FTDP-17 Mutations Compromise the Ability of Tau to Regulate Microtubule Dynamics in Cells
2006; Elsevier BV; Volume: 281; Issue: 17 Linguagem: Inglês
10.1074/jbc.m509420200
ISSN1083-351X
AutoresJanis Bunker, Kathy Kamath, Leslie Wilson, Mary Ann Jordan, Stuart C. Feinstein,
Tópico(s)Cellular transport and secretion
ResumoThe neural microtubule-associated protein Tau binds directly to microtubules and regulates their dynamic behavior. In addition to being required for normal development, maintenance, and function of the nervous system, Tau is associated with several neurodegenerative diseases, including Alzheimer disease. One group of neurodegenerative dementias known as FTDP-17 (fronto-temporal dementia with Parkinsonism linked to chromosome 17) is directly linked genetically to mutations in the tau gene, demonstrating that Tau misfunction can cause neuronal cell death and dementia. These mutations result either in amino acid substitutions in Tau or in altered Tau mRNA splicing that skews the expression ratio of wild-type 3-repeat and 4-repeat Tau isoforms. Because wild-type Tau regulates microtubule dynamics, one possible mechanism underlying Tau-mediated neurodegeneration is aberrant regulation of microtubule behavior. In this study, we microinjected normal and mutated Tau protein into cultured cells expressing fluorescent tubulin and measured the effects on the dynamic instability of individual microtubules. We found that the FTDP-17 amino acid substitutions G272V (in both 3-repeat and 4-repeat Tau contexts), ΔK280, and P301L all exhibited markedly reduced abilities to regulate dynamic instability relative to wild-type Tau. In contrast, the FTDP-17 R406W mutation (which maps in a regulatory region outside the microtubule binding domain of Tau) did not significantly alter the ability of 3-repeat or 4-repeat Tau to regulate microtubule dynamics. Overall, these data are consistent with a loss-of-function model in which both amino acid substitutions and altered mRNA splicing in Tau lead to neurodegeneration by diminishing the ability of Tau to properly regulate microtubule dynamics. The neural microtubule-associated protein Tau binds directly to microtubules and regulates their dynamic behavior. In addition to being required for normal development, maintenance, and function of the nervous system, Tau is associated with several neurodegenerative diseases, including Alzheimer disease. One group of neurodegenerative dementias known as FTDP-17 (fronto-temporal dementia with Parkinsonism linked to chromosome 17) is directly linked genetically to mutations in the tau gene, demonstrating that Tau misfunction can cause neuronal cell death and dementia. These mutations result either in amino acid substitutions in Tau or in altered Tau mRNA splicing that skews the expression ratio of wild-type 3-repeat and 4-repeat Tau isoforms. Because wild-type Tau regulates microtubule dynamics, one possible mechanism underlying Tau-mediated neurodegeneration is aberrant regulation of microtubule behavior. In this study, we microinjected normal and mutated Tau protein into cultured cells expressing fluorescent tubulin and measured the effects on the dynamic instability of individual microtubules. We found that the FTDP-17 amino acid substitutions G272V (in both 3-repeat and 4-repeat Tau contexts), ΔK280, and P301L all exhibited markedly reduced abilities to regulate dynamic instability relative to wild-type Tau. In contrast, the FTDP-17 R406W mutation (which maps in a regulatory region outside the microtubule binding domain of Tau) did not significantly alter the ability of 3-repeat or 4-repeat Tau to regulate microtubule dynamics. Overall, these data are consistent with a loss-of-function model in which both amino acid substitutions and altered mRNA splicing in Tau lead to neurodegeneration by diminishing the ability of Tau to properly regulate microtubule dynamics. Microtubules are dynamic polymers with growing and shortening behaviors that are exquisitely regulated. The dynamic behaviors of microtubules are both temporally and spatially regulated, even within individual cells (1Akhmanova A. Hoogenraad C.C. Curr. Opin. 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Biochemistry. 