Cryo-EM structure of I domain-containing integrin αEß7.

The integrin family is a transmembrane receptor that plays critical roles in the cell-cell and cell-extracellular matrix adhesion, signal transduction such as cell cycle regulation, organization of the intracellular cytoskeleton, and immune responses. Consequently, dysfunction of integrins is associated with a wide range of human diseases, including cancer and immune diseases, which makes integrins therapeutic targets for drug discovery. Here we report the cryo-EM structure of the human α-I domain-containing full-length integrin αEß7, which is expressed in the leukocytes of the immune system and a drug target for inflammatory bowel disease (IBD). The structure reveals the half-bent conformation, an intermediate between the close and the open conformation, while the α-I domain responsible for the ligand binding covers the headpiece domain by a unique spatial arrangement. Our results provide the structural information for the drug design targeting IBD.

Mapping transmembrane binding partners for E-cadherin ectodomains.

We combine proximity labeling and single molecule binding assays to discover transmembrane protein interactions in cells. We first screen for candidate binding partners by tagging the extracellular and cytoplasmic regions of a "bait" protein with BioID biotin ligase and identify proximal proteins that are biotin tagged on both their extracellular and intracellular regions. We then test direct binding interactions between proximal proteins and the bait, using single molecule atomic force microscope binding assays. Using this approach, we identify binding partners for the extracellular region of E-cadherin, an essential cell-cell adhesion protein. We show that the desmosomal proteins desmoglein-2 and desmocollin-3, the focal adhesion protein integrin-α2ß1, the receptor tyrosine kinase ligand ephrin-B1, and the classical cadherin P-cadherin, all directly interact with E-cadherin ectodomains. Our data shows that combining extracellular and cytoplasmic proximal tagging with a biophysical binding assay increases the precision with which transmembrane ectodomain interactors can be identified.

Membrane receptor activation mechanisms and transmembrane peptide tools to elucidate them.

Single-pass membrane receptors contain extracellular domains that respond to external stimuli and transmit information to intracellular domains through a single transmembrane (TM) α-helix. Because membrane receptors have various roles in homeostasis, signaling malfunctions of these receptors can cause disease. Despite their importance, there is still much to be understood mechanistically about how single-pass receptors are activated. In general, single-pass receptors respond to extracellular stimuli via alterations in their oligomeric state. The details of this process are still the focus of intense study, and several lines of evidence indicate that the TM domain (TMD) of the receptor plays a central role. We discuss three major mechanistic hypotheses for receptor activation: ligand-induced dimerization, ligand-induced rotation, and receptor clustering. Recent observations suggest that receptors can use a combination of these activation mechanisms and that technical limitations can bias interpretation. Short peptides derived from receptor TMDs, which can be identified by screening or rationally developed on the basis of the structure or sequence of their targets, have provided critical insights into receptor function. Here, we explore recent evidence that, depending on the target receptor, TMD peptides cannot only inhibit but also activate target receptors and can accommodate novel, bifunctional designs. Furthermore, we call for more sharing of negative results to inform the TMD peptide field, which is rapidly transforming into a suite of unique tools with the potential for future therapeutics.

Structure and mechanics of integrin-based cell adhesion.

Integrins are alpha/beta heterodimeric adhesion glycoprotein receptors that regulate a wide variety of dynamic cellular processes such as cell migration, phagocytosis, and growth and development. X-ray crystallography of the integrin ectodomain revealed its modular architecture and defined its metal-dependent interaction with extracellular ligands. This interaction is regulated from inside the cell (inside-out activation), through the short cytoplasmic alpha and beta integrin tails, which also mediate biochemical and mechanical signals transmitted to the cytoskeleton by the ligand-occupied integrins, effecting major changes in cell shape, behavior, and fate. Recent advances in the structural elucidation of integrins and integrin-binding cytoskeleton proteins are the subjects of this review.

Ischemia-induced apoptosis of intestinal epithelial cells correlates with altered integrin distribution and disassembly of F-actin triggered by calcium overload.

