Tgfbi/Bigh3 silencing activates ERK in mouse retina.

BIGH3 is a secreted protein, part of the extracellular matrix where it interacts with collagen and integrins on the cell surface. BIGH3 can play opposing roles in cancer, acting as either tumor suppressor or promoter, and its mutations lead to different forms of corneal dystrophy. Although many studies have been carried out, little is known about the physiological role of BIGH3. Using the cre-loxP system, we generated a mouse model with disruption of the Bigh3 genomic locus. Bigh3 silencing did not result in any apparent phenotype modifications, the mice remained viable and fertile. We were able to determine the presence of BIGH3 in the retinal pigment epithelium (RPE). In the absence of BIGH3, a transient decrease in the apoptotic process involved in retina maturation was observed, leading to a transient increase in the INL thickness at P15. This phenomenon was accompanied by an increased activity of the pro-survival ERK pathway.

Dpp spreading is required for medial but not for lateral wing disc growth.

Drosophila Decapentaplegic (Dpp) has served as a paradigm to study morphogen-dependent growth control. However, the role of a Dpp gradient in tissue growth remains highly controversial. Two fundamentally different models have been proposed: the 'temporal rule' model suggests that all cells of the wing imaginal disc divide upon a 50% increase in Dpp signalling, whereas the 'growth equalization model' suggests that Dpp is only essential for proliferation control of the central cells. Here, to discriminate between these two models, we generated and used morphotrap, a membrane-tethered anti-green fluorescent protein (GFP) nanobody, which enables immobilization of enhanced (e)GFP::Dpp on the cell surface, thereby abolishing Dpp gradient formation. We find that in the absence of Dpp spreading, wing disc patterning is lost; however, lateral cells still divide at normal rates. These data are consistent with the growth equalization model, but do not fit a global temporal rule model in the wing imaginal disc.

AMP-activated protein kinase plays an important evolutionary conserved role in the regulation of glucose metabolism in fish skeletal muscle cells

AMPK, a master metabolic switch, mediates the observed increase of glucose uptake in locomotory muscle of mammals during exercise. AMPK is activated by changes in the intracellular AMP:ATP ratio when ATP consumption is stimulated by contractile activity but also by AICAR and metformin, compounds that increase glucose transport in mammalian muscle cells. However, the possible role of AMPK in the regulation of glucose metabolism in skeletal muscle has not been investigated in other vertebrates, including fish. In this study, we investigated the effects of AMPK activators on glucose uptake, AMPK activity, cell surface levels of trout GLUT4 and expression of GLUT1 and GLUT4 as well as the expression of enzymes regulating glucose disposal and PGC1α in trout myotubes derived from a primary muscle cell culture. We show that AICAR and metformin significantly stimulated glucose uptake (1.6 and 1.3 fold, respectively) and that Compound C completely abrogated the stimulatory effects of the AMPK activators on glucose uptake. The combination of insulin and AMPK activators did not result in additive nor synergistic effects on glucose uptake. Moreover, exposure of trout myotubes to AICAR and metformin resulted in an increase in AMPK activity (3.8 and 3 fold, respectively). We also provide evidence suggesting that stimulation of glucose uptake by AMPK activators in trout myotubes may take place, at least in part, by increasing the cell surface and mRNA levels of trout GLUT4. Finally, AICAR increased the mRNA levels of genes involved in glucose disposal (hexokinase, 6-phosphofructokinase, pyruvate kinase and citrate synthase) and mitochondrial biogenesis (PGC-1α) and did not affect glycogen content or glycogen synthase mRNA levels in trout myotubes. Therefore, we provide evidence, for the first time in non-mammalian vertebrates, suggesting a potentially important role of AMPK in stimulating glucose uptake and utilization in the skeletal muscle of fish.

Genome-wide Association Studies Identify Genetic Loci Associated With Albuminuria in Diabetes.

