FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2090775206> ?p ?o ?g. }

• W2090775206 endingPage "25397" @default.
• W2090775206 startingPage "25388" @default.
• W2090775206 abstract "Phosphorylation of Tau protein and binding to microtubules is complex in neurons and was therefore studied in the less complicated model of humanized yeast. Human Tau was readily phosphorylated at pathological epitopes, but in opposite directions regulated by kinases Mds1 and Pho85, orthologues of glycogen synthase kinase-3β and cdk5, respectively (1Vandebroek T. Vanhelmont T. Terwel D. Borghgraef P. Lemaire K. Snauwaert J. Wera S. Van Leuven F. Winderickx J. Biochemistry. 2005; 44: 11466-11475Crossref PubMed Scopus (57) Google Scholar). We isolated recombinant Tau-4R and mutant Tau-P301L from wild type, Δmds1 and Δpho85 yeast strains and measured binding to Taxol-stabilized mammalian microtubules in relation to their phosphorylation patterns. Tau-4R isolated from yeast lacking mds1 was less phosphorylated and bound more to microtubules than Tau-4R isolated from wild type yeast. Paradoxically, phosphorylation of Tau-4R isolated from kinase Pho85-deficient yeast was dramatically increased resulting in very poor binding to microtubules. Dephosphorylation promoted binding to microtubules to uniform high levels, excluding other modifications. Isolated hyperphosphorylated, conformationally altered Tau-4R completely failed to bind microtubules. In parallel to Tau-4R, we expressed, isolated, and analyzed mutant Tau-P301L. Total dephosphorylated Tau-4R and Tau-P301L bound to microtubules very similarly. Surprisingly, Tau-P301L isolated from all yeast strains bound to microtubules more extensively than Tau-4R. Atomic force microscopy demonstrated, however, that the high apparent binding of Tau-P301L was due to aggregation on the microtubules, causing their deformation and bundling. Our data explain the pathological presence of granular Tau aggregates in neuronal processes in tauopathies. Phosphorylation of Tau protein and binding to microtubules is complex in neurons and was therefore studied in the less complicated model of humanized yeast. Human Tau was readily phosphorylated at pathological epitopes, but in opposite directions regulated by kinases Mds1 and Pho85, orthologues of glycogen synthase kinase-3β and cdk5, respectively (1Vandebroek T. Vanhelmont T. Terwel D. Borghgraef P. Lemaire K. Snauwaert J. Wera S. Van Leuven F. Winderickx J. Biochemistry. 2005; 44: 11466-11475Crossref PubMed Scopus (57) Google Scholar). We isolated recombinant Tau-4R and mutant Tau-P301L from wild type, Δmds1 and Δpho85 yeast strains and measured binding to Taxol-stabilized mammalian microtubules in relation to their phosphorylation patterns. Tau-4R isolated from yeast lacking mds1 was less phosphorylated and bound more to microtubules than Tau-4R isolated from wild type yeast. Paradoxically, phosphorylation of Tau-4R isolated from kinase Pho85-deficient yeast was dramatically increased resulting in very poor binding to microtubules. Dephosphorylation promoted binding to microtubules to uniform high levels, excluding other modifications. Isolated hyperphosphorylated, conformationally altered Tau-4R completely failed to bind microtubules. In parallel to Tau-4R, we expressed, isolated, and analyzed mutant Tau-P301L. Total dephosphorylated Tau-4R and Tau-P301L bound to microtubules very similarly. Surprisingly, Tau-P301L isolated from all yeast strains bound to microtubules more extensively than Tau-4R. Atomic force microscopy demonstrated, however, that the high apparent binding of Tau-P301L was due to aggregation on the microtubules, causing their deformation and bundling. Our data explain the pathological presence of granular Tau aggregates in neuronal processes in tauopathies. Transport along microtubules (MT) 3The abbreviations used are: MT, microtubules; MAP, microtubule-associated protein; AD, Alzheimer disease; AFM, atomic force microscopy; mAb, monoclonal antibody; GSK-3β, glycogen synthase kinase-3β; MES, 4-morpholineethanesulfonic acid; Pipes, 1,4-piperazinediethanesulfonic acid.3The abbreviations used are: MT, microtubules; MAP, microtubule-associated protein; AD, Alzheimer disease; AFM, atomic force microscopy; mAb, monoclonal antibody; GSK-3β, glycogen synthase