Nephrin Promotes Cell-Cell Adhesion through Homophilic Interactions
2003; Elsevier BV; Volume: 163; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)63590-0
ISSN1525-2191
AutoresJamshid Khoshnoodi, Kristmundur Sigmundsson, Lars‐Göran Öfverstedt, Ulf Skoglund, Björn Öbrink, Jorma Wartiovaara, Karl Tryggvason,
Tópico(s)Complement system in diseases
ResumoNephrin is a type-1 transmembrane protein and a key component of the podocyte slit diaphragm, the ultimate glomerular plasma filter. Genetic and acquired diseases affecting expression or function of nephrin lead to severe proteinuria and distortion or absence of the slit diaphragm. Here, we showed by using a surface plasmon resonance biosensor that soluble recombinant variants of nephrin, containing the extracellular part of the protein, interact with each other in a specific and concentration-dependent manner. This molecular interaction was increased by twofold in the presence of physiological Ca2+concentration, indicating that the binding is not dependent on, but rather promoted by Ca2+. Furthermore, transfected HEK293 cells and an immortalized mouse podocyte cell line overexpressing full-length human nephrin formed cellular aggregates, with cell-cell contacts staining strongly for nephrin. The distance between plasma membranes at the nephrin-containing contact sites was shown by electron microscopy to be 40 to 50 nm, similar to the width of glomerular slit diaphragm. The cell contacts could be dissociated with antibodies reacting with the first two extracellular Ig-like domains of nephrin. Wild-type HEK293 cells were shown to express slit diaphragm components CD2AP, P-cadherin, FAT, and NEPH1. The results show that nephrin molecules exhibit homophilic interactions that could promote cellular contacts through direct nephrin-nephrin interactions, and that the other slit diaphragm components expressed could contribute to that interaction. Nephrin is a type-1 transmembrane protein and a key component of the podocyte slit diaphragm, the ultimate glomerular plasma filter. Genetic and acquired diseases affecting expression or function of nephrin lead to severe proteinuria and distortion or absence of the slit diaphragm. Here, we showed by using a surface plasmon resonance biosensor that soluble recombinant variants of nephrin, containing the extracellular part of the protein, interact with each other in a specific and concentration-dependent manner. This molecular interaction was increased by twofold in the presence of physiological Ca2+concentration, indicating that the binding is not dependent on, but rather promoted by Ca2+. Furthermore, transfected HEK293 cells and an immortalized mouse podocyte cell line overexpressing full-length human nephrin formed cellular aggregates, with cell-cell contacts staining strongly for nephrin. The distance between plasma membranes at the nephrin-containing contact sites was shown by electron microscopy to be 40 to 50 nm, similar to the width of glomerular slit diaphragm. The cell contacts could be dissociated with antibodies reacting with the first two extracellular Ig-like domains of nephrin. Wild-type HEK293 cells were shown to express slit diaphragm components CD2AP, P-cadherin, FAT, and NEPH1. The results show that nephrin molecules exhibit homophilic interactions that could promote cellular contacts through direct nephrin-nephrin interactions, and that the other slit diaphragm components expressed could contribute to that interaction. Podocyte foot processes cover the outer aspect of the glomerular capillaries in an interdigitating manner. The slit between adjacent foot processes contains a highly ordered thin structure referred to as slit diaphragm (SD). The SD is thought to