Elevated concentrations of albumin in the urine, albuminuria, are a hallmark of diabetic kidney disease and are associated with an increased risk for end-stage renal disease and cardiovascular events. To gain insight into the pathophysiological mechanisms underlying albuminuria, we conducted meta-analyses of genome-wide association studies and independent replication in up to 5,825 individuals of European ancestry with diabetes and up to 46,061 without diabetes, followed by functional studies. Known associations of variants in CUBN, encoding cubilin, with the urinary albumin-to-creatinine ratio (UACR) were confirmed in the overall sample (P = 2.4 × 10(-10)). Gene-by-diabetes interactions were detected and confirmed for variants in HS6ST1 and near RAB38/CTSC. Single nucleotide polymorphisms at these loci demonstrated a genetic effect on UACR in individuals with but not without diabetes. The change in the average UACR per minor allele was 21% for HS6ST1 (P = 6.3 × 10(-7)) and 13% for RAB38/CTSC (P = 5.8 × 10(-7)). Experiments using streptozotocin-induced diabetic Rab38 knockout and control rats showed higher urinary albumin concentrations and reduced amounts of megalin and cubilin at the proximal tubule cell surface in Rab38 knockout versus control rats. Relative expression of RAB38 was higher in tubuli of patients with diabetic kidney disease compared with control subjects. The loci identified here confirm known pathways and highlight novel pathways influencing albuminuria.

Caracterização ácido-base da superfície de espécies mistas da alga Spirulina através de titulação potenciométrica e modelo de distribuição de sítios discretos

Acid base properties of mixed species of the microalgae Spirulina were studied by potentiometric titration in medium of 0.01 and 0.10 mols L-1 NaNO3 at 25.0±0.10 C using modified Gran functions or nonlinear regression techniques for data fitting. The discrete site distribution model was used, permitting the characterization of five classes of ionizable sites in both ionic media. This fact suggests that the chemical heterogeneity of the ionizable sites on the cell surface plays a major role on the acid-base properties of the suspension in comparison to electrostatic effects due to charge-charge interactions. The total of ionizable sites were 1.75±0.10 and 1.86±0.20 mmolsg-1 in ionic media of 0.01 and 0.10 mols L-1 NaNO3, respectively. A major contribution of carboxylic groups was observed with an average 34 and 22% of ionizable sites being titrated with conditional pcKa of 4.0 and 5.4, respectively. The remaining 44% of ionizable sites were divided in three classes with averaged conditional pcKa of 6.9, 8.7 and 10.12, which may be assigned respectively to imidazolic, aminic, and phenolic functionalities.

Immune Evasion by Borrelia burgdorferi – With Special Reference to CD38-mediated Chemotaxis of Neutrophils and Dendritic Cells

Lyme borreliosis is a tick-transmitted infection caused by the spirochete bacterium Borrelia burgdorferi sensu lato. The tick injects bacteria into host skin, where a first line defence, mainly the complement system, neutrophils, dendritic cells and macrophages are ready to attack foreign intruders. However, in the case of Lyme borreliosis, the original immune response in the skin is untypically mild among bacterial infections. A further untypical feature is the ability of B. burgdorferi to disseminate to distant organs, where, in some patients, symptoms appear after years after the original infection. This study aimed at uncovering some of the immune evasion mechanisms utilized by B. burgdorferi against the complement system, neutrophils and dendritic cells. B. burgdorferi was shown to inhibit chemotaxis of human neutrophils towards nformyl- methyl-leucyl-phenylalanine (fMLP). Outer surface protein B (OspB) of B. burgdorferi was shown to promote resistance to the attack of the complement system and neutrophil phagocytosis at low complement concentrations. B. burgdorferi was shown to inhibit migration of dendritic cells in vitro towards CCL19 and CCL21 and also in an in vivo model. This effect was shown to be due to the absence of CD38 on the borrelia-stimulated dendritic cell surface. A defect in p38 mitogen-activated-protein-kinase (p38) signaling was linked to defective CD38 expression. A defect in CD38 expression on B. burgdorferi-stimulated neutrophils was also observed. In this study, a number of novel immune evasion strategies utilized by B burgdorferi were chracterized. However, further studies are needed as other immune evasion mechanisms await to be uncovered.

Potential of caveolae in the therapy of cardiovascular and neurological diseases.

Caveolae are membrane micro-domains enriched in cholesterol, sphingolipids and caveolins, which are transmembrane proteins with a hairpin-like structure. Caveolae participate in receptor-mediated trafficking of cell surface receptors and receptor-mediated signaling. Furthermore, caveolae participate in clathrin-independent endocytosis of membrane receptors. On the one hand, caveolins are involved in vascular and cardiac dysfunction. Also, neurological abnormalities in caveolin-1 knockout mice and a link between caveolin-1 gene haplotypes and neurodegenerative diseases have been reported. The aim of this article is to present the rationale for considering caveolae as potential targets in cardiovascular and neurological diseases.

