Proteins In MDCK Cells Research Articles

Quantitative proteomic analysis of MDCK cell adhesion.

MDCK cells are a key reagent in modern vaccine production. As MDCK cells are normally adherent, creation of suspension cells for vaccine production using genetic engineering approaches is highly desirable. However, little is known regarding the mechanisms and effectors underlying MDCK cell adhesion. In this study, we performed a comparative analysis of whole protein levels between MDCK adhesion and suspension cells using an iTRAQ-based (isobaric tags for relative and absolute quantitation) proteomics approach. We found that expression of several proteins involved in cell adhesion exhibit reduced expression in suspension cells, including at the mRNA level. Proteins whose expression was reduced in suspension cells include cadherin 1 (CDH1), catenin beta-1 (CTNNB1), and catenin alpha-1 (CTNNA1), which are involved in intercellular adhesion; junction plakoglobin (JUP), desmoplakin (DSP), and desmoglein 3 (DSG3), which are desmosome components; and transglutaminase 2 (TGM2) and alpha-actinin-1 (ACTN1), which regulate the adhesion between cells and the extracellular matrix. A functional verification experiment showed that inhibition of E-cadherin significantly reduced intercellular adhesion of MDCK cells. E-Cadherin did not significantly affect the proliferation of MDCK cells and the replication of influenza virus. These findings reveal possible mechanisms underlying adhesion of MDCK cells and will guide the creation of MDCK suspension cells by genetic engineering.

Manipulation of Actin Cytoskeleton and Tight Junction Protein Knockdown Differentially Interact to Modulate Basal and H2O2‐Induced Paracellular Permeability of Renal Epithelial Cells

Renal epithelial tight junction permeability to large molecules (leak pathway) is increased by hydrogen peroxide (H2O2). Previous studies have suggested a role for the actin cytoskeleton in regulation of epithelial paracellular permeability but its possible involvement in H2O2‐induced regulation of renal epithelial paracellular permeability is unknown. To assess the role of the actin cytoskeleton, we examined the effect of two agents that manipulate differentially the actin cytoskeleton, blebbistatin (inhibits myosin II ATPase) and jasplakinolide (enhances F‐actin nucleation), on basal and H2O2‐induced renal epithelial leak pathway permeability, measured as calcein flux rate. The effects of these compounds was examined in wild type MDCK cells and in MDCK cells in which either occludin protein or ZO‐1 protein has been knocked down to determine if either of these tight junction proteins might be important in mediating actin network actions. Under basal conditions, treatment with 40 mM blebbistatin produced little or no change in calcein flux rate in wild type MDCK cells. H2O2 increased calcein flux rate, as expected. The combination of H2O2 and blebbistatin produced a synergistic increase in calcein flux rate. Jasplakinolide (200 nM) produced a marked increase in calcein flux rate. H2O2 and jasplakinolide did not increase calcein flux rate beyond the rate produced by jasplakinolide alone. Knockdown of occludin protein in MDCK cells did not affect basal calcein flux rate but enhanced the H2O2‐induced increase. In the absence of H‐2O2, blebbistatin produced little or no change in calcein flux rate. H2O2 and blebbistatin did not increase calcein flux rate further than that produced by H2O2 alone. Jasplakinolide increased the calcein flux rate but attenuated the increase produced by H2O2 when cells were incubated with both compounds. Knockdown of ZO‐1 protein increased basal calcein flux rate approximately two‐fold. Treatment with H2O2 did not further increase flux rate. Treatment of ZO‐1 knockdown cells with blebbistatin increased the calcein flux rate, in contrast to its lack of effect on wild type MDCK cells. Treatment with the combination of H2O2 and blebbistatin did not increase flux rate beyond that produced by blebbistatin alone. Jasplakinolide alone increased calcein flux rate in ZO‐1‐knockdown cells. The combination of H2O2 and jasplakinolide did not increase flux rate beyond that produced by jasplakinolide alone. These results suggest a complex role of the actin cytoskeleton in regulation of basal and H2O2‐stimulated leak pathway permeability in renal epithelial cells. First, they suggest that an intact F‐actin network is necessary to maintain low basal leak pathway permeability. Second, they suggest the tight junction is under inward actomyosin‐mediated tension, via the ZO‐1 protein, which contributes to maintenance of low leak pathway permeability, particularly in stress conditions. Third, they suggest that H2O2 treatment produces a state in MDCK cells that is functionally similar to knockdown of ZO‐1 protein. Fourth, they suggest that the effect of blebbistatin is mediated via occludin protein.Support or Funding InformationNIDDK and institutional fundsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SAFETY AND IMMUNOGENICITY OF COLD-ADAPTED RECOMBINANT INFLUENZA VECTOR EXPRESSING ESAT-6 AND AG85А ANTIGENS OF M. TUBERCULOSIS.

