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• W2061655823 abstract "The axonal microtubule-associated phosphoprotein tau interacts with neural plasma membrane (PM) components during neuronal development (Brandt, R., Léger, J., and Lee, G. (1995)J. Cell Biol. 131, 1327–1340). To analyze the mechanism and potential regulation of tau's PM association, a method was developed to isolate PM-associated tau using microsphere separation of surface-biotinylated cells. We show that tau's PM association requires an intact membrane cortex and that PM-associated tau and cytosolic tau are differentially phosphorylated at sites detected by several Alzheimer's disease (AD) diagnostic antibodies (Ser199/Ser202, Thr231, and Ser396/Ser404). In polar neurons, the association of endogenous tau phosphoisoforms with the membrane cortex correlates with an enrichment in the axonal compartment. To test for a direct effect of AD-specific tau modifications in determining tau's interactions, a phosphomutant that simulates an AD-like hyperphosphorylation of tau was produced by site-directed mutagenesis of Ser/Thr residues to negatively charged amino acids (Glu). These mutations completely abolish tau's association with the membrane cortex; however, the construct retains its capability to bind to microtubules. The data suggest that a loss of tau's association with the membrane cortex as a result of phosphorylation at sites that are modified during disease contributes to somatodendritic tau accumulation, axonal microtubule disintegration, and neuronal death characteristic for AD. The axonal microtubule-associated phosphoprotein tau interacts with neural plasma membrane (PM) components during neuronal development (Brandt, R., Léger, J., and Lee, G. (1995)J. Cell Biol. 131, 1327–1340). To analyze the mechanism and potential regulation of tau's PM association, a method was developed to isolate PM-associated tau using microsphere separation of surface-biotinylated cells. We show that tau's PM association requires an intact membrane cortex and that PM-associated tau and cytosolic tau are differentially phosphorylated at sites detected by several Alzheimer's disease (AD) diagnostic antibodies (Ser199/Ser202, Thr231, and Ser396/Ser404). In polar neurons, the association of endogenous tau phosphoisoforms with the membrane cortex correlates with an enrichment in the axonal compartment. To test for a direct effect of AD-specific tau modifications in determining tau's interactions, a phosphomutant that simulates an AD-like hyperphosphorylation of tau was produced by site-directed mutagenesis of Ser/Thr residues to negatively charged amino acids (Glu). These mutations completely abolish tau's association with the membrane cortex; however, the construct retains its capability to bind to microtubules. The data suggest that a loss of tau's association with the membrane cortex as a result of phosphorylation at sites that are modified during disease contributes to somatodendritic tau accumulation, axonal microtubule disintegration, and neuronal death characteristic for AD. plasma membrane Alzheimer's disease paired helical filament Dulbecco's modified Eagle's medium phosphate-buffered saline 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein 1,4-piperazinediethanesulfonic acid human transferrin receptor The axonal tau proteins represent a family of closely related low molecular weight phosphoproteins that copolymerize with microtubules and that modulate the dynamic instability of tubulin assembly in cell-free reactions (1.Weingarten M.D. Lockwood A.H. Hwo S.Y. Kirschner M.W. