Photoactivatable substrates show diverse phenotypes of leader cells in collective migration when moving along different extracellular matrix proteins.

In cancer metastasis, collectively migrating clusters are discriminated into leader and follower cells that move through extracellular matrices (ECMs) with different characteristics. The impact of changes in ECM protein types on leader cells and migrating clusters is unknown. To address this, we investigated the response of leader cells and migrating clusters upon moving from one ECM protein to another using a photoactivatable substrate bearing photocleavable PEG (PCP), whose surface changes from protein-repellent to protein-adhesive in response to light. We chose laminin and collagen I for our study since they are abundant in two distinct regions in living tissues, namely basement membrane and connective tissue. Using the photoactivatable substrates, the precise deposition of the first ECM protein in the irradiated areas was achieved, followed by creating well-defined cellular confinements. Secondary irradiation enabled the deposition of the second ECM protein in the new irradiated regions, resulting in region-selective heterogeneous and homogenous ECM protein-coated surfaces. Different tendencies in leader cell formation from laminin into laminin compared to those migrating from laminin into collagen were observed. The formation of focal adhesion and actin structures for cells within the same cluster in the ECM proteins responded according to the underlying ECM protein type. Finally, integrin ß1 was crucial for the appearance of leader cells for clusters migrating from laminin into collagen. However, when it came to laminin into laminin, integrin ß1 was not responsible. This highlights the correlation between leader cells in collective migration and the biochemical signals that arise from underlying extracellular matrix proteins.

Site-specific N-glycan profiles of α5 ß1 integrin from rat liver.

BACKGROUND INFORMATION: Like most other cell surface proteins, α5 ß1 integrin is glycosylated, which is required for its various activities in ways that mostly remain to be determined. RESULTS: Here, we have established the first comprehensive site-specific glycan map of α5 ß1 integrin that was purified from a natural source, that is, rat liver. This analysis revealed striking site selective variations in glycan composition. Complex bi, tri, or tetraantennary N-glycans were predominant at various proportions at most potential N-glycosylation sites. A few of these sites were nonglycosylated or contained high mannose or hybrid glycans, indicating that early N-glycan processing was hindered. Almost all complex N-glycans had fully galactosylated and sialylated antennae. Moderate levels of core fucosylation and high levels of O-acetylation of NeuAc residues were observed at certain sites. An O-linked HexNAc was found in an EGF-like domain of ß1 integrin. The extensive glycan information that results from our study was projected onto a map of α5 ß1 integrin that was obtained by homology modeling. We have used this model for the discussion of how glycosylation might be used in the functional cycle of α5 ß1 integrin. A striking example concerns the involvement of glycan-binding galectins in the regulation of the molecular homeostasis of glycoproteins at the cell surface through the formation of lattices or endocytic pits according to the glycolipid-lectin (GL-Lect) hypothesis. CONCLUSION: We expect that the glycoproteomics data of the current study will serve as a resource for the exploration of structural mechanisms by which glycans control α5 ß1 integrin activity and endocytic trafficking. SIGNIFICANCE: Glycosylation of α5 ß1 integrin has been implicated in multiple aspects of integrin function and structure. Yet, detailed knowledge of its glycosylation, notably the specific sites of glycosylation, is lacking. Furthermore, the α5 ß1 integrin preparation that was analyzed here is from a natural source, which is of importance as there is not a lot of literature in the field about the glycosylation of "native" glycoproteins.

New insights into the phosphorylation of the threonine motif of the ß1 integrin cytoplasmic domain.

Integrins require an activation step before ligand binding and signaling that is mediated by talin and kindlin binding to the ß integrin cytosolic domain (ß-tail). Conflicting reports exist about the contribution of phosphorylation of a conserved threonine motif in the ß1-tail (ß1-pT788/pT789) to integrin activation. We show that widely used and commercially available antibodies against ß1-pT788/pT789 integrin do not detect specific ß1-pT788/pT789 integrin signals in immunoblots of several human and mouse cell lysates but bind bi-phosphorylated threonine residues in numerous proteins, which were identified by mass spectrometry experiments. Furthermore, we found that fibroblasts and epithelial cells expressing the phospho-mimicking ß1-TT788/789DD integrin failed to activate ß1 integrins and displayed reduced integrin ligand binding, adhesion initiation and cell spreading. These cellular defects are specifically caused by the inability of kindlin to bind ß1-tail polypeptides carrying a phosphorylated threonine motif or phospho-mimicking TT788/789DD substitutions. Our findings indicate that the double-threonine motif in ß1-class integrins is not a major phosphorylation site but if phosphorylated would curb integrin function.