Recombinant viral vectors represent one of the most promising platforms for creating a new generation of vaccines against tuberculosis. We constructed a vaccine candidate based on a cold-adapted influenza vector with a truncated NS1 protein containing an insert of tuberculosis ESAT-6 and Ag85A antigens. The recombinant virus possessed a cold-adapted and temperature-sensitive phenotype and was attenuated for mice when administered intranasally. Immunofluorescent staining and Western blot showed the expression of ESAT-6 protein in MDCK cells infected by recombinant virus. After intranasal administration to mice, the recombinant virus stimulated a specific anti-tuberculosis CD4 + Th1-type response with the formation of polyfunctional antigen-specific T cells.

Remodeling the zonula adherens in response to tension and the role of afadin in this response.

Morphogenesis requires dynamic coordination between cell-cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell-cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions.

Alterations of proteins in MDCK cells during acute potassium deficiency

Chronic K+ deficiency can cause hypokalemic nephropathy associated with metabolic alkalosis, polyuria, tubular dilatation, and tubulointerstitial injury. However, effects of acute K+ deficiency on the kidney remained unclear. This study aimed to explore such effects by evaluating changes in levels of proteins in renal tubular cells during acute K+ deficiency. MDCK cells were cultivated in normal K+ (NK) (K+=5.3mM), low K+ (LK) (K+=2.5mM), or K+ depleted (KD) (K+=0mM) medium for 24h and then harvested. Cellular proteins were resolved by two-dimensional gel electrophoresis (2-DE) and visualized by SYPRO Ruby staining (5 gels per group). Spot matching and quantitative intensity analysis revealed a total 48 protein spots that had significantly differential levels among the three groups. Among these, 46 and 30 protein spots had differential levels in KD group compared to NK and LK groups, respectively. Comparison between LK and NK groups revealed only 10 protein spots that were differentially expressed. All of these differentially expressed proteins were successfully identified by Q-TOF MS and/or MS/MS analyses. The altered levels of heat shock protein 90 (HSP90), ezrin, lamin A/C, tubulin, chaperonin-containing TCP1 (CCT1), and calpain 1 were confirmed by Western blot analysis. Global protein network analysis showed three main functional networks, including 1) cell growth and proliferation, 2) cell morphology, cellular assembly and organization, and 3) protein folding in which the altered proteins were involved. Further investigations on these networks may lead to better understanding of pathogenic mechanisms of low K+-induced renal injury.

The large GTPase Mx1 is involved in apical transport in MDCK cells.

In epithelial cells apical proteins are transported by specific transport carriers to the correct membrane domain. The composition of these carriers is heterogeneous and comprises components such as motor proteins, annexins, lectins, Rab GTPases and cargo molecules. Here, we provide biochemical and fluorescence microscopic data to show that the dynamin-related large GTPase Mx1 is a component of post-Golgi vesicles carrying the neurotrophin receptor p75(NTR) . Moreover, siRNA-mediated depletion of Mx1 significantly decreased the transport efficiency of apical proteins in MDCK cells. In conclusion, Mx1 plays a crucial role in the delivery of cargo molecules to the apical membrane of epithelial cells.