act as the ultimate albumin-excluding ultrafilter critical in the formation of primary urine.1Tisher CC Madsen KM Anatomy of the kidney.in: Brenner BM Brenner and Rector's The Kidney ed 6. WB Saunders Company, Philadelphia2000: 3-67Google Scholar Based on electron microscopic studies of perfusion-fixed rodent kidneys, the SD has been suggested to have a zipper-like porous structure.2Rodewald R Karnovsky MJ Porous substructure of the glomerular slit diaphragm in the rat and mouse.J Cell Biol. 1974; 60: 423-433Crossref PubMed Scopus (434) Google Scholar The SD is affected in a variety of genetic and acquired diseases with proteinuria and nephrotic syndrome as a result.1Tisher CC Madsen KM Anatomy of the kidney.in: Brenner BM Brenner and Rector's The Kidney ed 6. WB Saunders Company, Philadelphia2000: 3-67Google Scholar, 3Khoshnoodi J Tryggvason K Congenital nephrotic syndromes.Curr Opin Genet Dev. 2001; 11: 322-327Crossref PubMed Scopus (29) Google Scholar Nephrin is a recently described protein4Kestilä M Lenkkeri U Männikkö M Lamerdin J McCready P Putaala H Ruotsalainen V Morita T Nissinen M Herva R Kashtan CE Peltonen L Holmberg C Olsen A Tryggvason K Positionally cloned gene for a novel glomerular protein—nephrin—is mutated in congenital nephrotic syndrome.Mol Cell. 1998; 1: 575-582Abstract Full Text Full Text PDF PubMed Scopus (1554) Google Scholar and the first molecule to be localized to the SD.5Ruotsalainen V Ljungberg P Wartiovaara J Lenkkeri U Kestilä M Jalanko H Holmberg C Tryggvason K Nephrin is specifically located at the slit diaphragm of glomerular podocytes.Proc Natl Acad Sci USA. 1999; 96: 7962-7967Crossref PubMed Scopus (600) Google Scholar, 6Holzman LB St. John PL Kovari IA Verma R Holthöfer H Abrahamson DR Nephrin localizes to the slit pore of the glomerular epithelial cell.Kidney Int. 1999; 56: 1481-1491Crossref PubMed Scopus (257) Google Scholar, 7Holthöfer H Ahola H Solin M-L Wang S Palmen T Luimula P Miettinen A Kerjaschki D Nephrin localizes at the podocyte filtration slit area and is characteristically spliced in the human kidney.Am J Pathol. 1999; 155: 1681-1687Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar In the SD, nephrin molecules of adjacent podocytes could possibly interact with each other and, thus contribute to the filter structure.8Tryggvason K Unraveling the mechanisms of glomerular ultrafiltration: nephrin, a key component of the slit diaphragm.J Am Soc Nephrol. 1999; 10: 2440-2445Crossref PubMed Google ScholarCongenital nephrotic syndrome of the Finnish type (CNF or NPHS1, MIM 256300) is a rare, but one of the most severe forms of genetic kidney disorders. Development of the clinical syndrome in CNF is closely correlated with major structural and morphological changes in podocyte foot processes and absence of the SD.3Khoshnoodi J Tryggvason K Congenital nephrotic syndromes.Curr Opin Genet Dev. 2001; 11: 322-327Crossref PubMed Scopus (29) Google Scholar, 9Holmberg C Jalanko H Tryggvason K Rapola J Congenital nephrotic syndrome.in: Barratt TM Avner ED Harmon WE Pediatric Nephrology. ed 4. Lippincott Williams & Wilkins, Baltimore1999: 765-777Google Scholar The nephrin gene (NPHS1), mutated in CNF, encodes a type-1 transmembrane glycoprotein belonging to the immunoglobulin superfamily (IgSF).4Kestilä M Lenkkeri U Männikkö M Lamerdin J McCready P Putaala H Ruotsalainen V Morita T Nissinen M Herva R Kashtan CE Peltonen L Holmberg C Olsen A Tryggvason K Positionally cloned gene for a novel glomerular protein—nephrin—is mutated in congenital nephrotic syndrome.Mol Cell. 1998; 1: 575-582Abstract Full Text Full Text PDF PubMed Scopus (1554) Google Scholar, 10Lenkkeri U Männikkö M McCready P Lamerdin J Gribouval O Niaudet PM Antignac CK Kashtan E Holmberg C Olsen A Kestilä M Tryggvason K Structure of the gene for congenital nephrotic syndrome of the Finnish type (NPHS1) and characterization of mutations.Am J Hum Genet. 