kinase-3β; MES, 4-morpholineethanesulfonic acid; Pipes, 1,4-piperazinediethanesulfonic acid. is essential for normal neuronal functions by providing synapses with necessary protein complexes, vesicles, and organelles. MT-associated proteins (MAP) regulate the structural and functional organization of the MT network in relation to the rest of the cytoskeleton, to allow axonal transport. Tau protein is an important active MAP, as negatively exemplified by the diverse group of neurodegenerative diseases termed tauopathies, including frontotemporal dementia and Alzheimer disease (AD) (for reviews see Refs. 2Hirokawa N. Takemura R. Nat. Rev. Neurosci. 2005; 6: 201-214Crossref PubMed Scopus (646) Google Scholar, 3Heutink P. Hum. Mol. Genet. 2000; 9: 979-986Crossref PubMed Scopus (98) Google Scholar, 4Ingram E.M. Spillantini M.G. Trends Mol. Med. 2002; 8: 555-562Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 5Terwel D. Dewachter I. Van Leuven F. Neuromol. Med. 2002; 2: 151-165Crossref PubMed Scopus (128) Google Scholar, 6Geschwind D.H. Neuron. 2003; 40: 457-460Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 7Drewes G. Trends Biochem. Sci. 2004; 29: 548-555Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 8Stoothoff W.H. Johnson G.V. Biochim. Biophys. Acta. 2005; 1739: 280-297Crossref PubMed Scopus (348) Google Scholar, 9Baas P.W. Qiang L. Trends Cell Biol. 2005; 15: 183-187Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 10Iqbal K. Grundke-Iqbal I. Acta Neuropathol (Berl.). 2005; 109: 25-31Crossref PubMed Scopus (85) Google Scholar, 11Dehmelt L. Halpain S. Genome Biol. 2005; 6: 204Crossref PubMed Scopus (543) Google Scholar, 12D'Souza I. Schellenberg G.D. J. Biol. Chem. 2006; 281: 2460-2469Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). In all tauopathies, hyperphosphorylation and aggregation of Tau protein appear invariably associated. Whereas it can be assumed that in normal conditions all Tau protein is bound to MT, it is not known whether detachment of Tau protein from MT is a prerequisite for phosphorylation to take place. Whereas Tau-MT binding depends critically on phosphorylation, the complexity and variability of the phosphorylation pattern of neuronal Tau precludes precise definition of the relative or absolute importance of individual phosphorylation sites. We have generated humanized yeast strains by expressing human Tau protein, different isoforms and mutants, to develop a less complex, eukaryotic cell-based model (1Vandebroek T. Vanhelmont T. Terwel D. Borghgraef P. Lemaire K. Snauwaert J. Wera S. Van Leuven F. Winderickx J. Biochemistry. 2005; 44: 11466-11475Crossref PubMed Scopus (57) Google Scholar). Phosphorylation of human Tau at specific residues, known to be pathological in the Tau aggregates in clinical tauopathies, was already important in normal yeast cultures probably due to the fact that human Tau did not bind appreciably to yeast MT. Significantly, phosphorylation was regulated in opposite directions by kinases Mds1 and Pho85, the functional homologues of GSK-3β and cdk5, respectively (1Vandebroek T. Vanhelmont T. Terwel D. Borghgraef P. Lemaire K. Snauwaert J. Wera S. Van Leuven F. Winderickx J. Biochemistry. 2005; 44: 11466-11475Crossref PubMed Scopus (57) Google Scholar). Thereby this model recapitulated our observations in vivo in brain of transgenic mice expressing human Tau protein and Tau kinases GSK-3β and cdk5/p35 (5Terwel D. Dewachter I. Van Leuven F. Neuromol. Med. 2002; 2: 151-165Crossref PubMed Scopus (128) Google Scholar, 13Spittaels K. Van den Haute C. Van Dorpe J. Bruynseels K. Vandezande K. Laenen I. Geerts H. Mercken M. Sciot R. Van Lommel A. Loos R. Van Leuven F. Am. J. Pathol. 1999; 155: 2153-2165Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 14Spittaels K. Van den Haute C. Van Dorpe J. Geerts H. Mercken M. Bruynseels K. Lasrado R. Vandezande K. Laenen I. Boon T. Van Lint J. Vandenheede J. Moechars D. Loos R. Van Leuven F. J. Biol. Chem. 2000; 275: 41340-41349Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 15Van den Haute C. Spittaels K. Van Dorpe J. Lasrado R. Vandezande K. Laenen I. Geerts H. Van Leuven F. Neurobiol. Dis. 2001; 8: 32-44Crossref PubMed Scopus (69) Google Scholar, 16Terwel D. Lasrado R. Snauwaert J. Vandeweert E. Van Haesendonck C. Borghgraef P. Van Leuven F. J. Biol. Chem. 2005; 280: 3963-3973Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 17Boekhoorn K. Terwel D. Biemans B. Borghgraef P. Wiegert O. Ramakers G. de Vos K. Krugers H. Tomiyama T. Mori H. Joels M. Van Leuven F. Lucassen P. J. Neurosci. 