complexes via ABS3 promotes R3 unfolding and vinculin binding, activating ABS2 and locking talin into an actin-binding configuration that stabilizes FAs.

Definition of a consensus integrin adhesome and its dynamics during adhesion complex assembly and disassembly.

Integrin receptor activation initiates the formation of integrin adhesion complexes (IACs) at the cell membrane that transduce adhesion-dependent signals to control a multitude of cellular functions. Proteomic analyses of isolated IACs have revealed an unanticipated molecular complexity; however, a global view of the consensus composition and dynamics of IACs is currently lacking. Here, we have integrated several IAC proteomes and generated a 2,412-protein integrin adhesome. Analysis of this dataset reveals the functional diversity of proteins in IACs and establishes a consensus adhesome of 60 proteins. The consensus adhesome likely represents a core cell adhesion machinery, centred around four axes comprising ILK-PINCH-kindlin, FAK-paxillin, talin-vinculin and α-actinin-zyxin-VASP, and includes underappreciated IAC components such as Rsu-1 and caldesmon. Proteomic quantification of IAC assembly and disassembly detailed the compositional dynamics of the core cell adhesion machinery. The definition of this consensus view of integrin adhesome components provides a resource for the research community.

FAK is required for tension-dependent organization of collective cell movements in Xenopus mesendoderm

Collective cell movements are integral to biological processes such as embryonic development and wound healing and also have a prominent role in some metastatic cancers. In migrating Xenopus mesendoderm, traction forces are generated by cells through integrin-based adhesions and tension transmitted across cadherin adhesions. This is accompanied by assembly of a mechanoresponsive cadherin adhesion complex containing keratin intermediate filaments and the catenin-family member plakoglobin. We demonstrate that focal adhesion kinase (FAK), a major component of integrin adhesion complexes, is required for normal morphogenesis at gastrulation, closure of the anterior neural tube, axial elongation and somitogenesis. Depletion of zygotically expressed FAK results in disruption of mesendoderm tissue polarity similar to that observed when expression of keratin or plakoglobin is inhibited. Both individual and collective migrations of mesendoderm cells from FAK depleted embryos are slowed, cell protrusions are disordered, and cell spreading and traction forces are decreased. Additionally, keratin filaments fail to organize at the rear of cells in the tissue and association of plakoglobin with cadherin is diminished. These findings suggest that FAK is required for the tension-dependent assembly of the cadherin adhesion complex that guides collective mesendoderm migration, perhaps by modulating the dynamic balance of substrate traction forces and cell cohesion needed to establish cell polarity.

Multimeric assembly of host-pathogen adhesion complexes involved in apicomplexan invasion.

Apicomplexan parasites are the causative agents of diseases that include malaria, toxoplasmosis, and coccidiosis. These obligate intracellular parasites have evolved to use a conserved mechanism for host-cell invasion. The apicomplexan phylum is defined by the presence of micronemes and rhoptries, which are distinct organelles located at the apical end of the parasite. These organelles secrete molecules necessary for host-cell invasion [1]. Apicomplexan parasites can invade disparate cell types, including hepatocytes, erythrocytes, lymphocytes, macrophages, and cells lining the digestive tract. Unlike viruses and intracellular bacteria, apicomplexans actively invade host cells without relying on host uptake pathways. As such, host-cell sensing and subsequent invasion are driven entirely by the parasite in a dynamic and rapid process. Intracellular residence protects the parasite from immune attack and enables parasite replication prior to host-cell lysis and subsequent invasion of neighboring host cells. The repertoire of ligand-receptor complexes utilized by parasites for entry into host cells is diverse. Some interactions occur through cell-specific receptors resulting in high-affinity interactions, while others occur through multiple lower-affinity interactions via surface moieties found on several cell types. Receptor-specific and general cell binding may explain host-cell tropism of different pathogens, although additional factors are important. There is growing evidence that multimeric assembly of parasite ligands and host surface molecules strengthens the host-pathogen interactions necessary for invasion. We discuss recent work that has advanced our knowledge of the assembly of adhesive complexes from two critical apicomplexan pathogens and highlight areas of research that require further investigation.