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 1858-1862Crossref PubMed Scopus (2234) Google Scholar, 2.Cleveland D.W. Hwo S.Y. Kirschner M.W. J. Mol. Biol. 1977; 116: 227-247Crossref PubMed Scopus (635) Google Scholar, 3.Drechsel D.N. Hyman A.A. Cobb M.H. Kirschner M.W. Mol. Biol. Cell. 1992; 3: 1141-1154Crossref PubMed Scopus (774) Google Scholar, 4.Trinczek B. Biernat J. Baumann K. Mandelkow E.-M. Mandelkow E. Mol. Biol. Cell. 1995; 6: 1887-1902Crossref PubMed Scopus (264) Google Scholar). When transfected in non-neuronal cells, tau induces microtubule polymerization and bundle formation, which is thought to result from the stabilization of cellular microtubules (5.Kanai Y. Takemura R. Oshima T. Mori H. Ihara Y. Yanagisawa M. Masaki T. Hirokawa N. J. Cell Biol. 1989; 109: 1173-1184Crossref PubMed Scopus (297) Google Scholar,6.Lee G. Rook S.L. J. Cell Sci. 1992; 102: 227-237Crossref PubMed Google Scholar). In cultured neurons, tau is characteristically enriched in the distal axon, which contains the most dynamic and least stable microtubule population (7.Black M.M. Slaughter T. Moshiach S. Obrocka M. Fischer I. J. Neurosci. 1996; 16: 3601-3619Crossref PubMed Google Scholar, 8.Kempf M. Clement A. Faissner A. Lee G. Brandt R. J. Neurosci. 1996; 16: 5583-5592Crossref PubMed Google Scholar, 9.Mandell J.W. Banker G.A. J. Neurosci. 1996; 16: 5727-5740Crossref PubMed Google Scholar). This is opposite to what would be expected for a microtubule-stabilizing protein and may suggest that tau has other or additional functions in neurons that distinguish it from other neuronal microtubule-associated proteins. Previously, we have identified two types of tau interactions in neural cells: binding to microtubules and interaction with plasma membrane (PM)1 components (10.Brandt R. Léger J. Lee G. J. Cell Biol. 1995; 131: 1327-1340Crossref PubMed Scopus (534) Google Scholar). Microtubule binding requires the carboxyl-terminal half of the protein containing tau's microtubule-binding repeat domain, whereas tau's PM association is mediated by its amino-terminal domain, which protrudes from the microtubule surface when tau is bound to microtubules. Thus, these interactions are mediated by distinct domains, raising the possibility that tau functions as a linker protein between the microtubule system and axonal PM components. This idea is consistent with the previous finding that a growth cone- and membrane-associated protein requires tau for its localization (11.DiTella M. Feiguin F. Morfini G. Cáceres A. Cell Motil. Cytoskeleton. 1994; 29: 117-130Crossref PubMed Scopus (66) Google Scholar). In addition, tau close to the plasma membrane would be well positioned to participate in signal transduction mechanisms that may involve the recently identified interaction with Src family non-receptor tyrosine kinases (12.Lee G. Newman S.T. Gard D.L. Band H. Panchamoorthy G. J. Cell Sci. 1998; 111: 3167-3177Crossref PubMed Google Scholar). As a phosphoprotein, tau is a substrate for several kinases and phosphatases in vitro and in cells (for reviews, see Refs.13.Mandelkow E.-M. Schweers O. Drewes G. Biernat J. Gustke N. Trinczek B. Mandelkow E. Ann. N. Y. Acad. Sci. 1996; 777: 96-106Crossref PubMed Scopus (119) Google Scholar and 14.Billingsley M.L. Kincaid R.L. Biochem. J. 1997; 323: 577-591Crossref PubMed Scopus (371) Google Scholar). The stoichiometry of tau phosphorylation is high in fetal brain and substantially decreased in the adult animal. However, tau isolated from patients with Alzheimer's disease (AD) has a high stoichiometry of phosphorylation that resembles tau modification during fetal development more than adult-specific phosphorylation. In AD, tau is redistributed from the axon toward the somatodendritic compartment, where it forms characteristic paired helical filaments (PHFs) (for a review, see Ref. 15.Spillantini M.G. Goedert M. Trends Neurosci. 