2006; 26: 3514-3523Crossref PubMed Scopus (132) Google Scholar, 18Hallows J.L. Chen K. DePinho R.A. Vincent I. J. Neurosci. 2003; 23: 10633-10644Crossref PubMed Google Scholar). Overexpression of wild type Tau-4R in neurons of transgenic mice triggered a severe axonopathy due to excessive binding of Tau-4R to MT. The resulting blockage of bidirectional axonal transport in turn caused axonopathy with severe wallerian degeneration and motor defects (13Spittaels K. Van den Haute C. Van Dorpe J. Bruynseels K. Vandezande K. Laenen I. Geerts H. Mercken M. Sciot R. Van Lommel A. Loos R. Van Leuven F. Am. J. Pathol. 1999; 155: 2153-2165Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). GSK-3β, co-expressed with Tau-4R rescued the axonopathy as well as the motor impairment completely (14Spittaels K. Van den Haute C. Van Dorpe J. Geerts H. Mercken M. Bruynseels K. Lasrado R. Vandezande K. Laenen I. Boon T. Van Lint J. Vandenheede J. Moechars D. Loos R. Van Leuven F. J. Biol. Chem. 2000; 275: 41340-41349Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar), illustrating the importance of phosphorylation of Tau-4R by GSK-3β for MT binding in vivo. The other major Tau kinase cdk5 was, however, not capable of phosphorylating Tau-4R or rescue the axonopathy, not even when co-expressed with its neuron-specific activator p35 in triple transgenic mice (15Van den Haute C. Spittaels K. Van Dorpe J. Lasrado R. Vandezande K. Laenen I. Geerts H. Van Leuven F. Neurobiol. Dis. 2001; 8: 32-44Crossref PubMed Scopus (69) Google Scholar). This indication that cdk5 acted unlike GSK-3β, and actually completely opposite was underlined by the increased phosphorylation of cytoskeletal proteins in brain of p35–/– mice (18Hallows J.L. Chen K. DePinho R.A. Vincent I. J. Neurosci. 2003; 23: 10633-10644Crossref PubMed Google Scholar), and joins seamlessly with our observations in yeast (1Vandebroek T. Vanhelmont T. Terwel D. Borghgraef P. Lemaire K. Snauwaert J. Wera S. Van Leuven F. Winderickx J. Biochemistry. 2005; 44: 11466-11475Crossref PubMed Scopus (57) Google Scholar). Mutations in the microtubule-associated protein Tau gene coding for Tau protein on chromosome 17 cause a varied group of dominantly inherited diseases, collectively known as frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). Both exonic and intronic mutations are known, giving rise to either mutant Tau protein or to augmented Tau-4R isoform expression, respectively, which apparently shares the propensity to become phosphorylated and aggregate into filaments and tangles, the common pathological defect in all tauopathies (for reviews see Refs. 3Heutink P. Hum. Mol. Genet. 2000; 9: 979-986Crossref PubMed Scopus (98) Google Scholar, 4Ingram E.M. Spillantini M.G. Trends Mol. Med. 2002; 8: 555-562Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 5Terwel D. Dewachter I. Van Leuven F. Neuromol. Med. 2002; 2: 151-165Crossref PubMed Scopus (128) Google Scholar, 6Geschwind D.H. Neuron. 2003; 40: 457-460Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 7Drewes G. Trends Biochem. Sci. 2004; 29: 548-555Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 8Stoothoff W.H. Johnson G.V. Biochim. Biophys. Acta. 2005; 1739: 280-297Crossref PubMed Scopus (348) Google Scholar, 9Baas P.W. Qiang L. Trends Cell Biol. 2005; 15: 183-187Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 10Iqbal K. Grundke-Iqbal I. Acta Neuropathol (Berl.). 2005; 109: 25-31Crossref PubMed Scopus (85) Google Scholar, 11Dehmelt L. Halpain S. Genome Biol. 2005; 6: 204Crossref PubMed Scopus (543) Google Scholar, 12D'Souza I. Schellenberg G.D. J. Biol. Chem. 2006; 281: 2460-2469Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Conflicting information concerns the influence of expressed mutations in Tau protein on its binding to MT and self-assembly (19Hasegawa M. Smith M.J. Goedert M. FEBS Lett. 1998; 437: 207-210Crossref PubMed Scopus (412) Google Scholar, 20Arrasate M. Perez M. Armas-Portela R. Avila J. FEBS Lett. 1999; 446: 199-202Crossref PubMed Scopus (101) Google Scholar, 21Goedert M. Jakes R. Crowther R.A. FEBS Lett. 1999; 450: 306-311Crossref PubMed Scopus (215) Google Scholar, 22Barghorn S. Zheng-Fischhöfer Q. Ackmann M. Biernat J. von Bergen M. Mandelkow E.M. Mandelkow E. Biochemistry. 