The expression of essential components for human influenza virus internalisation in Vero and MDCK cells

MDCK and Vero cell lines have been used as substrates for influenza virus replication. However, Vero cells produced lower influenza virus titer yield compared to MDCK. Influenza virus needs molecules for internalisation of the virus into the host cell, such as influenza virus receptor and clathrin. Human influenza receptor is usually a membrane protein containing Sia(α2,6) Gal, which is added into the protein in the golgi apparatus by α2,6 sialyltransferase (SIAT1). Light clathrin A (LCA), light clathrin B (LCB) and heavy clathrin (HC) are the main components needed for virus endocytosis. Therefore, it is necessary to compare the expression of SIAT1 and clathrin in Vero and MDCK cells. This study is reporting the expression of SIAT1 and clathrin observed in both cells with respect to the levels of (1) RNA by using RT-PCR, (2) protein by using dot blot analysis and confocal microscope. The results showed that Vero and MDCK cells expressed both SIAT1 and clathrin proteins, and the expression of SIAT1 in MDCK was higher compared to Vero cells. On the other hand, the expressions of LCA, LCB and HC protein in MDCK cells were not significantly different to Vero cells. This result showed that the inability of Vero cells to internalize H1N1 influenza virus was possibly due to the lack of transmembrane protein receptor which contained Sia(α2,6) Gal.

Most Influenza A Virions Fail To Express at Least One Essential Viral Protein

Segmentation of the influenza A virus (IAV) genome enables rapid gene reassortment at the cost of complicating the task of assembling the full viral genome. By simultaneously probing for the expression of multiple viral proteins in MDCK cells infected at a low multiplicity with IAV, we observe that the majority of infected cells lack detectable expression of one or more essential viral proteins. Consistent with this observation, up to 90% of IAV-infected cells fail to release infectious progeny, indicating that many IAV virions scored as noninfectious by traditional infectivity assays are capable of single-round infection. This fraction was not significantly affected by target or producer cell type but varied widely between different IAV strains. These data indicate that IAV exists primarily as a swarm of complementation-dependent semi-infectious virions, and thus traditional, propagation-dependent assays of infectivity may drastically misrepresent the true infectious potential of a virus population.

The Clathrin Adaptor AP-1A Mediates Basolateral Polarity

Clathrin and the epithelial-specific clathrin adaptor AP-1B mediate basolateral trafficking in epithelia. However, several epithelia lack AP-1B, and mice knocked out for AP-1B are viable, suggesting the existence of additional mechanisms that control basolateral polarity. Here, we demonstrate a distinct role of the ubiquitous clathrin adaptor AP-1A in basolateral protein sorting. Knockdown of AP-1A causes missorting of basolateral proteins in MDCK cells, but only after knockdown of AP-1B, suggesting that AP-1B can compensate for lack of AP-1A. AP-1A localizes predominantly to the TGN, and its knockdown promotes spillover of basolateral proteins into common recycling endosomes, the site of function of AP-1B, suggesting complementary roles of both adaptors in basolateral sorting. Yeast two-hybrid assays detect interactions between the basolateral signal of transferrin receptor and the medium subunits of both AP-1A and AP-1B. The basolateral sorting function of AP-1A reported here establishes AP-1 as a major regulator of epithelial polarity.

Activation of the Nipah Virus Fusion Protein in MDCK Cells Is Mediated by Cathepsin B within the Endosome-Recycling Compartment

Proteolytic activation of the fusion protein of the highly pathogenic Nipah virus (NiV F) is a prerequisite for the production of infectious particles and for virus spread via cell-to-cell fusion. Unlike other paramyxoviral fusion proteins, functional NiV F activation requires endocytosis and pH-dependent cleavage at a monobasic cleavage site by endosomal proteases. Using prototype Vero cells, cathepsin L was previously identified to be a cleavage enzyme. Compared to Vero cells, MDCK cells showed substantially higher F cleavage rates in both NiV-infected and NiV F-transfected cells. Surprisingly, this could not be explained either by an increased F endocytosis rate or by elevated cathepsin L activities. On the contrary, MDCK cells did not display any detectable cathepsin L activity. Though we could confirm cathepsin L to be responsible for F activation in Vero cells, inhibitor studies revealed that in MDCK cells, cathepsin B was required for F-protein cleavage and productive replication of pathogenic NiV. Supporting the idea of an efficient F cleavage in early and recycling endosomes of MDCK cells, endocytosed F proteins and cathepsin B colocalized markedly with the endosomal marker proteins early endosomal antigen 1 (EEA-1), Rab4, and Rab11, while NiV F trafficking through late endosomal compartments was not needed for F activation. In summary, this study shows for the first time that endosomal cathepsin B can play a functional role in the activation of highly pathogenic NiV.