The present study examined intestinal epithelial cell (IEC) integrin distribution and disassembly of actin cytoskeleton in response to ischemia-anoxia. Protective effects of calcium channel blocker(CCB) were further examined to explore underlying mechanisms of cellular injury. Materials and Methods. Primary cultures of rat IECs and an in vitro model of ischemia/anoxia were established. IECs were exposed to ischemia/anoxia in the presence and absence of verapamil. The extent of exfoliation was determined using light microscopy while apoptosis rate was measured using flow cytometry. Changes in intracellular calcium, the distribution of integrins and the morphology of F-actin were assessed by confocal microscopy. Results. Detachment and apoptosis of IECs increased following ischemia/anoxia-induced injury. Treatment with verapamil inhibited the detachment and apoptosis. Under control conditions, the strongest fluorescent staining for integrins appeared on the basal surface of IECs while this re-distributed to the apical membrane in response to ischemic injury. Depolymerization of F-actin was also observed in the injured cells. Verapamil attenuated both changes of integrins and F-actin. Conclusions. Redistribution of integrins and disruption of F-actin under ischemia/anoxia injury is associated with IEC detachment and increased apoptosis. These events appeared to be triggered by an increase in Ca(2+)(i) suggesting a potential use for CCB in prevention and treatment of intestinal injury.

Comparing dendritic and self-assembly strategies to multivalency--RGD peptide-integrin interactions.

This paper compares covalent and non-covalent approaches for the organisation of ligand arrays to bind integrins. In the covalent strategy, linear RGD peptides are conjugated to first and second generation dendrons, and using a fluorescence polarisation competition assay, the first generation compound is demonstrated to show the most effective integrin binding, with an EC(50) of 125 µM (375 µM per peptide unit). As such, this dendritic compound is significantly more effective than a monovalent ligand, which does not bind integrin, even at concentrations as high as 1 mM. However, the second generation compound is significantly less effective, demonstrating that there is an optimum ligand density for multivalency in this case. In the non-covalent approach to multivalency, the same RGD peptide is functionalised with a hydrophobic C12 chain, giving rise to a lipopeptide which is demonstrated to be capable of self-assembly. This lipopeptide is capable of effective integrin binding at concentrations of 200 µM. These results therefore demonstrate that covalent (dendritic) and non-covalent (micellar self-assembly) approaches have, in this case, comparable efficiency in terms of achieving multivalent organisation of a ligand array.

Dynamics and Physics of Integrin Activation in Tumor Cells by Nano-Sized Extracellular Ligands and Electromagnetic Fields.

Integrins are stress-sensing proteins expressed on the surface of cells. They regulate bidirectional signal transduction during cell-cell or cell-extracellular matrix (ECM) contacts. Integrins link the ECM with the cytoplasm through interaction with their ligands. Biophysically, such interactions can be understood as changes in stress fields at specific integrin stress-sensing domains, such as the MIDAS and ADMIDAS domains. Stress changes between ligands and cytoskeletal structures are involved in cancer cell growth by altering signal transduction pathways dependent on integrin activation. In this chapter, previous results regarding integrin activation and tumor cell growth using nanoparticles (NPs) of different materials, sizes and shapes are placed within a framework of polarized NPs in the ECM by external electromagnetic fields, in which the synergic action between polarized NPs and electromagnetic fields activates the integrins. Small size NPs activate integrins via the polar component of the dipole force between NPs and integrin sensing stress sites, while large size NPs exercise a similar action via the radial component. A quantum electrodynamic model also accounts for ECM overstressing by electromagnetic mode trapping between coherent symmetric and antisymmetric quantum states.

Cytoplasmic and transmembrane domains of integrin beta 1 and beta 3 subunits are functionally interchangeable.

Integrin beta subunits combine with specific sets of alpha subunits to form functional adhesion receptors. The structure and binding properties of integrins suggest the presence of domains controlling at least three major functions: subunit association, ligand binding, and cytoskeletal interactions. To more carefully define structure/function relationships, a cDNA construct consisting of the extracellular domain of the avian beta 1 subunit and the cytoplasmic and transmembrane domains of the human beta 3 subunit was prepared and expressed in murine 3T3 cells. The resulting chimeric beta 1/3 subunit formed heterodimers with alpha subunits from the beta 1 subfamily, could not interact with alpha IIb from the beta 3 subfamily, was targeted to focal contacts, and formed functional complexes within the focal contacts. A second cDNA construct was prepared that coded for an avian beta 1 subunit without a transmembrane or cytoplasmic domain. This subunit was not found in association with an accompanying alpha subunit, nor was it found expressed on the cell surface. Instead, it accumulated in vesicles within the cytoplasm and was eventually shed from the cell. The results from studies of the behavior of these two cDNA constructs demonstrate that the transmembrane and cytoplasmic domains play no role in alpha subunit selection, that the cytoplasmic domain of beta 3 is capable of functioning in the context of alpha subunits with which it is not normally paired, and that both integrin subunits must be membrane associated for normal assembly and transport to cell surface adhesive structures.