A Novel Missense Mutation, I890T, in the Pore Region of Cardiac Sodium Channel Causes Brugada Syndrome

Brugada syndrome (BrS) is a life-threatening, inherited arrhythmogenic syndrome associated with autosomal dominant mutations in SCN5A, the gene encoding the cardiac Na₊ channel alpha subunit (Naᵥ1.5). The aim of this work was to characterize the functional alterations caused by a novel SCN5A mutation, I890T, and thus establish whether this mutation is associated with BrS. The mutation was identified by direct sequencing of SCN5A from the proband’s DNA. Wild-type (WT) or I890T Naᵥ1.5 channels were heterologously expressed in human embryonic kidney cells. Sodium currents were studied using standard whole cell patch-clamp protocols and immunodetection experiments were performed using an antibody against human Naᵥ1.5 channel. A marked decrease in current density was observed in cells expressing the I890T channel (from -52.0 ± 6.5 pA/pF, n=15 to 35.9 ± 3.4 pA/pF, n = 22, at -20 mV, WT and I890T, respectively). Moreover, a positive shift of the activation curve was identified (V½ =-32.0 ± 0.3 mV, n = 18, and -27.3 ± 0.3 mV, n = 22, WT and I890T, respectively). No changes between WT and I890T currents were observed in steady-state inactivation, time course of inactivation, slow inactivation or recovery from inactivation parameters. Cell surface protein biotinylation analyses confirmed that Nav1.5 channel membrane expression levels were similar in WT and I890T cells. In summary, our data reveal that the I890T mutation, located within the pore of Nav1.5, causes an evident loss-of-function of the channel. Thus, the BrS phenotype observed in the proband is most likely due to this mutation

Differential Regulation of c-FLIP Isoforms Through Post-translational Modifications

Cells are constantly responding to signals from the surrounding tissues and the environment. To dispose of infected and potentially dangerous cells, to ensure the optimal execution of developmental processes and to maintain tissue homeostasis, a multicellular organism needs to tightly control both the number and the quality of its cells. Apoptosis is a form of active cellular self-destruction that enables an organism to regulate its cell number by deleting damaged or potentially dangerous cells. Apoptosis can be induced by death ligands, which bind to death receptors on the cell surface. Ligation of the receptors leads to the formation of an intracellular death inducing signaling complex (DISC). One of the DISC components is caspase-8, a protease that triggers the caspase cascade and is thereby a key initiator of programmed cell death. The activation of caspase-8 is controlled by the cellular FLICE-inhibitory proteins (c-FLIPs). Consequently, sensitivity towards receptor-mediated apoptosis is determined by the amount of c-FLIP, and the c-FLIP levels are actively regulated for example during erythroid differentiation of K562 erythroleukemia cells and by hyperthermia in Jurkat leukemia cells. The aim of my thesis was to investigate how c-FLIP is regulated during these processes. We found that c-FLIP isoforms are short-lived proteins, although c-FLIPS had an even shorter half-life than c-FLIPL. In both experimental models, increased death receptor sensitivity correlated with induced ubiquitylation and consequent proteasomal degradation of c-FLIP. Furthermore, we elucidated how phosphorylation regulates the biological functions and the turnover of c-FLIP, thereby contributing to death receptor sensitivity. We mapped the first phosphorylation sites on c-FLIP and dissected how their phosphorylation affects c-FLIP. Moreover, we demonstrated that phosphorylation of serine 193, a phosphorylated residue common to all c-FLIPs, is primarily mediated by the classical PKC. Furthermore, we discovered a novel connection between the phosphorylation and ubiquitylation of c-FLIP: phosphorylation of S193 protects c-FLIP from ubiquitylation. Surprisingly, although all c-FLIP isoforms are phosphorylated on this conserved residue, the biological outcome is different for the long and short isoforms, since S193 specifically prolongs the half-lives of the short c-FLIP isoforms, but not c-FLIPL. To summarize, we show that c-FLIP proteins are modified by ubiquitylation and phosphorylation, and that the biological outcomes of these modifications are isoform-specifically determined.