Myosin-X and talin modulate integrin activity at filopodia tips.

Filopodia assemble unique integrin-adhesion complexes to sense the extracellular matrix. However, the mechanisms of integrin regulation in filopodia are poorly defined. Here, we report that active integrins accumulate at the tip of myosin-X (MYO10)-positive filopodia, while inactive integrins are uniformly distributed. We identify talin and MYO10 as the principal integrin activators in filopodia. In addition, deletion of MYO10's FERM domain, or mutation of its ß1-integrin-binding residues, reveals MYO10 as facilitating integrin activation, but not transport, in filopodia. However, MYO10's isolated FERM domain alone cannot activate integrins, potentially because of binding to both integrin tails. Finally, because a chimera construct generated by swapping MYO10-FERM by talin-FERM enables integrin activation in filopodia, our data indicate that an integrin-binding FERM domain coupled to a myosin motor is a core requirement for integrin activation in filopodia. Therefore, we propose a two-step integrin activation model in filopodia: receptor tethering by MYO10 followed by talin-mediated integrin activation.

Receptor Switching in Newly Evolved Adeno-associated Viruses.

Adeno-associated viruses utilize different glycans and the AAV receptor (AAVR) for cellular attachment and entry. Directed evolution has yielded new AAV variants; however, structure-function correlates underlying their improved transduction are generally overlooked. Here, we report that infectious cycling of structurally diverse AAV surface loop libraries yields functionally distinct variants. Newly evolved variants show enhanced cellular binding, uptake, and transduction, but through distinct mechanisms. Using glycan-based and genome-wide CRISPR knockout screens, we discover that one AAV variant acquires the ability to recognize sulfated glycosaminoglycans, while another displays receptor switching from AAVR to integrin ß1 (ITGB1). A previously evolved variant, AAVhum.8, preferentially utilizes the ITGB1 receptor over AAVR. Visualization of the AAVhum.8 capsid by cryoelectron microscopy at 2.49-Å resolution localizes the newly acquired integrin recognition motif adjacent to the AAVR footprint. These observations underscore the new finding that distinct AAV surface epitopes can be evolved to exploit different cellular receptors for enhanced transduction. IMPORTANCE Understanding how viruses interact with host cells through cell surface receptors is central to discovery and development of antiviral therapeutics, vaccines, and gene transfer vectors. Here, we demonstrate that distinct epitopes on the surface of adeno-associated viruses can be evolved by infectious cycling to recognize different cell surface carbohydrates and glycoprotein receptors and solve the three-dimensional structure of one such newly evolved AAV capsid, which provides a roadmap for designing viruses with improved attributes for gene therapy applications.

Expression and purification of a novel single-chain diabody (scDb-hERG1/ß1) from Pichia pastoris transformants.

In the last decades, protein engineering has developed particularly in biotechnology and pharmaceutical field. In particular, the engineered antibody subclass has arisen. The single chain diabody format (scDb), conjugating small size with antigen specificity, offers versatility representing a gold standard for a variety of applications, spacing from research to diagnostics and therapy. Along with such advantages, comes the challenge of optimizing their production, improving expression systems, purification procedures and stability. All such parameters are detrimental for protein production in general and above all for recombinant antibody expression, which has to be fine-tuned, choosing a proper protein-expression host and adjusting expression protocols accordingly. In the present paper, we present data regarding the production and purification of a single chain diabody directed against the macromolecular complex hERG1/ß1 integrin. We focus on the expression of clones deriving from the transformation of Pichia pastoris yeast cells. In particular, we compare two different clones arose from two separate transformation processes, demonstrating that both are suitable for proper protein expression. Moreover, we have set up an expression protocol and compared the yields obtained using two purification machines: Akta Pure and Akta Start, with a positive outcome.

Quantitative single-protein imaging reveals molecular complex formation of integrin, talin, and kindlin during cell adhesion.