2000; 39: 11714-11721Crossref PubMed Scopus (283) Google Scholar, 23Alonso A.C. Mederlyova A. Novak M. Grundke-Iqbal I. Iqbal K. J. Biol. Chem. 2004; 279: 34873-34881Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Overexpression of different frontotemporal dementia mutants of Tau in transgenic mice results in the development of tauopathy (24Lewis J. McGowan E. Rockwood J. Melrose H. Nacharaju P. Van Slegtenhorst M. Gwinn-Hardy K. Murphy P.M. Baker M. Yu X. Duff K. Hardy J. Corral A. Lin W.L. Yen S.H. Dickson D.W. Davies P. Hutton M. Nat. Genet. 2000; 25: 402-405Crossref PubMed Scopus (1113) Google Scholar, 25Lim F. Hernandez F. Lucas J.J. Gomez-Ramos P. Moran M.A. Avila J. Mol. Cell. Neurosci. 2001; 18: 702-714Crossref PubMed Scopus (177) Google Scholar, 26Santacruz K. Lewis J. Spires T. Paulson J. Kotilinek L. Ingelsson M. Guimaraes A. DeTure M. Ramsden M. McGowan E. Forster C. Yue M. Orne J. Janus C. Mariash A. Kuskowski M. Hyman B. Hutton M. Ashe K.H. Science. 2005; 309: 476-481Crossref PubMed Scopus (1512) Google Scholar). We generated Tau-P301L mice and extensively compared them to Tau-4R transgenic mice (16Terwel D. Lasrado R. Snauwaert J. Vandeweert E. Van Haesendonck C. Borghgraef P. Van Leuven F. J. Biol. Chem. 2005; 280: 3963-3973Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 17Boekhoorn K. Terwel D. Biemans B. Borghgraef P. Wiegert O. Ramakers G. de Vos K. Krugers H. Tomiyama T. Mori H. Joels M. Van Leuven F. Lucassen P. J. Neurosci. 2006; 26: 3514-3523Crossref PubMed Scopus (132) Google Scholar). The contrast in pathology was evident because beyond 8 months of age, Tau-P301L mice developed a typical tauopathy, i.e. intra-neuronal aggregates and fibrils evolving into authentic NFT containing Tau with all major pathological epitopes (16Terwel D. Lasrado R. Snauwaert J. Vandeweert E. Van Haesendonck C. Borghgraef P. Van Leuven F. J. Biol. Chem. 2005; 280: 3963-3973Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Here we extrapolate studies on transgenic mice to humanized yeast cells expressing Tau-4R or mutant Tau-P301L. In addition, we isolated recombinant human Tau from wild type yeast and strains lacking yeast kinases Mds1 or Pho85, the orthologues of mammalian GSK-3β and cdk5, respectively (Ref. 1Vandebroek T. Vanhelmont T. Terwel D. Borghgraef P. Lemaire K. Snauwaert J. Wera S. Van Leuven F. Winderickx J. Biochemistry. 2005; 44: 11466-11475Crossref PubMed Scopus (57) Google Scholar and references therein). By an optimized filter assay, we measured binding of different Tau isoforms to Taxol-stabilized mammalian MT, in direct relation to the physiological phosphorylation status of Tau. The data demonstrate a close parallel between phosphorylation of Ser396/Ser404 and Ser409, recognized by monoclonal antibodies (mAbs) AD2 and PG5 (Refs. 1Vandebroek T. Vanhelmont T. Terwel D. Borghgraef P. Lemaire K. Snauwaert J. Wera S. Van Leuven F. Winderickx J. Biochemistry. 2005; 44: 11466-11475Crossref PubMed Scopus (57) Google Scholar and 16Terwel D. Lasrado R. Snauwaert J. Vandeweert E. Van Haesendonck C. Borghgraef P. Van Leuven F. J. Biol. Chem. 2005; 280: 3963-3973Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar and references therein), respectively, with loss of physiological binding to MT and pathological aggregation of Tau at the MT surface. Also clear, Mds1 and Pho85 control in opposite directions both the phosphorylation and MT-binding of Tau-4R. Surprisingly, de-phosphorylated Tau-4R and Tau-P301L bound with equal high affinity to MT, proving that the mutation did not directly affect MT binding per se, but indirectly affected the phosphorylation of mutant Tau-P301L. Moreover, phosphorylated Tau-P301L actually aggregated on the MT surface as measured by the MT binding assays and observed directly by atomic force microscopy (AFM), including pronounced deformation and bundling of MT in vitro. This novel combination of cellular and in vitro modeling provide direct links of specific phosphorylation, aggregation, and MT binding of Tau protein, and we believe these models can now be adapted to analyze very precisely the effect of single phosphoepitopes on the biochemical behavior of human Tau protein. Strains and Culture Conditions—Yeast cells were grown exactly as described before (1Vandebroek T. Vanhelmont T. Terwel D. Borghgraef P. Lemaire K. Snauwaert J. Wera S. Van Leuven F. Winderickx J. Biochemistry. 