Beta 3-endonexin, a novel polypeptide that interacts specifically with the cytoplasmic tail of the integrin beta 3 subunit.

The adhesive and signaling functions of integrins are regulated through their cytoplasmic domains. We identified a novel 111 residue polypeptide, designated beta 3-endonexin, that interacted with the cytoplasmic tail of the beta 3 integrin subunit in a yeast two-hybrid system. This interaction is structurally specific, since it was reduced by 64% by a point mutation in the beta 3 cytoplasmic tail (S752-->P) that disrupts integrin signaling. Moreover, this interaction is integrin subunit specific since it was not observed with the cytoplasmic tails of the alpha IIb, beta 1, or beta 2 subunits. beta 3-Endonexin fusion proteins bound selectively to detergent-solubilized beta 3 from platelets and human umbilical vein endothelial cells, and beta 3-endonexin mRNA and protein were detected in platelets and other tissues. A related mRNA encoded a larger polypeptide that failed to bind to beta integrin tails. The apparent specificity of beta 3-endonexin for the beta 3 integrin subunit suggests potential mechanisms for selective modulation of integrin functions.

A peptide isolated from phage display libraries is a structural and functional mimic of an RGD-binding site on integrins.

Many integrins recognize short RGD-containing amino acid sequences and such peptide sequences can be identified from phage libraries by panning with an integrin. Here, in a reverse strategy, we have used such libraries to isolate minimal receptor sequences that bind to fibronectin and RGD-containing fibronectin fragments in affinity panning. A predominant cyclic motif, *CWDDG/LWLC*, was obtained (the asterisks denote a potential disulfide bond). Studies using the purified phage and the corresponding synthetic cyclic peptides showed that *CWDDGWLC*-expressing phage binds specifically to fibronectin and to fibronectin fragments containing the RGD sequence. The binding did not require divalent cations and was inhibited by both RGD and *CWDDGWLC*-containing synthetic peptides. Conversely, RGD-expressing phage attached specifically to immobilized *CWDDGWLC*-peptide and the binding could be blocked by the respective synthetic peptides in solution. Moreover, fibronectin bound to a *CWDDGWLC*-peptide affinity column, and could be eluted with an RGD-containing peptide. The *CWDDGWLC*-peptide inhibited RGD-dependent cell attachment to fibronectin and vitronectin, but not to collagen. A region of the beta subunit of RGD-binding integrins that has been previously demonstrated to be involved in ligand binding includes a polypeptide stretch, KDDLW (in beta 3) similar to WDDG/LWL. Synthetic peptides corresponding to this region in beta 3 were found to bind RGD-displaying phage and conversion of its two aspartic residues into alanines greatly reduced the RGD binding. Polyclonal antibodies raised against the *CWDDGWLC*-peptide recognized beta 1 and beta 3 in immunoblots. These data indicate that the *CWDDGWLC*-peptide is a functional mimic of ligand binding sites of RGD-directed integrins, and that the structurally similar site in the integrin beta subunit is a binding site for RGD.

Crystal structures of Rea1-MIDAS bound to its ribosome assembly factor ligands resembling integrin-ligand-type complexes.

The Rea1 AAA+-ATPase dislodges assembly factors from pre-60S ribosomes upon ATP hydrolysis, thereby driving ribosome biogenesis. Here, we present crystal structures of Rea1-MIDAS, the conserved domain at the tip of the flexible Rea1 tail, alone and in complex with its substrate ligands, the UBL domains of Rsa4 or Ytm1. These complexes have structural similarity to integrin α-subunit domains when bound to extracellular matrix ligands, which for integrin biology is a key determinant for force-bearing cell-cell adhesion. However, the presence of additional motifs equips Rea1-MIDAS for its tasks in ribosome maturation. One loop insert cofunctions as an NLS and to activate the mechanochemical Rea1 cycle, whereas an additional ß-hairpin provides an anchor to hold the ligand UBL domains in place. Our data show the versatility of the MIDAS fold for mechanical force transmission in processes as varied as integrin-mediated cell adhesion and mechanochemical removal of assembly factors from pre-ribosomes.