1999; 64: 51-61Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar The mature polypeptide consists of 1241 amino acid residues with a calculated molecular mass of 132,532. However, because of posttranslational glycosylation, the mature polypeptide migrates as a 180- to 200-kd protein when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).11Yan K Khoshnoodi J Ruotsalainen V Tryggvason K N-linked glycosylation is critical for the plasma membrane localization of nephrin.J Am Soc Nephrol. 2002; 13: 1385-1389Crossref PubMed Scopus (77) Google Scholar Nephrin consists of eight extracellular immunoglobulin-like domains followed by a fibronectin type III domain, a short transmembrane part, and a cytoplasmic C-terminal domain. In the kidney, nephrin expression is restricted to the glomerular podocytes, and by immunoelectron microscopy nephrin has been localized to the SD area.5Ruotsalainen V Ljungberg P Wartiovaara J Lenkkeri U Kestilä M Jalanko H Holmberg C Tryggvason K Nephrin is specifically located at the slit diaphragm of glomerular podocytes.Proc Natl Acad Sci USA. 1999; 96: 7962-7967Crossref PubMed Scopus (600) Google Scholar, 6Holzman LB St. John PL Kovari IA Verma R Holthöfer H Abrahamson DR Nephrin localizes to the slit pore of the glomerular epithelial cell.Kidney Int. 1999; 56: 1481-1491Crossref PubMed Scopus (257) Google Scholar, 7Holthöfer H Ahola H Solin M-L Wang S Palmen T Luimula P Miettinen A Kerjaschki D Nephrin localizes at the podocyte filtration slit area and is characteristically spliced in the human kidney.Am J Pathol. 1999; 155: 1681-1687Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar The central role of nephrin in the formation and function of the SD has been demonstrated in CNF patients12Ruotsalainen V Patrakka J Tissari P Reponen P Hess M Kestilä M Holmberg C Salonen R Heikinheimo M Wartiovaara J Tryggvason K Jalanko H Role of nephrin in cell junction formation in human nephrogenesis.Am J Pathol. 2000; 157: 1905-1916Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar and in nephrin-deficient mice,13Putaala H Soininen R Kilpeläinen P Wartiovaara J Tryggvason K The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death.Hum Mol Genet. 2001; 10: 1-8Crossref PubMed Scopus (419) Google Scholar as both cases lead to a lack of the SD and massive proteinuria. Nephrotic syndrome has also been reported in mice lacking CD2AP, an adapter actin-binding protein,14Shih N-Y Li J Karpitskii V Nguyen A Dustin ML Kanagawa O Miner JH Shaw AS Congenital nephrotic syndrome in mice lacking CD2-associated protein.Science. 1999; 286: 312-315Crossref PubMed Scopus (693) Google Scholar and in patients with mutations in the NPHS2 gene-encoding podocin,15Boute N Gribouval O Roselli S Benessy F Lee H Fuchshuber A Dahan K Gubler M-C Niaudet P Antignac C NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome.Nat Genet. 2000; 24: 349-354Crossref PubMed Scopus (1186) Google Scholar a new member of the stomatin family with hairpin-like membrane-integrated protein topology. Also, inactivation of the novel SD proteins NEPH1 and FAT results in the development of congenital nephrotic syndrome and neonatal death, respectively.16Mitchell KJ Pinson KI Kelly OG Brennan J Zupicich J Scherz P Leighton PA Goodrich LV Lu X Avery BJ Tate P Dill K Pangilinan E Wakenight P Tessier-Lavigne M Skarnes WC Functional analysis of secreted and transmembrane proteins critical to mouse development.Nat Genet. 