2005; 44: 11466-11475Crossref PubMed Scopus (57) Google Scholar). cDNA coding for the human Tau isoform 2N/4R (1Vandebroek T. Vanhelmont T. Terwel D. Borghgraef P. Lemaire K. Snauwaert J. Wera S. Van Leuven F. Winderickx J. Biochemistry. 2005; 44: 11466-11475Crossref PubMed Scopus (57) Google Scholar) and human Tau-P301L 2N/4R (16Terwel D. Lasrado R. Snauwaert J. Vandeweert E. Van Haesendonck C. Borghgraef P. Van Leuven F. J. Biol. Chem. 2005; 280: 3963-3973Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) were inserted into the pYX212 plasmid containing the constitutive TPI promoter (R&D systems, Minneapolis, MN). Yeast transformants expressing Tau-4R or Tau-P301L were obtained in wild type, in Δmds1 and Δpho85 strains, and selected as described (1Vandebroek T. Vanhelmont T. Terwel D. Borghgraef P. Lemaire K. Snauwaert J. Wera S. Van Leuven F. Winderickx J. Biochemistry. 2005; 44: 11466-11475Crossref PubMed Scopus (57) Google Scholar). Purification of Tau—Tau-4R and Tau-P301L were purified from wild type, Δmds1, or Δpho85 yeast strains as described (1Vandebroek T. Vanhelmont T. Terwel D. Borghgraef P. Lemaire K. Snauwaert J. Wera S. Van Leuven F. Winderickx J. Biochemistry. 2005; 44: 11466-11475Crossref PubMed Scopus (57) Google Scholar). Purified Tau was concentrated on 10-kDa centricon devices (Millipore Corp., Bedford, MA) equilibrated with 10 mm MES buffer (pH 6.4). The amount of Tau was quantified by Western blotting with mAb Tau-5 and recombinant His-tagged Tau (Merck, Darmstadt, Germany) as standard (1Vandebroek T. Vanhelmont T. Terwel D. Borghgraef P. Lemaire K. Snauwaert J. Wera S. Van Leuven F. Winderickx J. Biochemistry. 2005; 44: 11466-11475Crossref PubMed Scopus (57) Google Scholar). Phosphorylation was assessed using phosphorylation-specific mAbs with specificity summarized in Table 1.TABLE 1Overview of the specificity of the monoclonal antibodies used in this projectAntibodySpecificitySourceTau-5All isoformsBD PharmingenAD-2Phosphorylated Ser396/Ser404Bio-RadAT8Phosphorylated Ser202/Ser205Innogenetics, Gent, BelgiumAT100Phosphorylated Thr212/Ser214Innogenetics, Gent, BelgiumAT180Phosphorylated Thr231/Ser235Innogenetics, Gent, BelgiumAT270Phosphorylated Thr181Innogenetics, Gent, BelgiumPG-5Phosphorylated Ser409Gift from P. DaviesYol1/34α-TubulinAbcam, Paris, France Open table in a new tab Dephosphorylation of Purified Tau—Purified Tau was concentrated in 50 mm Tris (pH 8.6) and quantified. Dephosphorylation was performed by incubating with alkaline phosphatase (Roche, Darmstadt, Germany), using 1.7 units/μg of Tau at 37 °C for 2 h. Preparation of Taxol-stabilized MT—Pig tubulin was isolated as described previously (27Shelanski M.L. Gaskin F. Cantor C.R. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 765-768Crossref PubMed Scopus (1963) Google Scholar). Tubulin aliquots were centrifuged (100,000 × g, 30 min, 4 °C) prior to analysis. MT were obtained by incubation of purified tubulin (10 μm) in assembly buffer (80 mm Pipes, 1 mm MgCl2, 1 mm EGTA (pH 6.8)) adjusted gradually to 10 μm Paclitaxel, 1 mm GTP and incubated at 37 °C for 30 min. Tau-MT Binding Filter Assay—Reaction mixtures in the same assembly buffer described above were prepared containing 10 nm Tau, which was centrifuged (100,000 × g, 30 min, 4 °C) prior to addition, with increasing concentrations of Taxol-stabilized MT (equivalent to up to 6 μm tubulin), further containing 0.05% bovine serum albumin and 1 mm GTP. After incubation at 37 °C for the indicated time periods, the mixture was transferred to pre-warmed 200-nm centrifuge devices (Nanosep MF, PALL, Michigan, MI) and centrifuged (4,000 × g, 10 min, 37 °C). Filtrates and retentates were adjusted and solubilized, respectively, in 50 mm Tris (pH 8.0), 10 mm β-mercaptoethanol, 2% SDS, 0.1% bromphenol blue, 10% glycerol, and boiled. SDS-PAGE and Western blotting (1Vandebroek T. Vanhelmont T. Terwel D. Borghgraef P. Lemaire K. Snauwaert J. Wera S. Van Leuven F. Winderickx J. Biochemistry. 