In silico docking of urokinase plasminogen activator and integrins.

BACKGROUND: Urokinase, its receptor and the integrins are functionally associated and involved in regulation of cell signaling, migration, adhesion and proliferation. No structural information is available on this potential multimolecular complex. However, the tri-dimensional structure of urokinase, urokinase receptor and integrins is known. RESULTS: We have modeled the interaction of urokinase on two integrins, alphaIIbbeta3 in the open configuration and alphavbeta3 in the closed configuration. We have found that multiple lowest energy solutions point to an interaction of the kringle domain of uPA at the boundary between alpha and beta chains on the surface of the integrins. This region is not far away from peptides that have been previously shown to have a biological role in urokinase receptor/integrins dependent signaling. CONCLUSIONS: We demonstrated that in silico docking experiments can be successfully carried out to identify the binding mode of the kringle domain of urokinase on the scaffold of integrins in the open and closed conformation. Importantly we found that the binding mode was the same on different integrins and in both configurations. To get a molecular view of the system is a prerequisite to unravel the complex protein-protein interactions underlying urokinase/urokinase receptor/integrin mediated cell motility, adhesion and proliferation and to design rational in vitro experiments.

Reconstituted skin from murine embryonic stem cells.

Embryonic stem (ES) cell lines can be expanded indefinitely in culture while maintaining their potential to differentiate into any cell type. During embryonic development, the skin forms as a result of reciprocal interactions between mesoderm and ectoderm. Here, we report the in vitro differentiation and enrichment of keratinocytes from murine ES cells seeded on extracellular matrix (ECM) in the presence of Bone Morphogenic Protein-4 (BMP-4) or ascorbate. The enriched preparation of keratinocytes was able to form an epidermal equivalent composed of a stratified epithelium when cultured at the air-liquid interface on a collagen-coated acellular substratum. Interestingly, an underlying cellular compartment that belongs to the fibroblast lineage was systematically formed between the reconstituted epidermis and the inert membrane. The resulting tissue displayed morphological patterns similar to normal embryonic skin, as evidenced by light and transmission electron microscopy. Immunohistochemical studies revealed expression patterns of cytokeratins, basement membrane (BM) proteins and late differentiation markers of epidermis, as well as fibroblast markers, similar to native skin. The results demonstrate the capacity of ES cells to reconstitute in vitro a fully differentiated skin. This ES-derived bioengineered skin provides a powerful tool for studying the molecular mechanisms controlling epidermal and dermal commitments.

Electron microscopy of integrins.

Integrins are a family of heterodimeric, cell-surface receptors that mediate interactions between the cytoskeleton and the extracellular matrix. We have used electron microscopy and single-particle image analysis combined with molecular modeling to investigate the structures of the full-length integrin alpha(IIb)beta(3) and the ectodomain of alpha(V)beta(3) in a complex with fibronectin. The full-length integrin alpha(IIb)beta(3) is purified from human platelets by ion exchange and gel filtration chromatography in buffers containing the detergent octyl-beta-D-glucopyranoside, whereas the recombinant ectodomain of alpha(V)beta(3) is soluble in aqueous buffer. Transmission electron microscopy is performed either in negative stain, where the protein is embedded in a heavy metal such as uranyl acetate, or in the frozen-hydrated state, where the sample is flash-frozen such that the buffer is vitrified and native conditions are preserved. Individual integrin particles are selected from low-dose micrographs, either by manual identification or an automated method using a cross-correlation search of the micrograph against a set of reference images. Due to the small size of integrin heterodimers (approximately 250 kDa) and the low electron dose required to minimize beam damage, the signal-to-noise level of individual particles is quite low, both by negative-stain electron microscopy and electron cryomicroscopy. Consequently, it is necessary to average many particle images with equivalent views. The particle images are subjected to reference-free alignment and classification, in which the particles are aligned to a common view and further grouped by statistical methods into classes with common orientations. Assessment of the structure from a set of two-dimensional averaged projections is often difficult, and a further three-dimensional (3D) reconstruction analysis is performed to classify each particle as belonging to a specific projection from a single 3D model. The 3D reconstruction algorithm is an iterative projection-matching routine in which the classified particles are used to construct a new, 3D map for the next iteration. Docking of known high-resolution structures of individual subdomains within the molecular envelope of the 3D EM map is used to derive a pseudoatomic model of the integrin complex. This approach of 3D EM image analysis and pseudoatomic modeling is a powerful strategy for exploring the structural biology of transmembrane signaling by integrins because it is likely that multiple conformational states will be difficult to crystallize, whereas the different states should be amenable to electron cryomicroscopy.