2001; 28: 241-249Crossref PubMed Scopus (347) Google Scholar, 17Sellin L Huber TB Gerke P Quack I Pavenstädt H Walz G NEPH1 defines a novel family of podocin-interacting proteins.EMBO J. 2003; 17: 115-117Google ScholarIt has recently been demonstrated that both CD2AP and podocin interact directly with the intracellular C-terminal part of nephrin and, together with nephrin, can be isolated as a raft-associated component of the podocyte SD.18Shih NY Li J Cotran R Mundel P Miner JH Shaw AS CD2AP localizes to the slit diaphragm and binds to nephrin via a novel C-terminal domain.Am J Pathol. 2001; 159: 2303-2308Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 19Saleem MA Ni L Witherden I Tryggvason K Ruotsalainen V Mundel P Mathieson PW Co-localisation of nephrin, podocin and the actin cytoskeleton. Evidence for a role in podocyte foot process formation.Am J Pathol. 2002; 161: 1459-1466Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 20Schwarz K Simons M Reiser J Saleem MA Faul C Kriz W Shaw AS Holzman LB Mundel P Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin.J Clin Invest. 2001; 108: 1621-1629Crossref PubMed Scopus (510) Google Scholar Based on its specific location in the SD and the fact that nephrin is a member of the IgSF, a hypothetical model has been proposed that nephrin molecules emerging from adjacent foot processes interact with each other in a homophilic manner and form the backbone of the SD structure;8Tryggvason K Unraveling the mechanisms of glomerular ultrafiltration: nephrin, a key component of the slit diaphragm.J Am Soc Nephrol. 1999; 10: 2440-2445Crossref PubMed Google Scholar however, evidence for such interactions has, thus far, not been presented.In the present study, we have shown by using a surface plasmon resonance technology that the extracellular part of soluble nephrin exhibits homophilic interactions. We also demonstrated that cells transfected with full-length nephrin cDNA aggregate to form cell-cell junctions with a spacing of similar width as that of native SD. Furthermore, the cellular aggregates can be dissociated with antibodies against the extracellular domain of nephrin. The results suggest that nephrin molecules could also in vivo, contribute to the SD filter structure through homophilic interactions.Materials and MethodsConstruction of cDNA Clones Encoding Human Nephrin VariantsThe construction of the full-length cDNA clone encoding the human nephrin has been reported earlier.21Liu L Cotta Doné S Khoshnoodi J Bertorello A Wartiovaara J Berggren P-O Tryggvason K Defective nephrin trafficking caused by missense mutations in the NPHS1 gene: insight into the mechanisms of congenital nephrotic syndrome.Hum Mol Genet. 2001; 10: 2637-2644Crossref PubMed Scopus (119) Google Scholar For expression of Fc-fused nephrin chimera (NphFc), a cDNA clone encoding the entire extracellular part of human nephrin was cloned into a modified mammalian expression vector pCR-3 (Invitrogen, Carlsbad, CA) with a SalI/NotI cDNA cassette encoding the Fc part (the hinge, CH2, and CH3 domains) of the human IgG1 (the pCR3Fc vector was kindly provided by Dr. Pascal Schneider, Institute of Biochemistry, University of Lausanne, Switzerland). Using polymerase chain reaction (PCR) and 3′-end primer (FNSalI 5′-AGTCAGTCGACCCCCGAGGGTCCT-3′), complementary to the sequence encoding the last four amino acid residues (GPSG) in the fibronectin type III domain of nephrin, followed by a SalI site (underlined), and an upstream primer, UP1 5′-AAGGTTGTGAGTCTGACCCCAC-3′, was used to amplify a short 3′-end fragment. The 3′-end fragment was cleaved with AccI and SalI to generate the AccI/SalI 3′-end fragment. A large EcoRI/AccI fragment corresponding to the middle part of nephrin cDNA together with the HindIII/EcoR1 5′-end fragment (see the full-length