Single-molecule localization microscopy (SMLM) enabling the investigation of individual proteins on molecular scales has revolutionized how biological processes are analysed in cells. However, a major limitation of imaging techniques reaching single-protein resolution is the incomplete and often unknown labeling and detection efficiency of the utilized molecular probes. As a result, fundamental processes such as complex formation of distinct molecular species cannot be reliably quantified. Here, we establish a super-resolution microscopy framework, called quantitative single-molecule colocalization analysis (qSMCL), which permits the identification of absolute molecular quantities and thus the investigation of molecular-scale processes inside cells. The method combines multiplexed single-protein resolution imaging, automated cluster detection, in silico data simulation procedures, and widely applicable experimental controls to determine absolute fractions and spatial coordinates of interacting species on a true molecular level, even in highly crowded subcellular structures. The first application of this framework allowed the identification of a long-sought ternary adhesion complex-consisting of talin, kindlin and active ß1-integrin-that specifically forms in cell-matrix adhesion sites. Together, the experiments demonstrate that qSMCL allows an absolute quantification of multiplexed SMLM data and thus should be useful for investigating molecular mechanisms underlying numerous processes in cells.

Analyzing the Integrin Adhesome by In Situ Proximity Ligation Assay.

The in situ proximity ligation assay (PLA) is capable of detecting single protein events such as protein protein-interactions and posttranslational modifications (e.g., protein phosphorylation) in tissue and cell samples prepared for analysis by immunofluorescent or immunohistochemical microscopy. The targets are detected using two primary antibodies which must be from different host species. A pair of secondary antibodies (PLA probes) conjugated to complementary oligonucleotides is applied to the sample, and a signal is generated only when the two PLA probes are in close proximity by their binding to the two primary antibodies that have bound to their targets in close proximity. The signal from each pair of PLA probes is visualized as an individual fluorescent spot. These PLA signals can be quantified (counted) using image analysis software (ImageJ), and also assigned to a specific subcellular location based on microscopy image overlays. In principle, in situ PLA offers a relatively simple and sensitive technique to analyze interactions among any proteins for which suitable antibodies are available. Integrin-mediated focal adhesions (FAs) are large multiprotein complexes consisting of more than 150 proteins, also known as the integrin adhesome, which link the extracellular matrix (ECM) to the actin cytoskeleton and regulate the functioning of mechanosignaling pathways. The in situ PLA approach is well suited for examining the spatiotemporal aspects of protein posttranslational modifications and protein interactions occurring in dynamic multiprotein complexes such as integrin mediated focal adhesions.

Structural and biophysical characterization of the type VII collagen vWFA2 subdomain leads to identification of two binding sites.

Type VII collagen is an extracellular matrix protein, which is important for skin stability; however, detailed information at the molecular level is scarce. The second vWFA (von Willebrand factor type A) domain of type VII collagen mediates important interactions, and immunization of mice induces skin blistering in certain strains. To understand vWFA2 function and the pathophysiological mechanisms leading to skin blistering, we structurally characterized this domain by X-ray crystallography and NMR spectroscopy. Cell adhesion assays identified two new interactions: one with ß1 integrin via its RGD motif and one with laminin-332. The latter interaction was confirmed by surface plasmon resonance with a KD of about 1 mm. These data show that vWFA2 has additional functions in the extracellular matrix besides interacting with type I collagen.

Identification of cell context-dependent YAP-associated proteins reveals ß1 and ß4 integrin mediate YAP translocation independently of cell spreading.

Yes-associated protein (YAP) is a transcriptional regulator and mechanotransducer, relaying extracellular matrix (ECM) stiffness into proliferative gene expression in 2D culture. Previous studies show that YAP activation is dependent on F-actin stress fiber mediated nuclear pore opening, however the protein mediators of YAP translocation remain unclear. Here, we show that YAP co-localizes with F-actin during activating conditions, such as sparse plating and culturing on stiff 2D substrates. To identify proteins mediating YAP translocation, we performed co-immunoprecipitation followed by mass spectrometry (co-IP/MS) for proteins that differentially associated with YAP under activating conditions. Interestingly, YAP preferentially associates with ß1 integrin under activating conditions, and ß4 integrin under inactivating conditions. In activating conditions, CRISPR/Cas9 knockout (KO) of ß1 integrin (ΔITGB1) resulted in decreased cell area, which correlated with decreased YAP nuclear localization. ΔITGB1 did not significantly affect the slope of the correlation between YAP nuclear localization with area, but did decrease overall nuclear YAP independently of cell spreading. In contrast, ß4 integrin KO (ΔITGB4) cells showed no change in cell area and similarly decreased nuclear YAP. These results reveal proteins that differentially associate with YAP during activation, which may aid in regulating YAP nuclear translocation.