Predicted and experimental structures of integrins and beta-propellers.

Integrins and other cell surface receptors have been fertile grounds for structure prediction experiments. Recently determined structures show remarkable successes, especially with beta-propeller domain predictions, and also reveal how ligand binding by integrins is conformationally regulated.

Monitoring mesenchymal stromal cell developmental stage to apply on-time mechanical stimulation for ligament tissue engineering.

To evaluate the appropriate time frame for applying mechanical stimuli to induce mesenchymal stromal cell (MSC) differentiation for ligament tissue engineering, developmental cell phenotypes were monitored during a period of in vitro culture. MSCs were seeded onto surface-modified silk fibroin fiber matrices and cultured in Petri dishes for 15 days. Cell metabolic activity, morphology, and gene expression of extracellular matrix (ECM) proteins (collagen type I and III and fibronectin), ECM receptors (integrins alpha-2, alpha-5, and beta-1), and heat-shock protein 70 (HSP-70) were monitored during the culture of MSC. MSCs showed fluctuations in cell metabolic activity, ECM, integrin, and HSP-70 transcription potentially correlating to innate developmental processes. Cellular response to mechanical stimulation was dependent on the stage of cell development. At day 9, when levels of cell metabolic activity, ECM, integrin, and HSP-70 transcription peaked, mechanical stimulation increased MSC metabolic activity, alignment, and collagen production. Mechanical stimulation applied at day 1 and 3 showed detrimental effects on MSCs seeded on silk matrices. The results presented in this study identify a unique correlation between innate MSC development processes on a surface-modified silk matrix and dynamic environmental signaling.

Modeling the alpha IIb beta 3 integrin solution conformation.

The alpha IIb beta 3 platelet integrin is the prototypical member of a widely distributed class of transmembrane receptors formed by the noncovalent association of alpha and beta subunits. Electron microscopic (EM) images of the alpha IIb beta 3 complex show an asymmetric particle with a globular domain from which two extended regions protrude to contact the lipid bilayer. Distance constraints provided by disulfide bond patterns, epitope mapping, and ligand mimetic cross-linking studies rather suggest a somewhat more compact conformation for the alpha IIb beta 3 complex. We have studied the shape of detergent-solubilized alpha IIb beta 3 by employing a low-resolution modeling procedure in which each polypeptide has been represented as an array of interconnected, nonoverlapping spheres (beads) of various sizes. The number, size, and three-dimensional relationships among the beads were defined either solely by dimensions obtained from published EM images of integrin receptors (EM models, 21 beads), or solely by interdomain constraints derived from published biochemical data (biochemical model, 37 beads). Interestingly, although no EM data were employed in its construction, the resulting overall shape of the biochemical model was still compatible with the EM data. Both kinds of models were then evaluated for their calculated solution properties. The more elongated EM models have diffusion and sedimentation coefficients that differ, at best, by +2% and -18% from the experimental values, determined, respectively, in octyl glucoside and Triton X-100. On the other hand, the parameters calculated for the more compact biochemical model showed a more consistent agreement with experimental values, differing by -7% (octyl glucoside) to -6% (Triton X-100). Thus, it appears that using the biochemical constraints as a starting point has resulted in not only a more detailed model of the detergent-solubilized alpha IIb beta 3 complex, where the relative spatial location of specific domains the size of 5-10 kDa can be tentatively mapped, but in a model that can also reconcile the electron microscopy with the biochemical and the solution data.

Distribution of alpha 6 integrin subunit in developing mouse submandibular gland.