2005; 44: 11466-11475Crossref PubMed Scopus (57) Google Scholar) were used with mAb Tau-5 to detect Tau. The relative concentration of Tau was determined from a curve of optical density against dilution of a standard preparation of human Tau (10 nm). The amount of bound Tau and the concentration of tubulin were corrected for nonspecific binding of Tau to the membrane and the amount of unpolymerized tubulin, respectively. Curve fitting (SigmaPlot 9.0, Rockware Inc., Cureglia, Switzerland) yielded values for affinity constant and maximal binding (binding equation: y = Bmax·x/(Kd,app + x), in which y is the percentage binding and x is the concentration of MT expressed as tubulin monomer content). Statistical analysis was performed by one-way analysis of variance, followed by the multiple comparison test of Tukey. Ultrastructural Analysis by AFM—Mixtures of 250 nm Tau and 1.5 μm MT were fixed with 2% glutaraldehyde and deposited on silanized silicon supports for AFM analysis as described previously (1Vandebroek T. Vanhelmont T. Terwel D. Borghgraef P. Lemaire K. Snauwaert J. Wera S. Van Leuven F. Winderickx J. Biochemistry. 2005; 44: 11466-11475Crossref PubMed Scopus (57) Google Scholar). AFM low power images were taken with Point probes type FM cantilevers (Nanoworld, Neuchatel, Switzerland), whereas high power images were obtained with Data probe type cantilevers (DP18/HI'RES/AIBS) (MikroMasch, Madrid, Spain) (1Vandebroek T. Vanhelmont T. Terwel D. Borghgraef P. Lemaire K. Snauwaert J. Wera S. Van Leuven F. Winderickx J. Biochemistry. 2005; 44: 11466-11475Crossref PubMed Scopus (57) Google Scholar). Filter Assay of Binding of Recombinant Human Tau-4R Protein to Taxol-stabilized MT—Human Tau-4R protein was expressed in and isolated from wild type, Δmds1, and Δpho85 yeast strains as described (1Vandebroek T. Vanhelmont T. Terwel D. Borghgraef P. Lemaire K. Snauwaert J. Wera S. Van Leuven F. Winderickx J. Biochemistry. 2005; 44: 11466-11475Crossref PubMed Scopus (57) Google Scholar). As opposed to recombinant Tau isolated from bacterial sources, this procedure faithfully maintains the phosphorylation status of Tau protein as present intracellularly in wild type and manipulated yeast cells, a less complex but eukaryotic model (1Vandebroek T. Vanhelmont T. Terwel D. Borghgraef P. Lemaire K. Snauwaert J. Wera S. Van Leuven F. Winderickx J. Biochemistry. 2005; 44: 11466-11475Crossref PubMed Scopus (57) Google Scholar). The quality and phosphorylation status of the used preparations of isolated Tau were authenticated by Western blotting (Fig. 1, A and B). Binding of Tau protein to preformed, Taxol-stabilized MT was measured by varying the concentration of MT, expressed as the concentration of tubulin monomers measured spectrophotometrically and by Western blotting. A fixed relatively low concentration of recombinant Tau protein (10 nm) was used in these studies, allowing determination of the apparent dissociation constants of MT binding (28Butner K.A. Kirschner M.W. J. Cell Biol. 1991; 115: 717-730Crossref PubMed Scopus (439) Google Scholar). Binding of Tau-4R to MT was measured by an optimized filter assay (see “Experimental Procedures”). In brief, Tau/MT mixtures were incubated and centrifuged through disposable 200-nm filters, separating unbound Tau protein in the soluble pass-through fraction, although retaining all Tau-MT complexes. Bound and free Tau were measured in retentates and filtrates, respectively, by Western blotting with the pan-Tau monoclonal antibody Tau-5. This procedure allowed us to quantitatively differentiate MT-bound versus unbound Tau. In addition, as extra internal markers, the majority of tubulin retained on the filter as MT, and the small amount of monomeric tubulin in the filtrates, were measured by Western blotting with an anti-tubulin antibody (Fig. 1C). Binding of Isolated Tau-4R to MT Is Affected in Opposite Directions by Mds1 and Pho85 Inactivation—We first measured binding of isolated Tau-4R to Taxol-stabilized MT, compared with dephosphorylated Tau-4R treated with alkaline phosphatase after isolation from the BY4741 wild type yeast strain. More than 90% of dephospho-Tau-4R bound to MT with an apparent affinity of 0.49 ± 0.09 μm (Fig. 2A, Table 2). The original