Abrogation of EMILIN1-ß1 integrin interaction promotes experimental colitis and colon carcinogenesis.

Colon cancer is one of the first tumor types where a functional link between inflammation and tumor onset has been described; however, the microenvironmental cues affecting colon cancer progression are poorly understood. Here we demonstrate that the expression of the ECM molecule EMILIN-1 halts the development of AOM-DSS induced tumors. In fact, upon AOM-DSS treatment the Emilin1-/- (E1-/-) mice were characterized by a higher tumor incidence, bigger adenomas and less survival. Similar results were obtained with the E933A EMILIN-1 (E1-E933A) transgenic mouse model, expressing a mutant EMILIN-1 unable to interact with α4/α9ß1 integrins. Interestingly, upon chronic treatment with DSS, E1-/- and E1-E933A mice were characterized by the presence of increased inflammatory infiltrates, higher colitis scores and more severe mucosal injury respect to the wild type (E1+/+) mice. Since alterations of the intestinal lymphatic network are a well-established feature of human inflammatory bowel disease and EMILIN-1 is a key structural element in the maintenance of the integrity of lymphatic vessels, we assessed the lymphatic vasculature in this context. The analyses revealed that both E1-/- and E1-E933A mice displayed a higher density of LYVE-1 positive vessels; however, their functionality was severely compromised after colitis induction. Taken together, these results suggest that the loss of EMILIN-1 expression may cause the reduction of the inflammatory resolution during colon cancer progression due to a decreased lymph flow and impaired inflammatory cell drainage.

Anisotropic Ligand Nanogeometry Modulates the Adhesion and Polarization State of Macrophages.

Material implants trigger host reactions generated by cells, such as macrophages, which display dynamic adhesion and polarization including M1 inflammatory state and M2 anti-inflammatory state. Creating materials that enable diverse nanoscale display of integrin-binding groups, such as RGD ligand, can unravel nanoscale recruitment and ligation of integrin, which modulate cellular adhesion and activation. Here, we synthesized gold nanorods (GNRs) with various nanoscale anisotropies (i.e., aspect ratios, ARs), but in similar surface areas, and controlled their substrate conjugation to display an anisotropic ligand nanogeometry without modulating ligand density. Using nanoscale immunolabeling, we demonstrated that highly anisotropic ligand-coated GNRs ("AR4" and "AR7") facilitated the recruitment of integrin ß1 on macrophages to their nanoscale surfaces. Consequently, highly anisotropic GNRs (e.g., "AR4" and "AR7") elevated the adhesion and M2 state of macrophages, with the inhibition of their M1 state in the culture and mice, entailing rho-associated protein kinase. This nanoscale anisotropic nanogeometry provides a novel and critical parameter to be considered in the generation of biomaterials to potentially modulate host reactions to the implants for immunomodulatory tissue regeneration.

Biodegradable Anisotropic Microparticles for Stepwise Cell Adhesion and Preparation of Janus Cell Microparticles.

The biomimetic anisotropic particles have different physicochemical properties on the opposite two sides, enabling diverse applications in emulsion, photonic display, and diagnosis. However, the traditional anisotropic particles have a very small size, ranging from submicrons to a few microns. The design and fabrication of anisotropic macron-sized particles with new structures and properties is still challenging. In this study, anisotropic polycaprolactone (PCL) microparticles well separated with each other were prepared by crystallization from the dilute PCL solution in a porous 3D gelatin template. They had fuzzy and smooth surfaces on each side, and a size as large as 70 µm. The fuzzy surface of the particle adsorbed significantly larger amount of proteins, and was more cell-attractive regardless of the cell types. The particles showed stronger affinity toward fibroblasts over hepatocytes, which paved a new way for cell isolation merely based on the surface morphology. After a successive seeding process, Janus cell microparticles with fibroblasts and endothelial cells (ECs) on each side were designed and obtained by making use of the anisotropic surface morphology, which showed significant difference in EC functions in terms of prostacyclin (PGl2) secretion, demonstrating the unique and appealing functions of this type of anisotropic microspheres.

A systematic survey of conformational states in ß1 and ß4 integrins using negative-stain electron microscopy.