1998; 21: 428-433Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar). It is thought that an increased phosphorylation state of tau (“hyperphosphorylation”) represents an early and critical event during the development of tau pathology. In vitro, phosphorylation by several kinases, including protein kinase A and the neuronal kinase p110 microtubule affinity-regulating kinase, greatly reduces tau's activity to promote microtubule assembly and increases dynamic instability (4.Trinczek B. Biernat J. Baumann K. Mandelkow E.-M. Mandelkow E. Mol. Biol. Cell. 1995; 6: 1887-1902Crossref PubMed Scopus (264) Google Scholar, 16.Scott C.W. Spreen R.C. Herman J.L. Chow F.P. Davison M.D. Young J. Caputo C.B. J. Biol. Chem. 1993; 268: 1166-1173Abstract Full Text PDF PubMed Google Scholar, 17.Scott C.W. Vulliet P.R. Caputo C.B. Brain Res. 1993; 611: 237-242Crossref PubMed Scopus (35) Google Scholar, 18.Brandt R. Lee G. Teplow D.B. Shalloway D. Abdel-Ghany M. J. Biol. Chem. 1994; 269: 11776-11782Abstract Full Text PDF PubMed Google Scholar). In particular, phosphorylation of Ser262, which is located within tau's repeat domain (numbering refers to the adult human tau isoform containing 441 residues (19.Goedert M. Spillantini M.G. Potier M.C. Ulrich J. Crowther R.A. EMBO J. 1989; 8: 393-399Crossref PubMed Scopus (846) Google Scholar)), abolishes tau's interaction with microtubules and may have a critical role in regulating tau's interaction with microtubules (20.Drewes G. Trinczek B. Illenberger S. Biernat J. Schmitt-Ulms G. Meyer H.E. Mandelkow E.-M. Mandelkow E. J. Biol. Chem. 1995; 270: 7679-7688Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). Several AD diagnostic antibodies such as AT-8, AT-180, and PHF-1 detect epitopes containing phosphorylated Ser-Pro or Thr-Pro motifs, which could point to an involvement of proline-directed kinases, e.g.mitogen-activated protein kinase, glycogen synthase kinase-3β, and Cdk5, in the disease process. These kinases preferentially phosphorylate sites that flank the microtubule-binding domain of tau protein and have only a weak effect on the binding of tau to microtubules (21.Biernat J. Gustke N. Drewes G. Mandelkow E.-M. Mandelkow E. Neuron. 1993; 11: 153-163Abstract Full Text PDF PubMed Scopus (651) Google Scholar, 22.Schneider A. Biernat J. von Bergen M. Mandelkow E. Mandelkow E.-M. Biochemistry. 1999; 38: 3549-3558Crossref PubMed Scopus (450) Google Scholar). It will be important to know how phosphorylation affects tau's non-microtubule interactions and whether proline-directed sites are involved. To analyze the mechanism and potential regulation of tau's PM association, a fast and efficient method to separate PM-associated tau from cytosolic tau using microsphere separation of surface-biotinylated cells was developed. We demonstrate that tau phosphoisoforms are differentially distributed in the PM and cytosolic fractions using phosphorylation-sensitive tau antibodies that are directed against sites modified in PHFs. In addition, we show that tau's PM association requires an intact membrane cortex and is regulated by phosphorylation. In polar neurons, the association of endogenous tau phosphoisoforms with the membrane cortex correlates with an enrichment in the axonal compartment. Phosphomutants that simulate a PHF-like hyperphosphorylation of tau in regions that flank the microtubule-binding domain are defective in their association with the membrane cortex, but can still bind to microtubules. Chemicals were purchased from Sigma (Deisenhofen, Germany), cell culture media and supplements from Life Technologies, Inc., tissue culture flasks and dishes from Nunc (Roskilde, Denmark), and restriction enzymes and T4 DNA ligase from MBI Fermentas (St. Leon-Rot, Germany), unless stated otherwise. Tubulin was isolated from bovine brain by two assembly-disassembly cycles and phosphocellulose