construct) and the AccI/SalI 3′-end fragment were ligated into the HindIII/SalI-cleaved pCR3Fc vector to generate a construct (pCR3NphFc) encoding the entire extracellular nephrin followed by the Fc part of human IgG1. Finally, the ligated fragments were checked by sequencing.The cDNA clone encoding the soluble histidine-tagged nephrin (NphHis) was generated from the pCR3NphFc construct. Two synthetic oligonucleotides, SalIHis 5′-T CGACCATCATCACCATCACCATTGA-3′ with cohesive end SalI site (underlined) and a stop codon (italics and underlined), and NotIHis 5′-GGCCTCAATGGTGATGGTGATGATGG-3′ with a cohesive end NotI site (underlined), were used to construct a SalI/6His/NotI-linker fragment with complementary overlapping 3′-ends encoding six histidine residues and 5′-cohesive ends corresponding to SalI and NotI sites, respectively. After annealing, the linker was inserted into the pCR3NphFc construct cleaved with SalI and NotI. The final construct (pCR3NphHis) encoded the entire extracellular portion of nephrin followed by a 6-His tag and a stop codon.Cell Lines, Culturing Conditions, and Generation of Stable Transfected Cell LinesAll cell culture media, supplements (GibcoBRL, Grand Island, NY) and plates/flasks (Nunc, Naperville, IL) were supplied by Life Technologies Inc. The conditionally immortalized mouse podocyte cell line (MCP-5, here termed IMP) was kindly provided by Dr. Peter Mundel (Albert Einstein College of Medicine, Bronx, NY). The cells were maintained and propagated at 33°C in RPMI 1640 medium containing 10 U/ml interferon-γ (Sigma, St. Louis, MO) and 10% fetal bovine serum as described.22Mundel P Reiser J Zuniga Mejia Borja A Pavenstadt H Davidson GR Kriz W Zeller R Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines.Exp Cell Res. 1997; 236: 248-258Crossref PubMed Scopus (759) Google Scholar For differentiation the cells were cultured at 37°C in medium without the interferon-γ. When needed, cells were cultured on type I collagen (collagen-A; Biochrom KG, Berlin, Germany) for differentiation. The human embryonic kidney cell line QBI293A (Qbiogene, Carlsbad, CA), derived from the HEK293 cell line, was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, and 100 mg/ml of streptomycin. Transfections of both cell lines were performed by lipofectin (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer's protocol. Stable clones were selected for 2 weeks in medium containing 1 mg/ml of geneticin and were then cloned at that stage.Expression and Purification of Recombinant ProteinsStable HEK293 cell clones expressing the chimeric NphFc and NphHis recombinant proteins were cultured in triple flasks (Nunc). The cell clone expressing chimeric NphFc was cultured in Dulbecco's modified Eagle's medium containing fetal bovine serum to reduce co-purification of serum IgGs with the recombinant nephrin. After 3 days of confluency, the medium was harvested and phenylmethyl sulfonyl fluoride was added to a concentration of 2 mmol/L before filtration through a 0.22-μm membrane. One liter of the harvested medium was loaded circulating onto a 1-ml HiTrap protein A-Sepharose column (Amersham Biosciences, Uppsala, Sweden) equilibrated with phosphate-buffered saline (PBS) in a cold room overnight. The column was connected to a fast performance liquid chromatography (FPLC) system (ΔKTApurifier system; Amersham Biosciences), washed with 10 ml of PBS and the proteins were eluted with 50 mmol/L of citrate-NaOH, pH 3.0. The eluent was neutralized with 1 mol/L Tris-HCl buffer, pH 8.0, and dialyzed against a 20-mmol/L Tris-HCl buffer, pH 7.5. Proteins were then loaded onto an ion-exchange chromatography column (Mono Q HR 5/5, Amersham Biosciences). The NphFc recombinant protein