Elevated Expression of the Integrin-Associated Protein PINCH Suppresses the Defects of Drosophila melanogaster Muscle Hypercontraction Mutants

A variety of human diseases arise from mutations that alter muscle contraction. Evolutionary conservation allows genetic studies in Drosophila melanogaster to be used to better understand these myopathies and suggest novel therapeutic strategies. Integrin-mediated adhesion is required to support muscle structure and function, and expression of Integrin adhesive complex (IAC) proteins is modulated to adapt to varying levels of mechanical stress within muscle. Mutations in flapwing (flw), a catalytic subunit of myosin phosphatase, result in non-muscle myosin hyperphosphorylation, as well as muscle hypercontraction, defects in size, motility, muscle attachment, and subsequent larval and pupal lethality. We find that moderately elevated expression of the IAC protein PINCH significantly rescues flw phenotypes. Rescue requires PINCH be bound to its partners, Integrin-linked kinase and Ras suppressor 1. Rescue is not achieved through dephosphorylation of non-muscle myosin, suggesting a mechanism in which elevated PINCH expression strengthens integrin adhesion. In support of this, elevated expression of PINCH rescues an independent muscle hypercontraction mutant in muscle myosin heavy chain, MhcSamba1. By testing a panel of IAC proteins, we show specificity for PINCH expression in the rescue of hypercontraction mutants. These data are consistent with a model in which PINCH is present in limiting quantities within IACs, with increasing PINCH expression reinforcing existing adhesions or allowing for the de novo assembly of new adhesion complexes. Moreover, in myopathies that exhibit hypercontraction, strategic PINCH expression may have therapeutic potential in preserving muscle structure and function.

Estrogen-Dependent Uterine Secretion of Osteopontin Activates Blastocyst Adhesion Competence

Embryo implantation is a highly orchestrated process that involves blastocyst-uterine interactions. This process is confined to a defined interval during gestation referred to as the “window of embryo implantation receptivity”. In mice this receptive period is controlled by ovarian estrogen and involves a coordination of blastocyst adhesion competence and uterine receptivity. Mechanisms coordinating the acquisition of blastocyst adhesion competence and uterine receptivity are largely unknown. Here, we show that ovarian estrogen indirectly regulates blastocyst adhesion competence. Acquisition of blastocyst adhesion competence was attributed to integrin activation (e.g. formation of adhesion complexes) rather than de novo integrin synthesis. Osteopontin (OPN) was identified as an estrogen-dependent uterine endometrial gland secretory factor responsible for activating blastocyst adhesion competence. Increased adhesion complex assembly in OPN-treated blastocysts was mediated through focal adhesion kinase (FAK)- and phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathways. These findings define for the first time specific regulatory components of an estrogen-dependent pathway coordinating blastocyst adhesion competence and uterine receptivity.

An agent based model of integrin clustering: Exploring the role of ligand clustering, integrin homo-oligomerization, integrin–ligand affinity, membrane crowdedness and ligand mobility

Integrins are cell-surface protein heterodimers that coordinate cellular responses to mechanochemical cues from the extracellular matrix (ECM) and stimulate the assembly of small adhesion complexes, which are the initial sites of cell-ECM adhesion. Clustering of integrins is known to mediate signaling through a variety of signal transduction pathways. Yet, the molecular mechanisms of integrin clustering are poorly understood. In this paper, we develop computational models, using agent based modeling (ABM) techniques, to explore two key underlying mechanisms of integrin clustering, namely ligand organization and integrin homo-oligomerization. Our models help to shed light on the potential roles ligand clustering and integrin homo-oligomerization may play in controlling integrin clustering. A potential mechanism for the clustering of integrin is discussed and the effects of other parameters such as integrin–ligand affinity, membrane crowdedness and ligand mobility on integrin clustering are examined.

MiR-127 Protects Proximal Tubule Cells against Ischemia/Reperfusion: Identification of Kinesin Family Member 3B as miR-127 Target

Ischemia/reperfusion (I/R) is at the basis of renal transplantation and acute kidney injury. Molecular mechanisms underlying proximal tubule response to I/R will allow the identification of new therapeutic targets for both clinical settings. microRNAs have emerged as crucial and tight regulators of the cellular response to insults including hypoxia. Here, we have identified several miRNAs involved in the response of the proximal tubule cell to I/R. Microarrays and RT-PCR analysis of proximal tubule cells submitted to I/R mimicking conditions in vitro demonstrated that miR-127 is induced during ischemia and also during reperfusion. miR-127 is also modulated in a rat model of renal I/R. Interference approaches demonstrated that ischemic induction of miR-127 is mediated by Hypoxia Inducible Factor-1alpha (HIF-1α) stabilization. Moreover, miR-127 is involved in cell-matrix and cell-cell adhesion maintenance, since overexpression of miR-127 maintains focal adhesion complex assembly and the integrity of tight junctions. miR-127 also regulates intracellular trafficking since miR-127 interference promotes dextran-FITC uptake. In fact, we have identified the Kinesin Family Member 3B (KIF3B), involved in cell trafficking, as a target of miR-127 in rat proximal tubule cells. In summary, we have described a novel role of miR-127 in cell adhesion and its regulation by HIF-1α. We also identified for the first time KIF3B as a miR-127 target. Both, miR-127 and KIF3B appear as key mediators of proximal epithelial tubule cell response to I/R with potential al application in renal ischemic damage management.