phosphorylated Tau-4R preparation isolated from wild type yeast bound to MT with a lower apparent affinity of 2.15 ± 0.43 μm (Fig. 2A, Table 2).TABLE 2Kd,app (expressed in micromolar) of wild type and mutant Tau-4R monomers or dimers, isolated from different yeast strainsMonomerDimer, Tau-4RTau-4RTau-P301LDephosphorylation0.49 ± 0.09a0.68 ± 0.07a0.08 ± 0.04dBY47412.15 ± 0.43b0.75 ± 0.27a0.83 ± 0.47aΔmds12.48 ± 0.70b0.85 ± 0.15a0.63 ± 0.34aΔpho85>10c1.23 ± 0.45a,b1.58 ± 0.51b Open table in a new tab We then compared MT binding of Tau-4R isolated from wild type, Δmds1, and Δpho85 yeast strains. Tau-4R isolated from Δmds1 yeast bound to MT more extensively than Tau-4R isolated from wild type yeast (Fig. 2A, Table 2). In contrast, Tau-4R isolated from Δpho85 yeast strains showed a strongly reduced extent and affinity of MT binding (Fig. 2A, Table 2). Dimeric Tau-4R Protein Binds to MT with Higher Affinity—The preparations of recombinant Tau-4R isolated from yeast contained higher molecular weight species, tentatively identified and further referred to as Tau-4R dimers, based on their apparent Mr. Their origin or mode of apparent covalent linkage is unknown. Similar dimers were observed in the crude extracts from humanized Tau-4R yeast cultures (1Vandebroek T. Vanhelmont T. Terwel D. Borghgraef P. Lemaire K. Snauwaert J. Wera S. Van Leuven F. Winderickx J. Biochemistry. 2005; 44: 11466-11475Crossref PubMed Scopus (57) Google Scholar), and moreover also in brain extracts from Tau-4R transgenic mice (13Spittaels K. Van den Haute C. Van Dorpe J. Bruynseels K. Vandezande K. Laenen I. Geerts H. Mercken M. Sciot R. Van Lommel A. Loos R. Van Leuven F. Am. J. Pathol. 1999; 155: 2153-2165Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 14Spittaels K. Van den Haute C. Van Dorpe J. Geerts H. Mercken M. Bruynseels K. Lasrado R. Vandezande K. Laenen I. Boon T. Van Lint J. Vandenheede J. Moechars D. Loos R. Van Leuven F. J. Biol. Chem. 2000; 275: 41340-41349Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 16Terwel D. Lasrado R. Snauwaert J. Vandeweert E. Van Haesendonck C. Borghgraef P. Van Leuven F. J. Biol. Chem. 2005; 280: 3963-3973Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Tau-4R dimers, detected by Western blotting of bound and unbound fractions, were quantified in parallel to the Tau-4R monomers, eventually requiring somewhat longer exposure of the blots. Tau-4R dimers bound with higher apparent affinity to MT than monomeric Tau-4R (Fig. 2B, Table 2). Remarkably, Tau-4R dimers originating from Δpho85 yeast cultures bound to MT with much higher apparent affinity than the monomeric Tau-4R present in the same preparations (Fig. 2B, Table 2). When no other factors are involved, homodimeric proteins are expected to bind with affinity that is the square of that of the corresponding monomers. The experimental data approach this theoretical value fairly closely, indicating that dimerization of Tau-4R did not increase the affinity by another mechanism. The dimers are unlikely to be heterodimers of Tau-4R with unknown proteins, given the fact that they are observed in these widely different organisms of transgenic mice and yeast. Binding of Tau-4R to MT in Relation to Its Phosphorylation Status—Inspection of the Western blots developed with the pan-Tau antibody Tau-5 revealed important differences in electrophoretic mobility of Tau-4R present in bound and unbound fractions. Very conspicuously, electrophoretically faster migrating Tau subforms in all preparations bound preferably to MT, whereas electrophoretically slower migrating Tau isoforms were excluded from the MT-bound fractions. Because in the heterologous yeast expression system the electrophoretic mobility of Tau-4R reflects its phosphorylation status (Ref. 1Vandebroek T. Vanhelmont T. Terwel D. Borghgraef P. Lemaire K. Snauwaert J. Wera S" @default.
• W2090775206 created "2016-06-24" @default.
• W2090775206 creator A5011368490 @default.
• W2090775206 creator A5026407068 @default.
• W2090775206 creator A5053841150 @default.
• W2090775206 creator A5054385268 @default.
• W2090775206 creator A5061926187 @default.
• W2090775206 creator A5063402807 @default.
• W2090775206 creator A5081889455 @default.
• W2090775206 creator A5087618989 @default.
• W2090775206 date "2006-09-01" @default.