chromatography as described previously (23.Brandt R. Lee G. J. Biol. Chem. 1993; 268: 3414-3419Abstract Full Text PDF PubMed Google Scholar). PC12 cells were cultured in serum/DMEM (DMEM supplemented with 10% fetal bovine serum, 5% horse serum, 292 μg/ml glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin) and transfected using Lipofectin as described previously (10.Brandt R. Léger J. Lee G. J. Cell Biol. 1995; 131: 1327-1340Crossref PubMed Scopus (534) Google Scholar). For generation of stable lines, cells were selected with Geneticin, and individual clones were isolated as described previously (10.Brandt R. Léger J. Lee G. J. Cell Biol. 1995; 131: 1327-1340Crossref PubMed Scopus (534) Google Scholar). Stable lines were cultured in serum/DMEM supplemented with 250 μg/ml Geneticin. For immunocytochemistry, PC12 cells were grown on polylysine-coated glass coverslips. NT2 cells were grown, differentiated for 5 weeks with retinoic acid, replated, and cultured for another 2 weeks in the presence of mitotic inhibitors to yield terminally differentiated neurons (NT2-N cells) as described previously (24.Piontek J. Chen C.C. Kempf M. Brandt R. J. Neurochem. 1999; 73: 139-146Crossref PubMed Scopus (25) Google Scholar). For immunocytochemistry, the neurons were plated at 2000 cells/cm2 onto Matrigel (Becton Dickinson, Bedford, MA)-coated coverslips and cultured for another 3 days in serum-containing medium. Following this treatment, almost all neurons had established polarity as judged from axon-specific tau staining. Cells were fixed with 4% (w/v) paraformaldehyde in PBS (10 mm phosphate buffer, pH 7.4, 2.7 mm KCl, and 137 mm NaCl) containing 4% (w/v) sucrose for 20 min at room temperature. After washing with PBS, cells were incubated with 0.1m glycine in PBS for 20 min and permeabilized for 5 min in 0.2% (v/v) Triton X-100 in PBS. For detergent extraction, a combined Nonidet P-40 extraction-fixation procedure as described previously (6.Lee G. Rook S.L. J. Cell Sci. 1992; 102: 227-237Crossref PubMed Google Scholar) was used. Staining was performed essentially as described earlier (6.Lee G. Rook S.L. J. Cell Sci. 1992; 102: 227-237Crossref PubMed Google Scholar) in PBS containing 1% (w/v) bovine serum albumin using monoclonal mouse antibodies against tau (Tau-1 (25.Binder L.I. Frankfurter A. Rebhun L.I. J. Cell Biol. 1985; 101: 1371-1378Crossref PubMed Scopus (1249) Google Scholar) and PHF-1 (26.Greenberg S.G. Davies P. Schein J.D. Binder L.I. J. Biol. Chem. 1992; 267: 564-569Abstract Full Text PDF PubMed Google Scholar)) and FLAG (M5, Eastman Kodak Co.), monoclonal rat antibody against tubulin (YL1/2 (27.Kilmartin J.V. Wright B. Milstein C. J. Cell Biol. 1982; 93: 576-582Crossref PubMed Scopus (639) Google Scholar)), and fluorescein isothiocyanate-coupled donkey anti-rat and Cy3-coupled donkey anti-mouse antibodies (all secondary antibodies from Dianova, Hamburg, Germany). For total protein staining, cells were washed after the secondary antibody reaction five times for 2 min each with PBS, incubated for 15 min with 10 μg/ml 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein (DTAF), and washed five times for 2 min each with PBS. Coverslips were mounted in anti-fade medium as described previously (10.Brandt R. Léger J. Lee G. J. Cell Biol. 1995; 131: 1327-1340Crossref PubMed Scopus (534) Google Scholar). Tau-transfected PC12 cells were grown to ∼80% confluency on a collagen-coated 10-cm tissue culture dish; washed once with prewarmed PBS; incubated with prewarmed PBS containing 2 mm MgCl2 and 0.5 mmCaCl2 for 10 min at room temperature; transferred to 4 °C, and incubated for another 10 min. Cells were surface-labeled with 0.1 mg/ml sulfosuccinimidyl 2-(biotinamido)ethyl-1,3′-dithiopropionate (Pierce) in PBS containing 2 mm MgCl2 and 0.5 