Luminescence-based assay methods in drug discovery

The drug discovery process is facing new challenges in the evaluation process of the lead compounds as the number of new compounds synthesized is increasing. The potentiality of test compounds is most frequently assayed through the binding of the test compound to the target molecule or receptor, or measuring functional secondary effects caused by the test compound in the target model cells, tissues or organism. Modern homogeneous high-throughput-screening (HTS) assays for purified estrogen receptors (ER) utilize various luminescence based detection methods. Fluorescence polarization (FP) is a standard method for ER ligand binding assay. It was used to demonstrate the performance of two-photon excitation of fluorescence (TPFE) vs. the conventional one-photon excitation method. As result, the TPFE method showed improved dynamics and was found to be comparable with the conventional method. It also held potential for efficient miniaturization. Other luminescence based ER assays utilize energy transfer from a long-lifetime luminescent label e.g. lanthanide chelates (Eu, Tb) to a prompt luminescent label, the signal being read in a time-resolved mode. As an alternative to this method, a new single-label (Eu) time-resolved detection method was developed, based on the quenching of the label by a soluble quencher molecule when displaced from the receptor to the solution phase by an unlabeled competing ligand. The new method was paralleled with the standard FP method. It was shown to yield comparable results with the FP method and found to hold a significantly higher signal-tobackground ratio than FP. Cell-based functional assays for determining the extent of cell surface adhesion molecule (CAM) expression combined with microscopy analysis of the target molecules would provide improved information content, compared to an expression level assay alone. In this work, immune response was simulated by exposing endothelial cells to cytokine stimulation and the resulting increase in the level of adhesion molecule expression was analyzed on fixed cells by means of immunocytochemistry utilizing specific long-lifetime luminophore labeled antibodies against chosen adhesion molecules. Results showed that the method was capable of use in amulti-parametric assay for protein expression levels of several CAMs simultaneously, combined with analysis of the cellular localization of the chosen adhesion molecules through time-resolved luminescence microscopy inspection.

In vivo imaging of vascular adhesion protein 1 - preclinical studies with positron emission tomography

The golden standard in nuclear medicine imaging of inflammation is the use of radiolabeled leukocytes. Although their diagnostic accuracy is good, the preparation of the leukocytes is both laborious and potentially hazardous for laboratory personnel. Molecules involved in leukocyte migration could serve as targets for the development of inflammation imaging agents. An excellent target would be a molecule that is absent or expressed at low level in normal tissues, but is induced or up-regulated at the site of inflammation. Vascular adhesion protein-1 (VAP-1) is a very promising target for in vivo imaging, since it is translocated to the endothelial cell surface when inflammation occurs. VAP-1 functions as an endothelial adhesion molecule that participates in leukocyte recruitment to inflamed tissues. Besides being an adhesion molecule, VAP-1 also has enzymatic activity. In this thesis, the targeting of VAP-1 was studied by using Gallium-68 (68Ga) labeled peptides and an Iodine-124 (124I) labeled antibody. The peptides were designed based on molecular modelling and phage display library searches. The new imaging agents were preclinically tested in vitro, as well as in vivo in animal models. The most promising imaging agent appeared to be a peptide belonging to the VAP-1 leukocyte ligand, Siglec-9 peptide. The 68Ga-labeled Siglec-9 peptide was able to detect VAP-1 positive vasculature in rodent models of sterile skin inflammation and melanoma by positron emission tomography. In addition to peptides, the 124I-labeled antibody showed VAP-1 specific binding both in vitro and in vivo. However, the estimated human radiation dose was rather high, and thus further preclinical studies in disease models are needed to clarify the value of this imaging agent. Detection of VAP-1 on endothelium was demonstrated in these studies and this imaging approach could be used in the diagnosis of inflammatory conditions as well as melanoma. These studies provide a proof-of-concept for PET imaging of VAP-1 and further studies are warranted.