was eluted from the column with a linear 0- to 1-mol/L NaCl-gradient (20-column volume) in 20 mmol/L of Tris-HCl, pH 7.5, and desalted using Sephadex G-50 NICK columns (Amersham Biosciences) equilibrated with 20 mmol/L of Tris-HCl, pH 7.5.For purification of His-tagged NphHis protein, one liter of the harvested medium was loaded onto a DEAE-Sepharose column using an ΔKTApurifier system and proteins were eluted with 20 mmol/L of Tris-HCl and 1 mol/L of NaCl, pH 7.5. The eluate was loaded onto a Ni-NTA agarose column (Qiagen, Valencia, CA) equilibrated with the washing buffer (20 mmol/L Tris-HCl, 0.5 mol/L NaCl, 10% glycerol, 0.5% Tween 20, pH 8.0). The column was washed with at least 30-column volumes before elution with a stepwise gradient of imidazole 0 to 200 mmol/L in the washing buffer. Collected fractions were analyzed by SDS-PAGE and fractions with the highest amount of NphHis (eluted at 50 to 100 mmol/L of imidazole) were pooled, dialyzed against 20 mmol/L Tris-HCl, pH 7.5, and loaded onto a Mono Q HR 5/5 column. Proteins were eluted with a linear 0- to 1-mol/L NaCl gradient (40-column volume) in 20 mmol/L of Tris-HCl buffer, pH 7.5. The eluate was desalted on Sephadex G-50 NICK columns equilibrated with 20 mmol/L of Tris-HCl, pH 7.5, aliquoted, and frozen at −20°C. The purity of the recombinant proteins was determined by SDS-PAGE and silver staining.Sample Preparation and Western Blot AnalysisCell lysates were prepared by washing the cells twice before addition of hot SDS sample buffer (63 mmol/L Tris-HCl, 2% SDS, 10% glycerol, 0.1 mol/L dithiothreitol, pH 6.8). The cell lysates were collected with a rubber scraper and passed through 26-gauge needles and then transferred into Eppendorf tubes, boiled, and centrifuged for 10 minutes before loading. Human glomeruli were isolated by differential sieving through 400-μm and 200-μm mesh brass sieves and washed extensively with ice-cold PBS including protease inhibitors. Isolated glomeruli were collected by centrifugation at 10,000 × g and resuspended in PBS at a concentration of 20,000 glomeruli per ml. Fractions of the isolated glomeruli were lysed in equal volumes of hot 2× SDS sample buffer and centrifuged to remove unsolubilized material. To remove the carbohydrate moieties on nephrin, a N-glycosidase F deglycosylation kit was used according to the manufacturer protocol (catalog no. 1836552, Roche). All samples were subjected to SDS-PAGE and the proteins were transferred to polyvinylidene difluoride membranes. The membranes were blocked and incubated with affinity-purified pAb1 antibodies (0.2 μg/ml), washed, and incubated with horseradish peroxidase-conjugated goat anti-rabbit antibodies. The immunoreactivity was detected by a chemiluminescent kit (Life Science Products) according to the manufacturer's instructions. The prestained molecular standard used in the SDS-PAGE was from Bio-Rad (catalog number 161-0372; Precision Protein Standards). Protein measurements were performed using a protein assay according to the manufacturer's protocol (Bio-Rad, Hercules, CA).Surface Plasmon ResonanceProtein interaction analysis based on surface plasmon resonance technology was performed using a Biacore 2000 optical biosensor (Biacore AB, Uppsala, Sweden). Proteins were immobilized on carboxymethylated dextran surfaces of research-grade (CM5 sensor chips) using amine-coupling chemistry. Flow cells were activated with a 1:1 mixture of 0.1 mol/L N-hydroxysuccinimide and 0.4 mol/L 3-(N,N-dimethylamino)propyl-N-ethylcarbodiimide at a flow rate of 20 μl/min at 25°C. All proteins were chromatographed on a Sephadex G-25 column equilibrated in running buffer, HBS-P [10 mmol/L HEPES, 0.15 mol/L NaCl, 0.005% surfactant P20 (Biacore), pH 