mmCaCl2 for 30 min; quenched for 20 min with PBS containing 2 mm MgCl2, 0.5 mm CaCl2, and 50 mm glycine; and washed once with 0.25 msucrose, all at 4 °C. Streptavidin-coupled microspheres (∼1 μm in diameter) were washed once with PBS and twice with 0.25m sucrose using a magnetic bead attractor (Stratagene, Heidelberg, Germany) and added onto the cells, and incubation was continued for 1 h at 4 °C. Cells were washed with 0.25m sucrose, and 2.4 ml of separation buffer (0.25m sucrose containing 1 mm ATP, 1 mmMgCl2, 1 mm EGTA, and protease and phosphatase inhibitors (1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 1 mm sodium pyrophosphate, 1 mm sodium orthovanadate, and 20 mm NaF)) were added on ice. Then, the cells were scraped off, distributed into 1.5-ml Sarstedt tubes, and quick-frozen in liquid nitrogen. After thawing on ice, tubes were transferred into a magnetic bead attractor, and the unbound fraction (cytosol/crude organelles) was removed. The bound fraction (PM) was washed three times with 500 μl of separation buffer/tube. The S–S bridge of the biotin derivative was cleaved by incubation with 750 μl of separation buffer containing 0.1m 2-mercaptoethanol. The suspension was pressed five times through a 27-gauge needle, incubated for 30 min at 4 °C with vortexing, and separated using the bead attractor. The cleavage procedure was repeated once; the supernatants were pooled; and the PM was collected by ultracentrifugation (100,000 × g, 1 h, 4 °C). For comparative analysis, the unbound fraction was separated by ultracentrifugation (100,000 × g, 1 h, 4 °C) into a crude organelle pellet and the cytosol. The pellet fractions were resuspended in 100 μl of separation buffer. The cytosolic fraction was precipitated with trichloroacetic acid/deoxycholate as described previously (28.Bensadoun A. Weinstein D. Anal. Biochem. 1976; 70: 241-250Crossref PubMed Scopus (2740) Google Scholar). NT2-N cells were cultured on collagen-coated 6-cm tissue culture dishes, and all buffer volumes were adjusted according to the smaller area of the dishes. For separation under actin-stabilizing conditions, cells were cultured for 1 h in serum/DMEM containing 0.1 μmjasplakinolide (Molecular Probes, Inc., Leiden, Netherlands). The wash at room temperature was performed in the presence of 0.1 μm jasplakinolide, and all subsequent steps in separation buffer contained 5 μm phalloidin (AppliChem, Darmstadt, Germany). For separation under actin-depolymerizing conditions, cells were cultured for 1 h in serum/DMEM containing 0.35 μg/ml latrunculin B (Calbiochem-Novabiochem GmbH, Bad Soden, Germany). The wash at room temperature was performed in the presence of 0.35 μg/ml latrunculin B, and all subsequent steps in separation buffer contained 0.35 μg/ml latrunculin B in the absence of ATP, MgCl2, and EGTA. For separation after phosphatase treatment, cells were cultured for 1 h in serum/DMEM containing 10 or 100 nm okadaic acid. The wash at room temperature was performed in the presence of 10 or 100 nm okadaic acid, and all subsequent steps were carried out under standard conditions. Isolated plasma membranes were fixed for 3 h with 2% (v/v) paraformaldehyde and 2.5% (v/v) glutaraldehyde in 0.1 m sodium cacodylate buffer, pH 7.4, and then post-fixed for 1 h with 1% (w/v) OsO4(Herth, München, Germany) and 1.5% (w/v) K3(Fe(CN)6) (Merck, Darmstadt) in 0.1m sodium cacodylate buffer, pH 7.4. The membranes were washed with water, en bloc stained for 1 h with 1% (w/v) uranyl acetate (Merck), washed with water, and then dehydrated with graded ethanol followed by propylene oxide. Membranes were embedded in 1,2,3-propanetriol glycidyl ether (Serva, Heidelberg) by conventional methods, sectioned, and contrasted consecutively with uranyl acetate and lead citrate. Electron