Alpha2-Adrenoceptor-mediated vascular responses induced by dexmedetomidine

Alpha2-Adrenoceptors are cell-surface G protein coupled receptors that mediate many of the effects of the catecholamines noradrenaline and adrenaline. The three human α2-adrenoceptor subtypes are widely expressed in different tissues and organs, and they mediate many different physiological and pharmacological effects in the central and peripheral nervous system and as postsynaptic receptors in target organs. Previous studies have demonstrated that α2-adrenoceptors mediate both vascular constriction and dilatation in humans. Large inter-individual variation has been observed in the vascular responses to α2-adrenoceptor activation in clinical studies. All three receptor subtypes are potential drug targets. It was therefore considered important to further elucidate the details of adrenergic vascular regulation and its genetic variation, since such knowledge may help to improve the development of future cardiovascular drugs and intensive care therapies. Dexmedetomidine is the most selective and potent α2-adrenoceptor agonist currently available for clinical use. When given systemically, dexmedetomidine induces nearly complete sympatholysis already at low concentrations, and postsynaptic effects, such vasoconstriction, can be observed with increasing concentrations. Thus, local infusions of small doses of dexmedetomidine into dorsal hand veins and the application of pharmacological sympathectomy with brachial plexus block provide a means to assess drug-induced peripheral vascular responses without interference from systemic pharmacological effects and autonomic nervous system regulation. Dexmedetomidine was observed to have biphasic effects on haemodynamics, with an initial decrease in blood pressure at low concentrations followed by substantial increases in blood pressure and coronary vascular resistance at high concentrations. Plasma concentrations of dexmedetomidine that significantly exceeded the recommended therapeutic level did not reduce myocardial blood flow below the level that is observed with the usual therapeutic concentrations and did not induce any evident myocardial ischaemia in healthy subjects. Further, it was demonstrated that dexmedetomidine also had significant vasodilatory effects through activation of endothelial nitric oxide synthesis, and thus when the endothelial component of the blood vessel response to dexmedetomidine was inhibited, peripheral vasoconstriction was augmented. Hand vein constriction responses to α2-adrenoceptor activation by dexmedetomidine were only weakly associated with the constriction responses to α1-adrenoceptor activation, pointing to independent cellular regulation by these two adrenoceptor classes. Substantial inter-individual variation was noted in the venous constriction elicited by activation of α2-adrenoceptors by dexmedetomidine. In two study populations from two different continents, a single nucleotide polymorphism in the PRKCB gene was found to be associated with the dorsal hand vein constriction response to dexmedetomidine, suggesting that protein kinase C beta may have an important role in the vascular α2-adrenoceptor signalling pathways activated by dexmedetomidine.

JNK, a versatile regulator of kinesin-1 transport and cytoskeleton dynamics

C-Jun N-terminal kinase (JNK) is traditionally recognized as a crucial factor in stress response and inducer of apoptosis upon various stimulations. Three isoforms build the JNK subfamily of MAPK; generally expressed JNK1 and JNK2 and brain specific JNK3. Degenerative potency placed JNK in the spotlight as potential pharmacological option for intervention. Unfortunately, adverse effects of potential drugs and observation that expression of only JNK2 and JNK3 are induced upon stress, restrained initial enthusiasm. Notably, JNK1 demonstrated atypical high constitutive activity in neurons that is not responsive to cellular stresses and indicated existence of physiological activity. This thesis aimed at revealing the physiological functions of JNK1 in actin homeostasis through novel effector MARCKS-Like 1 (MARCKSL1) protein, neuronal trafficking mediated by major kinesin-1 motor protein and microtubule (MT) dynamics via STMN2/SCG10. The screen for novel physiological JNK substrates revealed specific phosphorylation of C-terminal end of MARCKSL1 at S120, T148 and T183 both ex vivo and in vitro. By utilizing site-specific mutagenesis, various actin dynamics and migrations assays we were able to demonstrate that JNK1 phosphorylation specifically facilitates F-actin bundling and thus filament stabilisation. Consecutively, this molecular mechanism was proved to enhance formation of filopodia; cell surface projections that allow cell sensing surrounding environment and migrate efficiently. Our results visualize JNK dependent and MARCKSL1 executed induction of filopodia in neurons and fibroblast indicating general mechanism. Subsequently, inactivation of JNK action on MARCKSL1 shifts cellular actin machinery into lamellipodial dynamic arrangement. Tuning of actin cytoskeleton inevitably melds with cell migration. We observed that both active JNK and JNK pseudo-phosphorylated form of MARCKSL1 reduce actin turnover in intact cells leading to overall diminished cell motility. We demonstrate that tumour transformed cells from breast, prostate, lung and muscle-derived cancers upregulate MARCKSL1. We showed on the example of prostate cancer PC-3 cell line that JNK phosphorylation negatively controls MARCKSL1 ability to induce migration, which precedes cancer cell metastasis. The second round of identification of JNK physiological substrates resulted in detection of predominant motor protein kinesin-1 (Kif5). Mass spectrometry detailed analysis showed evident endogenous phosphorylation of kinesin-1 on S176 within motor domain that interacts with MT. In vitro phosphorylation of bacterially expressed kinesin heavy chain by JNK isoforms displayed higher specificity of JNK1 when compared to JNK3. Since, JNK1 is constitutively active in neurons it signified physiological aspect of kinesin-1 regulation. Subsequent biochemical examination revealed that kinesin-1, when not phosphorylated on JNK site, exhibits much higher affinity toward MTs. Expression of the JNK non-phosphorable kinesin-1 mutant in intact cells as well as in vitro single molecule imaging using total internal reflection fluorescence microscopy indicated that the mutant loses normal speed and is not able to move processively into proper cellular compartments. We identify novel kinesin-1 cargo protein STMN2/SCG10, which along with known kinesin-1 cargo BDNF is showing impaired trafficking when JNK activity is inhibited. Our data postulates that constitutive JNK activity in neurons is crucial for unperturbed physiologically relevant transport of kinesin-1 dependant cargo. Additionally, my work helps to validate another novel physiological JNK1 effector STMN2/SCG10 as determinant of axodendritic neurites dynamics in the developing brain through regulation of MT turnover. We show successively that this increased MT dynamics is crucial during developmental radial migration when brain layering occurs. Successively, we are able to show that introduction of JNK phosphorylation mimicking STMN2/SCG10 S62/73D mutant rescues completely JNK1 genetic deletion migration phenotype. We prove that STMN2/SCG10 is predominant JNK effector responsible for MT depolymerising activity and neurite length during brain development. Summarizing, this work describes identification of three novel JNK substrates MARCKSL1, kinesin-1 and STMN2/SCG10 and investigation of their roles in cytoskeleton dynamics and cargo transport. This data is of high importance to understand physiological meaning of JNK activity, which might have an adverse effect during pharmaceutical intervention aiming at blocking pathological JNK action.