Structural analyses of ß2 and ß3 integrins have revealed that they generally assume a compact bent conformation in the resting state and undergo a global conformational transition involving extension during upregulation of ligand affinity, collectively called the 'switchblade model'. This hypothesis, however, has not been extensively tested for other classes of integrins. We prepared a set of recombinant integrin ectodomain fragments including αvß3, α2ß1, α3ß1, α5ß1, α6ß1 and α6ß4, and used negative-stain electron microscopy to examine their structures under various conditions. In contrast to αvß3 integrin, which exhibited a severely bent conformation in low-affinity 5 mM Ca2+ conditions, all ß1 integrin heterodimers displayed a mixed population of half-bent to fully extended conformations. Moreover, they did not undergo significant conformational change upon activation by Mn2+ Integrin α6ß4 was even more resistant to conformational regulation, showing a completely extended structure regardless of the buffer conditions. These results suggest that the mechanisms of conformational regulation of integrins are more diverse and complex than previously thought, requiring more experimental scrutiny for each integrin subfamily member.

Acidic pHe regulates cytoskeletal dynamics through conformational integrin ß1 activation and promotes membrane protrusion.

An acidic extracellular pH (pHe) in the tumor microenvironment has been suggested to facilitate tumor growth and metastasis. However, the molecular mechanisms by which tumor cells sense acidic signal to induce a transition to an aggressive phenotype remain elusive. Here, we showed that an acidic pHe (pH 6.5) stimulation resulted in protrusion and epithelial-mesenchymal transition (EMT) of cancer cells, which promoted migration and matrix degeneration. Using computational molecular dynamics simulations, we reported acidic pHe-induced opening of the Integrin dimers (α5ß1) headpiece which indicated the activation of integrin. Moreover, acidic pHe promoted maturation of focal adhesions, temporal activation of Rho GTPases and microfilament reorganization through integrin ß1-activated FAK signaling. Furthermore, mechanical balance of cytoskeleton (actin, tubulin and vimentin) contributed to acidic pHe-triggered protrusion and morphology change. Taken together, these findings revealed that integrin ß1 could be a novel pH-regulated sensitive molecule which confers protrusion and malignant phenotype of cancer cells.

The role of Protein Disulfide Isomerase and thiol bonds modifications in activation of integrin subunit alpha11.

Integrins belong to a family of transmembrane receptors that mediate cell migration and adhesion to ECM. Extracellular domains of integrin heterodimers contain cysteine-rich regions, which are potential sites of thiol-disulfide exchanges. Rearrangements of extracellular disulfide bonds regulate activation of integrin receptors by promoting transition from an inactive state into a ligand-binding competent state. Modifications of integrin disulfide bonds dependent on oxidation-reduction can be mediated by Protein Disulfide Isomerse (PDI). This paper provides evidences that binding to integrin ligands initiate changes in free thiol pattern on cell surface and that thiol-disulfide exchange mediated by PDI leads to activation of integrin subunit α11. By employing co-immunoprecipitation and confocal microscopy analysis we showed that α11ß1 and PDI create complexes bounded by disulfide bonds. Using surface plasmon resonance we provide biochemical evidence that PDI can interact directly with integrin subunit α11.

Integrin ß1 is bound to galectin-1 in human trophoblast.

Interaction of sugar binding proteins-galectins, with glycoconjugates is considered relevant for various reproductive processes. Galectin-1 (gal-1) is a molecule involved in trophoblast cell invasion, which is accomplished through interaction with cell surface and/or extracellular matrix glycoproteins. A possibility of interaction of endogenous gal-1 and trophoblast ß1 integrins, both previously shown relevant for trophoblast invasion, was investigated. Confocal microscopy showed overlap in gal-1 and ß1 integrin localization at the plasma membrane of isolated cytotrophoblast, HTR-8/SVneo extravillous trophoblast cell line and JAr choriocarcinoma cells. Immunoprecipitation confirmed an interaction of gal-1 with integrin ß1, but not with α1 or α5 integrin subunits. Nondenaturing electrophoresis and subcellular fractionation suggested association of gal-1 with ß1 integrin in intracellular and plasma membrane compartments of HTR-8/SVneo cells. Gal-1/ß1 integrin complex was sensitive to chemical and enzyme treatments, indicating carbohydrate dependent interaction. Down-regulation of gal-1 by siRNA, however, had no effect on level or distribution of ß1 integrin, as determined by qPCR and flow cytometry. These results suggest complex lectin type interaction of gal-1 with ß1 integrin at the trophoblast cell membrane, which could influence trophoblast cell adhesion, migration and invasion.