microscopy was performed on a Zeiss 10CR electron microscope. For co-sedimentation assays, Taxol-stabilized microtubules were prepared by polymerizing 30 μm purified tubulin in BRB80 (80 mmPipes/KOH, pH 6.8, 1 mm CaCl2, and 1 mm EGTA) containing 1 mm GTP by stepwise addition of 0.01 volume of 10 μm, 100 μm, and 1 mm Taxol for 5 min at 37 °C each. Tau-transfected PC12 cells were collected by centrifugation and washed once with PBS. Rat tau (2 × 106) or human tau (5 × 106) cells were suspended in 50 μl of PBS containing protease and phosphatase inhibitors, quick-frozen in liquid nitrogen, thawed on ice, and centrifuged at 100,000 × g for 1 h at 4 °C. The supernatant was adjusted to 1 mmGTP and 10 μm Taxol in a total volume of 70 μl with BRB80, prewarmed at 37 °C, and divided. One-half was mixed with 15 μl of Taxol-stabilized microtubules, and the other half was mixed with 15 μl of BRB80 containing 10 μm Taxol. After incubation for 10 min at 37 °C, the mixtures were centrifuged through 100 μl of 30% (w/v) sucrose cushions in BRB80 containing 1 mm GTP and 10 μm Taxol at 100,000 × g for 1 h at 20 °C. The pellets were resuspended in 150 μl of BRB80. 1% (v/v) (anti-tubulin antibody) or 89% (v/v) (anti-tau antibodies) of each fraction was separated by SDS-polyacrylamide gel electrophoresis with 10% acrylamide and analyzed by immunoblotting. SDS-polyacrylamide gel electrophoresis and immunoblotting were performed as described previously (23.Brandt R. Lee G. J. Biol. Chem. 1993; 268: 3414-3419Abstract Full Text PDF PubMed Google Scholar) using rainbow-colored protein molecular weight markers (high molecular weight range; Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). For dot-blot analysis, samples were diluted with 500 μl of dot-blot buffer (10 mm Tris-HCl, 0.9% (w/v) NaCl, 30 (w/v) methanol, and 0.5% (w/v) deoxycholate, pH 7.4) and immobilized on Immobilon NC Pure (Millipore Corp., Bedford, MA) using a dot-blot hybridization chamber (Loxo, Dossenheim, Germany). Detection was with monoclonal anti-tau (Tau-1, Tau-5 (Pharmingen, San Diego, CA), AT-8 and AT-180 (IC Chemikalien, Ismaning, Germany), and PHF-1), anti-FLAG (M2, Eastman Kodak Co.), anti-tubulin (DM1A) and anti-actin (IgM) (Amersham Pharmacia Biotech), human transferrin receptor (H68.4; WAK-Chemie, Bad Soden), and horseradish peroxidase-coupled goat anti-mouse (Dianova) antibodies. Detection and development using enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech) were performed as described previously (29.Ilschner S. Brandt R. Glia. 1996; 18: 129-140Crossref PubMed Scopus (43) Google Scholar). For lectin blot analysis, membranes were blocked for 1 h with 3% bovine serum albumin in TTBS (20% Tween 20, 50 mm Tris-HCl, pH 8.0, and 0.5 m NaCl); incubated for 1 h with horseradish peroxidase-coupled concanavalin A in 0.3% bovine serum albumin/TTBS; washed three times for 10 min each with TTBS; rinsed in 50 mm Tris-HCl, pH 8.0, and 0.5 m NaCl; washed with 9 g/liter NaCl, 1.21 g/liter Tris-HCl, pH 7.4, and 0.05% Tween 20; and developed using ECL. Quantitation was carried out with an Arcus II scanner (Agfa-Gevaert) and the program NIH Image 1.61/ppc. Confocal image analysis was performed with a Leica TCS 4 D True confocal scanner equipped with an argon/krypton laser as described previously (8.Kempf M. Clement A. Faissner A. Lee G. Brandt R. J. Neurosci. 1996; 16: 5583-5592Crossref PubMed Google Scholar). For determination of peripheral enrichment in tau-transfected PC12 cells, single optical sections were recorded from individual cells at 50% of their height (10–15 μm). The distribution of label in cellular compartments (axon and somatodendritic compartment) or in individual processes of human model neurons was determined as described previously (30.Kwei S.L. Clement A. Faissner A. Brandt R. Neuroreport. 