• W2090775206 modified "2023-09-28" @default.
• W2090775206 title "Microtubule Binding and Clustering of Human Tau-4R and Tau-P301L Proteins Isolated from Yeast Deficient in Orthologues of Glycogen Synthase Kinase-3β or cdk5" @default.
• W2090775206 cites W1480786153 @default.
• W2090775206 cites W1528790659 @default.
• W2090775206 cites W1970356391 @default.
• W2090775206 cites W1974581177 @default.
• W2090775206 cites W1988884999 @default.
• W2090775206 cites W1993878844 @default.
• W2090775206 cites W1994790971 @default.
• W2090775206 cites W1995895492 @default.
• W2090775206 cites W1996447834 @default.
• W2090775206 cites W2004133797 @default.
• W2090775206 cites W2009585248 @default.
• W2090775206 cites W2012405577 @default.
• W2090775206 cites W2014636427 @default.
• W2090775206 cites W2018365364 @default.
• W2090775206 cites W2022969837 @default.
• W2090775206 cites W2030390812 @default.
• W2090775206 cites W2030804652 @default.
• W2090775206 cites W2031418848 @default.
• W2090775206 cites W2033529907 @default.
• W2090775206 cites W2038523706 @default.
• W2090775206 cites W2039270622 @default.
• W2090775206 cites W2053652338 @default.
• W2090775206 cites W2054933404 @default.
• W2090775206 cites W2059501379 @default.
• W2090775206 cites W2067042124 @default.
• W2090775206 cites W2090027894 @default.
• W2090775206 cites W2092704615 @default.
• W2090775206 cites W2093879884 @default.
• W2090775206 cites W2094776926 @default.
• W2090775206 cites W2095577036 @default.
• W2090775206 cites W2103913931 @default.
• W2090775206 cites W2106379738 @default.
• W2090775206 cites W2112946437 @default.
• W2090775206 cites W2114991107 @default.
• W2090775206 cites W2122294268 @default.
• W2090775206 cites W2124922792 @default.
• W2090775206 cites W2127229268 @default.
• W2090775206 cites W2128121742 @default.
• W2090775206 cites W2135026720 @default.
• W2090775206 cites W2135309154 @default.
• W2090775206 cites W2139553041 @default.
• W2090775206 cites W2141653500 @default.
• W2090775206 cites W2159643400 @default.
• W2090775206 cites W2166555967 @default.
• W2090775206 cites W2168068084 @default.
• W2090775206 cites W2170741797 @default.
• W2090775206 cites W2309661748 @default.
• W2090775206 cites W2341233269 @default.
• W2090775206 doi "https://doi.org/10.1074/jbc.m602792200" @default.
• W2090775206 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16818492" @default.
• W2090775206 hasPublicationYear "2006" @default.
• W2090775206 type Work @default.
• W2090775206 sameAs 2090775206 @default.
• W2090775206 citedByCount "53" @default.
• W2090775206 countsByYear W20907752062012 @default.
• W2090775206 countsByYear W20907752062013 @default.
• W2090775206 countsByYear W20907752062014 @default.
• W2090775206 countsByYear W20907752062015 @default.
• W2090775206 countsByYear W20907752062016 @default.
• W2090775206 countsByYear W20907752062017 @default.
• W2090775206 countsByYear W20907752062018 @default.
• W2090775206 countsByYear W20907752062019 @default.
• W2090775206 countsByYear W20907752062020 @default.
• W2090775206 countsByYear W20907752062021 @default.
• W2090775206 countsByYear W20907752062022 @default.
• W2090775206 countsByYear W20907752062023 @default.
• W2090775206 crossrefType "journal-article" @default.
• W2090775206 hasAuthorship W2090775206A5011368490 @default.
• W2090775206 hasAuthorship W2090775206A5026407068 @default.
• W2090775206 hasAuthorship W2090775206A5053841150 @default.
• W2090775206 hasAuthorship W2090775206A5054385268 @default.
• W2090775206 hasAuthorship W2090775206A5061926187 @default.
• W2090775206 hasAuthorship W2090775206A5063402807 @default.
• W2090775206 hasAuthorship W2090775206A5081889455 @default.
• W2090775206 hasAuthorship W2090775206A5087618989 @default.
• W2090775206 hasBestOaLocation W20907752061 @default.
• W2090775206 hasConcept C104317684 @default.
• W2090775206 hasConcept C104629339 @default.
• W2090775206 hasConcept C112243037 @default.
• W2090775206 hasConcept C181199279 @default.
• W2090775206 hasConcept C182996813 @default.
• W2090775206 hasConcept C184235292 @default.
• W2090775206 hasConcept C192118531 @default.
• W2090775206 hasConcept C20418707 @default.
• W2090775206 hasConcept C2779222958 @default.

• next