Multiple Biosynthetic Trafficking Routes for Apically Secreted Proteins in MDCK Cells

Many newly synthesized membrane proteins traverse endocytic intermediates en route to the surface in polarized epithelial cells; however, the biosynthetic itinerary of secreted proteins has not been elucidated. We monitored the trafficking route of two secreted proteins with different apical sorting signals: the N-glycan-dependent cargo glycosylated growth hormone (gGH) and Ensol, a soluble version of endolyn whose apical sorting is independent of N-glycans. Both proteins were observed to colocalize in part with apical recycling endosome (ARE) markers. Cargo that lacks an apical targeting signal and is secreted in a nonpolarized manner did not localize to the ARE. Expression of a dominant-negative mutant of myosin Vb, which disrupts ARE export of glycan-dependent membrane proteins, selectively inhibited apical release of gGH but not Ensol. Fluorescence recovery after photobleaching (FRAP) measurements revealed that gGH in the ARE was less mobile than Ensol, consistent with tethering to a sorting receptor. However, knockdown of galectin-3 or galectin-4, lectins implicated in apical sorting, had no effect on the rate or polarity of gGH secretion. Together, our results suggest that apically secreted cargoes selectively access the ARE and are exported via differentially regulated pathways.

E14 Expression of Matrix Gla Protein in MDCK cells exposed to Oxalates, Calcium Oxalate Monohydrate and Hydroxyapatite crystals

E14 Expression of Matrix Gla Protein in MDCK cells exposed to Oxalates, Calcium Oxalate Monohydrate and Hydroxyapatite crystals

Expression of Human MDGA1 Increases Cell Motility and Cell-Cell Adhesion and Reduces Adhesion to Extracellular Matrix Proteins in MDCK Cells

Characterization of the novel human protein MDGA1 (MAM Domain containing Glycosylphosphatidylinositol Anchor-1) has been reported in our laboratory in the past few years. hMDGA1 is a glycoprotein containing 955 aminoacids (137kDa) attached to the eukaryotic cell membrane by a GPI (Glycosylphosphatidylinositol) anchor and localized specifically into membrane microdomains known as lipid rafts. Moreover, MDGA1 protein contains structural features found in different types of cell adhesion molecules (CAMs) such as the presence of immunoglobulin domains and a MAM domain (Meprin, A5 protein, receptor protein-tyrosine phosphatase μ), suggesting a role of MDGA1 in cell migration and/or adhesion. In order to investigate this aim, stable MDCK cell lines expressing MDGA1 or the truncated proteins IgGPI (lacking the MAM domain) and MAMGPI (lacking Ig domains) were generated. Our results reveal that MDGA1 increases the ability of MDCK cells to migrate, as it contains both Ig and MAM domains which have been implicated in cell motility. In addition, cell adhesion to extracellular matrix proteins, mainly to collagen IV, is reduced by MDGA1 and the IgGPI and MAMGPI truncated proteins. Accordingly, silencing MDGA1 by siRNA revealed a significant increase in adhesion to collagen IV. Furthermore, MDGA1 expression, through the intrinsic properties of the MAM domain, increases cell-cell adhesion independently of the cell monolayer used, suggesting that MDGA1 mediates cell-cell adhesiveness in a heterophilic manner.