Thymic atrophy in cattle poisoned with Solanum glaucophyllum

Solanum glaucophyllum (Sg) [= S. malacoxylon] is a calcinogenic plant inducing "Enzootic Calcinosis" in cattle. The 1,25-dihydroxyvitamin D3, its main toxic principle, regulates bone and calcium metabolism and also exerts immunomodulatory effects. Thymocyte precursors from bone marrow-derived progenitor cells differentiate into mature T-cells. Differentiation of most T lymphocytes is characterized not only by the variable expression of CD4/CD8 receptor molecules and increased surface density of the T cell antigen receptor, but also by changes in the glycosylation pattern of cell surface glycolipids or glycoproteins. Thymocytes exert a feedback influence on thymic non-lymphoid cells. Sg-induced modifications on cattle thymus T-lymphocytes and on non-lymphoid cells were analysed. Heifers were divided into 5 groups (control, intoxicated with Sg during 15, 30 or 60 days, and probably recovered group). Histochemical, immunohistochemical, lectinhistochemical and morphometric techniques were used to characterize different cell populations of the experimental heifers. Sg-poisoned heifers showed a progressive cortical atrophy that was characterized using the peanut agglutinin (PNA) lectin that recognizes immature thymocytes. These animals also increased the amount of non-lymphoid cells per unit area detected with the Picrosirius technique, WGA and DBA lectins, and pancytokeratin and S-100 antibodies. The thymus atrophy found in intoxicated animals resembled that of the physiological aging process. A reversal effect on these changes was observed after suppression of the intoxication. These findings suggest that Sg-intoxication induces either directly, through the 1,25-dihydroxyvitamin D3 itself, or indirectly through the hypercalcemia, the observed alteration of the thymus.

Receptors and endocytosis of coxsackievirus A9

Coxsackievirus A9 (CV-A9) belongs to human enteroviruses within family Picornaviridae, which are the main cause of aseptic meningitis. In addition, CV-A9 causes a wide range of other clinical manifestations of acute disease including respiratory infections, myocarditis, encephalitis and severe generalized infections in newborns. In this study, the functions of integrins αVβ6 and αVβ3 in the attachment and cellular entry of CV-A9 were analyzed. Further, virus and cell surface interactions and endocytosis of CV-A9 were studied in specific cell lines. Also, a method for production of GFP-expressing CV-A9 particles by long PCR-mediated mutagenesis and in vivo transcription was developed. The results indicated that RGD-motif (arginine-glycine-asparagine) that resides in the viral capsid is important for CV-A9 infection particularly in cell lines expressing integrin αVβ6 and that this integrin serves as a high affinity attachment receptor for the virus. CV-A9 is also capable of infecting certain cell lines independently of αV-integrins by binding to the cell surface HSPA5 protein. Regardless of the attachment stage, the internalization of the virus occurs via the same entry pathway and is dependent on β2M, dynamin, and Arf6 but independent of clathrin and caveolin-1. Furthermore, the virus internalization occurs within Arf6-containing vesicles suggesting that Arf6 is central mediator of CV-A9 endocytosis. While in this study the results of CV-A9 endocytosis were based on microscopical visualization within individual fixed cells, a rapid method for generation of a virus for real-time imaging was also described.