We examined the development of mouse submandibular gland by light and electron microscopy and determined the distribution of the alpha 6 integrin subunit and laminin in this process by immunofluorescence and immunoelectron microscopy. At Days 13.5 and 14 of gestation alpha 6 was localized over the entire cell surface of undifferentiated epithelial cells of the terminal cluster. On Day 15 the expression of alpha 6 could no longer be detected over central cells in the proximal portion of branched terminal cluster, whereas peripheral cells were stained over the entire surface. On Day 17 of gestation to day of birth, alpha 6 expression was restricted to the basolateral surface of the differentiated acinotubular structure. Its expression on acinar cells was uniformly distributed throughout the basolateral membrane, but on duct cells stronger staining towards the basal surface was noted. Similar expression was observed in adult acinar and duct cells. Expression of the alpha 6-subunit at the cell-cell contact in an early stage and its expression at the basolateral surface in an advanced stage indicate that integrin containing alpha 6 plays a significant role in cell-cell and cell-substrate interaction. Stage and cell type-specific change of integrin expression may be significant for submandibular development.

Confocal imaging of the alpha 6 and beta 4 integrin subunits in the human cornea with aging.

PURPOSE: The purpose of this study was to examine changes in the distribution of integrin subunits, alpha 6 and alpha 4, in the normal human cornea with age. METHODS: Thirty normal corneas were examined and divided into three groups; corneas from children younger than 2 years, corneas from adults 29 to 70 years, and corneas from adults older than 70 years. The corneas were frozen and the sections were cut, double-stained with monoclonal antibodies to the integrin subunits, and visualized with Texas Red or fluorescein using confocal laser scanning microscopy. Computer imaging was conducted to determine differences. RESULTS: The alpha 6 subunit was generally localized along the basal and lateral surfaces of basal epithelial cells and projected into Bowman's membrane. The beta 4 subunit was only present along the basal surface. Overall, the major age-related difference was the loss of continuous alpha 6 and beta 4 subunits along the basal surface of basal epithelial cells. When reconstructed images from corneas of individuals older than 70 years were optically sectioned en face, the alpha 6 subunit appeared discontinuous. If the same optical images were viewed from corneas of younger individuals, the staining was continuous. The number and distribution of hemidesmosomes along the basal lamina did not change with age in the corneas examined. CONCLUSIONS: Using computer imaging associated with confocal laser scanning microscopy, we have demonstrated that there is an age-related change in the localization of the alpha 6 and beta 4 subunits.

Ultrastructural localization of integrin subunits alpha3 and alpha6 in capillarized sinusoids of the human cirrhotic liver.

Normal liver sinusoids are not lined by a basement membrane (BM). In contrast, in the course of development of liver cirrhosis, a structured BM is formed de novo in the space of Disse. This BM contributes to the inhibition of the metabolic function of the liver but the pathogenic background of the formation of this perisinusoidal BM is still unclear. Integrins of the beta1-class are generally essential for BM stability and some of them (such as alpha2beta1, alpha3beta1 and alpha6beta1) appear de novo in the perisinusoidal space of the cirrhotic liver. Their cellular distribution in capillarized sinusoids as well as the correlation between their cellular distribution and the formation of the microvascular BM in the cirrhotic liver has not been shown at the ultrastructural level. In the present work we aimed to clarify this issue. We focused on integrins alpha3beta1 and alpha6beta1 and localised them ultrastructurally in human cirrhotic liver microvessels using postembedding immunogold which allows the ultrastructural localization of antigens with high resolution in the tissue. The newly formed basement membrane of capillarized sinusoids was visualized by means of fixation with addition of tannic acid, which enables the visualization of structures of the extracellular matrix with the highest resolution. Also, we carried out laminin detection using postembedding immunogold. Our results show that both alpha3beta1 and alpha6beta1 are expressed on the surface of both hepatocytes and endothelial cells, i.e. on both sides of the newly formed basement membrane. This latter shows zones of higher density both in close proximity to the endothelial and to the hepatocytic surfaces which resemble laminae densae. We propose that hepatocytes and endothelial cells may, therefore, by expressing such integrins, contribute to the formation of this pathological BM in the microvessels of the human cirrhotic liver. On stellate cells, which are major producers of BM components, both integrins alpha3beta1 and alpha6beta1 were also localized.