Identification of unusual truncated forms of nucleocapsid protein in MDCK cells infected by Avian influenza virus (H9N2)

Unusual truncated forms of nucleocapsid protein (NP) were identified in the lysate of MDCK cells infected by Avian influenza virus (H9N2) using MS-based proteomics approach. Moreover, O-sulfonation that was considered as an unusual modification was identified in one of the tryptic peptides from the truncated NP. The findings might have implications on better understanding on the role of nucleoprotein in Avian influenza virus-host interaction.

Absolute quantification of multidrug resistance-associated protein 2 (MRP2/ABCC2) using liquid chromatography tandem mass spectrometry

The multidrug resistance-associated protein 2 (MRP2/ABCC2) plays an important role in hepatobiliary efflux of many drugs and drug metabolites and has been reported to account for dramatic interspecies differences in the aspects of pharmacokinetics. In the present study, an absolute quantification method was developed to quantitatively measure MRP2/ABCC2 using LC-MS/MS for detection of a selective tryptic peptide. A unique 16-mer tryptic peptide was identified by conducting capillary LC nanospray ESI-Q-TOF analysis of the immunoprecipitation-enriched samples of MRP2/ABCC2 following proteolysis with trypsin. The lower limit of quantification was established to be 31.25 pM with the linearity of the standard curve spanned to 2500 pM. Both the accuracy (relative error) and the precision (coefficient of variation) of the method were below 15%. Using this method, we successfully determined the absolute amount of MRP2/ABCC2 protein in MRP2/ABCC2 gene-transfected MDCK cells as well as the basal levels of canine Mrp2/Abcc2 protein in MDCK cells. Our findings also demonstrate that the sensitivity of this method exceeds the sensitivity of immunoblotting assay which was not able to detect the basal levels of canine Mrp2/Abcc2 in MDCK cells. The method could be directly applicable to many current research needs related to MRP2/ABCC2 protein.

Nephrogenic diabetes insipidus in mice caused by deleting COOH-terminal tail of aquaporin-2

In mammals, the hormonal regulation of water homeostasis is mediated by the aquaporin-2 water channel (Aqp2) of the collecting duct (CD). Vasopressin induces redistribution of Aqp2 from intracellular vesicles to the apical membrane of CD principal cells, accompanied by increased water permeability. Mutations of AQP2 gene in humans cause both recessive and dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. In this study, we generated a line of mice with the distal COOH-terminal tail of the Aqp2 deleted (Aqp2(Delta230)), including the protein kinase A phosphorylation site (S256), but still retaining the putative apical localization signal (221-229) at the COOH-terminal. Mice heterozygous for the truncation appear normal. Homozygotes are viable to adulthood, with reduced urine concentrating capacity, increased urine output, decreased urine osmolality, and increased daily water consumption. Desmopressin increased urine osmolality in wild-type mice but had no effect on Aqp2(Delta230/Delta230) mice. Kidneys from affected mice showed CD and pelvis dilatation and papillary atrophy. By immunohistochemical and immunoblot analyses using antibody against the NH(2)-terminal region of the protein Aqp2(Delta230/Delta230) mice had a markedly reduced protein abundance. Expression of the truncated protein in MDCK cells was consistent with a small amount of functional expression but no stimulation. Thus we have generated a mouse model of NDI that may be useful in studying the physiology and potential therapy of this disease.

Altered Biogenesis of ΔF508-CFTR Following Treatment with Doxorubicin

Cystic fibrosis (CF) is caused by mutations to the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common of these mutations is deletion of a phenylalanine residue at position 508 (ΔF508), which accounts for ñ70% of all CF alleles. This mutation interferes with the biogenesis and maturation of ΔF508-CFTR to the plasma membrane. However, ΔF508-CFTR can partially function upon proper localization. Thus, pharmacological correction of ΔF508-CFTR maturation holds promise in CF therapy. Our previous studies indicate that a single non-cytotoxic dose of the anthracycline doxorubicin (Dox) significantly increase ΔF508-CFTR-associated chloride secretion in MDCK cells by increasing the expression of this protein at the apical plasma membrane. We report here that Dox alters the biogenesis of ΔF508-CFTR. Treatment with Dox increases the resistance of ΔF508-CFTR to trypsin digestion, possibly by expediting protein folding. Further, treatment with Dox reduces the amount of polyubiquitinated ΔF508-CFTR in cells and prolongs the half-life of this protein. Concomitantly, treatment with Dox decreases the association of ΔF508-CFTR with HSP70 but does not alter the expression of major HSP70 family members. Based on these results, we propose that Dox expedites the folding and maturation of ΔF508-CFTR by acting as a pharmacological chaperone, which consequently promotes the functional expression of this protein in MDCK cells.