Identification of the integrin-binding site on coagulation factor VIIa required for proangiogenic PAR2 signaling.

The tissue factor (TF) pathway serves both hemostasis and cell signaling, but how cells control these divergent functions of TF remains incompletely understood. TF is the receptor and scaffold of coagulation proteases cleaving protease-activated receptor 2 (PAR2) that plays pivotal roles in angiogenesis and tumor development. Here we demonstrate that coagulation factor VIIa (FVIIa) elicits TF cytoplasmic domain-dependent proangiogenic cell signaling independent of the alternative PAR2 activator matriptase. We identify a Lys-Gly-Glu (KGE) integrin-binding motif in the FVIIa protease domain that is required for association of the TF-FVIIa complex with the active conformer of integrin ß1. A point mutation in this motif markedly reduces TF-FVIIa association with integrins, attenuates integrin translocation into early endosomes, and reduces delayed mitogen-activated protein kinase phosphorylation required for the induction of proangiogenic cytokines. Pharmacologic or genetic blockade of the small GTPase ADP-ribosylation factor 6 (arf6) that regulates integrin trafficking increases availability of TF-FVIIa with procoagulant activity on the cell surface, while inhibiting TF-FVIIa signaling that leads to proangiogenic cytokine expression and tumor cell migration. These experiments delineate the structural basis for the crosstalk of the TF-FVIIa complex with integrin trafficking and suggest a crucial role for endosomal PAR2 signaling in pathways of tissue repair and tumor biology.

Talin regulates integrin ß1-dependent and -independent cell functions in ureteric bud development.

Kidney collecting system development requires integrin-dependent cell-extracellular matrix interactions. Integrins are heterodimeric transmembrane receptors consisting of α and ß subunits; crucial integrins in the kidney collecting system express the ß1 subunit. The ß1 cytoplasmic tail has two NPxY motifs that mediate functions by binding to cytoplasmic signaling and scaffolding molecules. Talins, scaffolding proteins that bind to the membrane proximal NPxY motif, are proposed to activate integrins and to link them to the actin cytoskeleton. We have defined the role of talin binding to the ß1 proximal NPxY motif in the developing kidney collecting system in mice that selectively express a Y-to-A mutation in this motif. The mice developed a hypoplastic dysplastic collecting system. Collecting duct cells expressing this mutation had moderate abnormalities in cell adhesion, migration, proliferation and growth factor-dependent signaling. In contrast, mice lacking talins in the developing ureteric bud developed kidney agenesis and collecting duct cells had severe cytoskeletal, adhesion and polarity defects. Thus, talins are essential for kidney collecting duct development through mechanisms that extend beyond those requiring binding to the ß1 integrin subunit NPxY motif.

The Rho ADP-ribosylating C3 exoenzyme binds cells via an Arg-Gly-Asp motif.

The Rho ADP-ribosylating C3 exoenzyme (C3bot) is a bacterial protein toxin devoid of a cell-binding or -translocation domain. Nevertheless, C3 can efficiently enter intact cells, including neurons, but the mechanism of C3 binding and uptake is not yet understood. Previously, we identified the intermediate filament vimentin as an extracellular membranous interaction partner of C3. However, uptake of C3 into cells still occurs (although reduced) in the absence of vimentin, indicating involvement of an additional host cell receptor. C3 harbors an Arg-Gly-Asp (RGD) motif, which is the major integrin-binding site, present in a variety of integrin ligands. To check whether the RGD motif of C3 is involved in binding to cells, we performed a competition assay with C3 and RGD peptide or with a monoclonal antibody binding to ß1-integrin subunit and binding assays in different cell lines, primary neurons, and synaptosomes with C3-RGD mutants. Here, we report that preincubation of cells with the GRGDNP peptide strongly reduced C3 binding to cells. Moreover, mutation of the RGD motif reduced C3 binding to intact cells and also to recombinant vimentin. Anti-integrin antibodies also lowered the C3 binding to cells. Our results indicate that the RGD motif of C3 is at least one essential C3 motif for binding to host cells and that integrin is an additional receptor for C3 besides vimentin.