7.4], containing 3.4 mmol/L ethylenediaminetetraacetic acid (EDTA) before use. The ligands, NphHis, NphFc, and human IgG1 (DAKO, Glostrup, Denmark), were diluted in immobilization buffer (10 mmol/L malate buffer, pH 6.0) to a final concentration of 10 μg/ml and immobilized in equal molar ratios, resulting in immobilization densities of 5000:5000:2500 response units for NphHis:NphFc:IgG1, respectively. After immobilization, the surfaces were blocked with 1 mol/L of ethanolamine, pH 8.0, followed by extensive wash with regeneration buffer (HBS-P containing 3.4 mmol/L EDTA and 1 mol/L NaCl). Recorded interactions were performed at 25°C with proteins diluted in HBS-P containing either 3.4 mmol/L EDTA or 1 mmol/L Ca2+. Binding of analytes to the immobilized ligands were measured in resonance units, response unit (1000 response units = 1 ng/mm2 bound protein). Binding data were analyzed as previously described,23Sigmundsson K Másson G Rice R Beauchemin N Öbrink B Determination of active concentrations and association/dissociation rate constants of interacting biomolecules: an analytical solution to the theory for kinetic and mass transport limitations in biosensor technology and its experimental verification.Biochemistry. 2002; 41: 8263-8276Crossref PubMed Scopus (72) Google Scholar using an algorithm for calculation of association and dissociation rate constants that corrected for mass transport effects.Immunofluorescence Staining and Antibody TreatmentRabbit polyclonal antibodies (pAb1) against the two first Ig-like domains (amino acids 22 to 240) of human nephrin have been described.5Ruotsalainen V Ljungberg P Wartiovaara J Lenkkeri U Kestilä M Jalanko H Holmberg C Tryggvason K Nephrin is specifically located at the slit diaphragm of glomerular podocytes.Proc Natl Acad Sci USA. 1999; 96: 7962-7967Crossref PubMed Scopus (600) Google Scholar, 12Ruotsalainen V Patrakka J Tissari P Reponen P Hess M Kestilä M Holmberg C Salonen R Heikinheimo M Wartiovaara J Tryggvason K Jalanko H Role of nephrin in cell junction formation in human nephrogenesis.Am J Pathol. 2000; 157: 1905-1916Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar For immunofluorescence staining, cells were cultured on glass coverslips (coated with type I collagen), then washed with PBS and fixed with 2% formaldehyde and 0.1% glutaraldehyde in PBS for 30 minutes. Cells were then washed twice with PBS blocked with 2% bovine serum albumin and 1% casein hydrolysate in PBS for 1 hour at room temperature. The cells were incubated with affinity-purified rabbit anti-nephrin antibodies pAb1 (10 μg/ml) for 1 hour at room temperature. After three washes, the cells were treated for 30 minutes with a secondary antibody (fluorescein isothiocyanate-conjugated swine anti-rabbit IgG, DAKO). Actin cytoskeleton was stained with rhodamine-phalloidin. The cells were finally washed three times, mounted, and examined by a DMRB Leica microscope and photographed with a digital camera (Hamamatsu C4742-95, Bridgewater, NJ).For experiments with antibody treatment, wild-type HEK293 and IMP cells and cells transfected with full-length nephrin were cultured on glass coverslips coated with type I collagen for 4 to 7 days. Thereafter, the medium was replaced with prewarmed fresh medium containing 1% fetal calf serum and affinity-purified pAb1 in a range of 0 to 40 μg/ml. An equal amount of preimmune rabbit IgG was used in a parallel experiment as negative control. Cells were incubated at 37°C for 1 hour and then either photographed immediately using a phase contrast microscope or fixed and stained for nephrin as described above.Electron Microscopy (EM) and Electron TomographyFor immuno-EM, nephrin-expressing HEK293 cells were fixed