1998; 9: 1035-1040Crossref PubMed Scopus (18) Google Scholar). For each condition, 10 representative neurons with no contact with other cells were evaluated, and the means ± S.E. were calculated. To compare processes with different lengths, fluorescence intensities were normalized to a maximum total fluorescence intensity of 1.0 of the respective label and plotted against the relative position in the axon (maximum length set as 1.0). Eukaryotic expression plasmids for human tau were constructed in fpRc/CMV as described previously (31.Léger J. Kempf M. Lee G. Brandt R. J. Biol. Chem. 1997; 272: 8441-8446Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The vector fpRc/CMV had been modified from pRc/CMV (Invitrogen, San Diego, CA) to express proteins with the sequence MDKDDDDK (FLAG) (32.Hopp T.P. Prickett K.S. Price V.L. Libby R.T. March C.J. Cerretti D.P. Urdal D.L. Conlon P.J. Bio/Technology. 1988; 6: 1204-1210Crossref Scopus (754) Google Scholar, 33.Prickett K.S. Amberg D.C. Hopp T. BioTechniques. 1989; 7: 580-589PubMed Google Scholar) fused to the amino-terminal end as an epitope tag. To construct a PHF-like tau mutant, codons for Ser198, Ser199, Ser202, Thr231, Ser235, Ser396, Ser404, Ser409, Ser413, and Ser422 were mutated to GAA (glutamate) by polymerase chain reaction or site-directed mutagenesis (Muta-GenTM phagemidin vitro mutagenesis kit, Bio-Rad). Constructs were verified by dideoxy sequencing using T7 sequenase (Amersham Pharmacia Biotech). Protein concentrations were determined by the method of Bradford (34.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) using bovine serum albumin as a standard. Alkaline phosphodiesterase-1 assays were essentially performed as described previously (35.Storrie B. Madden E.A. Methods Enzymol. 1990; 182: 203-225Crossref PubMed Scopus (497) Google Scholar). 25-μl samples were diluted with 25 μl of 0.25 m sucrose in a chilled 1.5-ml Sarstedt tube. 200 μl of reaction mixture (2.5 mm sodium thymidine 5′-monophosphate p-nitrophenyl ester and 250 mmTris-HCl, pH 9.0) were added, vortexed, and incubated at 37 °C. As soon as a color reaction was observed (from 2 h to overnight), 500 μl of 0.5 m glycine and 0.5 m sodium carbonate were added, and the absorbance was measured against a blank (50 μl of 0.25 m sucrose instead of sample) at 410 nm. Lactate dehydrogenase assays were essentially performed as described previously (35.Storrie B. Madden E.A. Methods Enzymol. 1990; 182: 203-225Crossref PubMed Scopus (497) Google Scholar). The samples were adjusted with 0.25 msucrose to 100 μl. 900 μl of reaction mixture (0.2 mmβ-NADH, 1 mm sodium pyruvate, and 0.1% (v/v) Triton X-100 in 0.2 m Tris-HCl, pH 7.3) were added, vortexed, and incubated at room temperature. Lactate dehydrogenase activity was read as a decrease in absorption at 340 nm measured against a blank (100 μl of 0.25 m sucrose instead of sample). Prior to the assay, the cytosolic fraction was concentrated by dialysis against 0.2m imidazole-HCl, pH 7.2, and lyophilization. In PC12 cells stably transfected with adult rat tau cDNA, tau was clearly enriched at the cellular periphery as judged by laser scanning microscopy of cells that had been processed for immunocytochemistry (Fig.1 A). The distribution of tau did not reflect the total protein distribution in the cells, which was, as determined from staining with the general protein stain DTAF, more concentrated at the center of the cell. No tau staining was observed in unpermeabilized cells, indicating that tau interacts with the cytosolic face of integral PM components or components of the membrane cortex. To analyze the mechanism and potential regulation of tau's association with the plasma membrane, a method to isolate a PM fraction including the submembranous