Identification of an AHNAK Binding Motif Specific for the Annexin2/S100A10 Tetramer

The Annexin2 tetramer (A2t), which consists of two Annexin2 molecules bound to a S100A10 dimer, is implicated in membrane-trafficking events. Here, we showed using a yeast triple-hybrid experiment and in vitro binding assay that Annexin2 is required for strong binding of S100A10 to the C-terminal domain of the protein Ahnak. We also revealed that this effect involves only the Annexin2 N-terminal tail, which is implicated in S100A10/Annexin2 tetramerization. The minimal A2t binding motif (A2tBP1) in Ahnak was mapped to a 20-amino acid peptide, and this peptide is highly specific for A2t. We also identified a second A2t binding motif (A2tBP2) present in the N-terminal domain of Ahnak, which binds to A2t, albeit with less affinity. When overexpressed as an EGFP fusion protein in MDCK cells, A2tBPs cofractionate in a calcium-dependent manner and co-immunoprecipitate with S100A10 and Annexin2. In living cells, A2tBPs target EGFP to the cytoplasm as does Annexin2. In response to oxidative and mechanical stress, EGFP-A2tBPs relocalize within minutes to the plasma membrane; a behavior shared with Annexin2-GFP. These results suggest that the A2t complex exists within the cytoplasm of resting living cells and that its localization at the plasma membrane relies on cellular signaling. Together, our data demonstrate that A2tBP1 is a specific A2t complex binding domain and may be a powerful tool to help elucidate A2t structure and cellular functions.

Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical domain during polarization of MDCK cells

Epithelial polarization involves the segregation of apical and basolateral membrane domains, which are stabilized and maintained by tight junctions and membrane traffic. We report that unlike most apical and basolateral proteins in MDCK cells, which separate only after junctions have formed, the apical marker gp135 signifies an early level of polarized membrane organization established already in single cells. We identified gp135 as the dog orthologue of podocalyxin. With a series of domain mutants we show that the COOH-terminal PSD-95/Dlg/ZO-1 (PDZ)–binding motif is targeting podocalyxin to the free surface of single cells as well as to a subdomain of the terminally polarized apical membrane. This special localization of podocalyxin is shared by the cytoplasmic PDZ-protein Na+/H+ exchanger regulatory factor (NHERF)-2. Depleting podocalyxin by RNA interference caused defects in epithelial polarization. Together, our data suggest that podocalyxin and NHERF-2 function in epithelial polarization by contributing to an early apical scaffold based on PDZ domain-mediated interactions.

Polarized trafficking of thyrocyte proteins in MDCK cells

Recent studies suggest striking similarities between polarized protein sorting in thyrocytes and MDCK epithelial cells, including apical trafficking of thyroglobulin (Tg), thyroid peroxidase, and aminopeptidase N; as well as basolateral targeting of heparan sulfate proteoglycans, thrombospondin 1 (TSP1), type 1 5'-deiodinase, sodium–potassium ATPase, and the thyrotropin receptor. In this report, we have firstly expressed in stably transfected MDCK II cells a range of truncation mutants lacking up to 78% of the C-terminus of TSP1; these studies indicate that the N-terminal region containing the heparin binding domain is sufficient for basolateral targeting of TSP1. Secondly, we have stably transfected MDCK II cells with both Tg and sodium-iodide symporter (NIS) cDNAs, obtaining clones that simultaneously express both thyroid-specific proteins at the apical and basolateral cell surfaces, respectively. These studies represent promising early steps towards designing artificial thyrocytes by thyroid gene transfer into MDCK cells.