with 3.5% paraformaldehyde alone or with different concentrations (0.01 to 0.1%) of glutaraldehyde, processed and immunostained as described.5Ruotsalainen V Ljungberg P Wartiovaara J Lenkkeri U Kestilä M Jalanko H Holmberg C Tryggvason K Nephrin is specifically located at the slit diaphragm of glomerular podocytes.Proc Natl Acad Sci USA. 1999; 96: 7962-7967Crossref PubMed Scopus (600) Google Scholar, 24Skoglund U Öfverstedt L-G Burnet RM Bricogne G Maximum-entropy three-dimensional reconstruction with deconvolution of the contrast transfer function: a test application with adenovirus.J Struct Biol. 1996; 117: 173-188Crossref PubMed Scopus (87) Google Scholar In short, LR-White thin sections on gold-nickel grids were incubated with primary antibodies in blocking solution and washed. After incubation with 5- or 10-nm gold-conjugated secondary antibodies, the sections were poststained in 1% uranyl acetate. Controls included use of nonimmune rabbit IgG as primary antibody. A Jeol 1200 EX electron microscope was used for examining the sections.For electron tomography, the immunoresin sections were prepared as for EM but immuno-marked only with 5-nm-gold and the grids were treated with 10-nm-gold protein A (Amersham Biosciences) for alignment purposes in image reconstruction. The electron tomography was performed essentially as described earlier24Skoglund U Öfverstedt L-G Burnet RM Bricogne G Maximum-entropy three-dimensional reconstruction with deconvolution of the contrast transfer function: a test application with adenovirus.J Struct Biol. 1996; 117: 173-188Crossref PubMed Scopus (87) Google Scholar, 25Miralles F Öfverstedt L-G Sabri N Aissouni Y Hellman U Skoglund U Visa N Electron tomography reveals posttranscriptional binding of pre-mRNPs to specific fibers in the nucleoplasm.J Cell Biol. 2000; 148: 271-282Crossref PubMed Scopus (51) Google Scholar using a Philips CEM 200 FEG transmission electron microscopy. Automatic low-dose tilt series were recorded with a slow-scan camera (2048 × 2048 CCD chip, pixel size 14 μm; TVIPS GmbH, Gauting, Germany) and using the EMMENU software. Images were recorded at 1 or 2° tilt intervals (−65 to + 60°, 26,700×, final pixel size of 5.24 Å). The total dose on the immunoresin sections was below 30 e−/Å.2Rodewald R Karnovsky MJ Porous substructure of the glomerular slit diaphragm in the rat and mouse.J Cell Biol. 1974; 60: 423-433Crossref PubMed Scopus (434) Google Scholar Geometrical image alignment was performed using gold markers (error usually under 1 pixel). Image refinement was done using the COMET technique.24Skoglund U Öfverstedt L-G Burnet RM Bricogne G Maximum-entropy three-dimensional reconstruction with deconvolution of the contrast transfer function: a test application with adenovirus.J Struct Biol. 1996; 117: 173-188Crossref PubMed Scopus (87) Google Scholar The reconstructions were visualized by isodensity contouring as surface rendered or wire-frame representation with the program XTV25Miralles F Öfverstedt L-G Sabri N Aissouni Y Hellman U Skoglund U Visa N Electron tomography reveals posttranscriptional binding of pre-mRNPs to specific fibers in the nucleoplasm.J Cell Biol. 2000; 148: 271-282Crossref PubMed Scopus (51) Google Scholar or by volume rendering with the program BOB (Ken Chin-Purcell, Minnesota Supercomputer Center Inc.).RNA Isolation, Primer Design, and Reverse Transcriptase (RT)-PCRTotal RNA was isolated from wild-type and nephrin-transfected HEK293 cells using an RNeasy kit according to the manufacturer's protocol (Qiagen). Forward and reverse primers were designed to examine the expression levels of human genes encoding the SD components, CD2AP, FAT1
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