cortex from cultured cells was developed. The method is based on microsphere separation of surface-biotinylated cells and yielded three fractions: a PM fraction that was highly enriched in plasma membrane components, a crude organelle fraction that contained all types of membranous organelles together with residual plasma membrane that did not bind to the beads, and a cytosolic fraction representing the supernatant of a high speed centrifugation of the unbound material (Fig. 1 B). The PM fraction contained ∼60% of high mannose-type glycoproteins frequently found in plasma membranes (36.Carraway C.A.C. Carraway K.L. Carraway C.A.C. The Cytoskeleton: A Practical Approach. IRL Press Ltd., Oxford1992: 123-150Google Scholar) as indicated by a concanavalin A dot-blot (Fig.1 C). About 20% of the total protein and ∼60% of the specific activity of the PM enzyme alkaline phosphodiesterase-1 were present in this fraction (Table I). Immunoblot analysis showed the presence of the transferrin receptor that is abundantly expressed on the cell surface of many cultured cell lines (37.White S. Miller K. Hopkins C. Trowbridge I.S. Biochem. Biophys. Acta. 1992; 1136: 28-34Crossref PubMed Scopus (59) Google Scholar) and actin (25–40% for independent isolations), as would be expected for a fraction containing plasma membranes and a submembranous cortex (Fig. 1 C). Tubulin, which behaves as a cytosolic protein under the conditions used for the fractionation (isolation at 4 °C), was almost completely absent from the PM fraction (<2% of total tubulin), indicating a very low degree of contamination with cytosolic proteins. In agreement with the latter result, the specific activity of the cytosolic enzyme lactate dehydrogenase was very low in the PM fraction (∼1%) (Table I). Electron microscopy of the isolated PM fraction revealed the presence of a large number of apparently empty vesicles with an irregular shape and a diameter ranging between 50 and 700 nm, probably representing plasma membrane vesicles (Fig.1 D).Table IDistribution of protein and alkaline phosphodiesterase-1 and lactate dehydrogenase activities in isolated fractions after microsphere separation of surface-biotinylated cellsFractionProteinaMean ± S.D. (n = 5).APDE-1 activitybMean ± S.D. (n = 3).LDH activitycMean ± range (n = 2).Cytosol60.5 ± 17.17.1 ± 5.797.0 ± 2.1Crude organelles18.6 ± 9.831.4 ± 5.31.7 ± 1.4Plasma membrane21.4 ± 9.261.5 ± 2.51.3 ± 0.7Surface biotinylation, microsphere separation, protein determination, and alkaline phosphodiesterase-1 and (APDE-1) and lactate dehydrogenase (LDH) assays were performed as described under “Experimental Procedures” with PC12 cells stably expressing rat tau. The amount of protein and specific activities in percent of the total in all fractions are given.a Mean ± S.D. (n = 5).b Mean ± S.D. (n = 3).c Mean ± range (n = 2). Open table in a new tab Surface biotinylation, microsphere separation, protein determination, and alkaline phosphodiesterase-1 and (APDE-1) and lactate dehydrogenase (LDH) assays were performed as described under “Experimental Procedures” with PC12 cells stably expressing rat tau. The amount of protein and specific activities in percent of the total in all fractions are given. About 20% of tau was present in the PM fraction as judged by i" @default.
• W2061655823 created "2016-06-24" @default.
• W2061655823 creator A5023704791 @default.
• W2061655823 creator A5037778101 @default.
• W2061655823 creator A5070946569 @default.
• W2061655823 date "2000-05-01" @default.
• W2061655823 modified "2023-09-27" @default.
• W2061655823 title "Interaction of Tau with the Neural Membrane Cortex Is Regulated by Phosphorylation at Sites That Are Modified in Paired Helical Filaments" @default.
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