2000; 39: 6136-6144Crossref PubMed Scopus (128) Google Scholar). Alternatively, dominant phenotypes could also be achieved by a loss-of-function mechanism in which mutations in just one Tau allele are sufficient to compromise the ability of the cell to properly regulate microtubule dynamics, leading to cell death. Here we sought to test a key prediction of the loss-of-function hypothesis, i.e. that FTDP-17 mutant Tau isoforms possess compromised abilities to regulate microtubule dynamics relative to wild-type Tau. To test this prediction, we injected identical amounts of either wild-type or FTDP-17 mutant Tau into living MCF-7 cells, and we analyzed the effects upon the dynamic instability behavior of individual microtubules in the thin lamellar peripheral region of the cells. Tau Protein Purification—pRK expression vectors containing the human cDNA sequences for the shortest 4-repeat and 3-repeat Tau isoforms (encoding 383 and 352 amino acids, respectively) were the kind gifts from Dr. Kenneth Kosik (University of California, Santa Barbara) and Dr. Gloria Lee (University of Iowa). FTDP-17 mutations were introduced into both 4-repeat and 3-repeat Tau constructs using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) (see Fig. 1A). The sequence of all constructs was verified by direct sequence analysis prior to use. Tau protein was expressed and purified as described previously (33Bunker J.M. Wilson L. Jordan M.A. Feinstein S.C. Mol. Biol. Cell. 2004; 15: 2720-2728Crossref PubMed Scopus (122) Google Scholar, 34Goode B.L. Denis P.E. Panda D. Radeke M.J. Miller H.P. Wilson L. Feinstein S.C. Mol. Biol. Cell. 1997; 8: 353-365Crossref PubMed Scopus (243) Google Scholar). Briefly, Tau expression was induced in BL21 (DE3) cells (Novagen, Madison, WI). Bacteria were lysed by sonication, and the lysate was clarified by centrifugation (12,000 × g, 15 min, 4 °C). Supernatants were boiled to precipitate heat-labile proteins and re-centrifuged. The heat-stable proteins were adsorbed to a phosphocellulose column and eluted with a 0.2 to 1.0 m NaCl gradient. Fractions containing Tau protein were pooled and further purified using reverse-phase liquid chromatography (DeltaPak-C18; Millipore, Billerica, MA). High pressure liquid chromatography fractions containing Tau were pooled, lyophilized, and resuspended in PBS 2The abbreviations used are: PBS, phosphate-buffered saline; GFP, green fluorescent protein. (Fig. 1B). The concentration of each Tau sample was determined by SDS-PAGE comparison with a "Tau mass standard," the concentration of which was established by amino acid analysis (35Panda D. Samuel J.C. Massie M. Feinstein S.C. Wilson L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9548-9553Crossref PubMed Scopus (226) Google Scholar). Cell Culture—MCF7 human breast cancer cells (ATCC, Manassas, VA) stably expressing the GFP-tubulin plasmid pEGFP-Tub (Clontech) (33Bunker J.M. Wilson L. Jordan M.A. Feinstein S.C. Mol. Biol. Cell. 2004; 15: 2720-2728Crossref PubMed Scopus (122) Google Scholar) were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with nonessential amino acids, 10% bovine serum, antibiotic-antimycotic (Invitrogen), and geneticin (400 μg/ml; Invitrogen) at 37 °C and 5.5% CO2. Cells were seeded 36-48 h before injection on 12-mm CellLocate coverslips (Eppendorf, Hamburg, Germany) coated with poly-d-lysine (100 μg/ml; Sigma) followed by human fibronectin (20 μg/ml; Invitrogen) and laminin (10 μg/ml; Sigma). To induce a more flattened morphology, cells were serum-starved in media containing 2% bovine serum 12 h before injection. Microinjection—Seeded cells were transferred to serum-free Dulbecco's modified Eagle's medium lacking bicarbonate and containing 25 mm HEPES and 4.5 g/liter glucose (recording media) (Invitrogen). All wild-type and mutant Tau proteins were diluted to a concentration of 13.3 μm in PBS plus 1.4 mm β-mercaptoethanol. Immediately prior to microinjection, the solution was centrifuged (50,000 × g, 15 min, 4 °C) to remove any aggregates or debris. Pressure microinjection was performed using an Eppendorf Transjector 5246 and Injectman. The injection volume was ∼10% of the cell volume (36Lamb N.J. Fernandez A. Methods Enzymol. 1997; 283: 72-83Crossref PubMed Scopus (11) Google Scholar), resulting in an ∼1.3 μm final Tau concentration in the cells. Injected cells were returned to normal media and incubated 2-3 h at 37 °C to allow equilibration of Tau within the cells. The quantity of Tau injected into cells was based on the following rationale. Based upon the work of Dhamodharan and Wadsworth (37Dhamodharan R. Wadsworth P. J. Cell Sci. 1995; 108: 1679-1689Crossref PubMed Google Scholar), we estimated that the total tubulin concentration in the cell was ∼20 μm (38Hiller G. Weber K. Cell. 1978; 14: 795-804Abstract Full Text PDF PubMed Scopus (200) Google Scholar) with 65% in polymer during interphase (39Zhai Y. Borisy G.G. J. Cell Sci. 1994; 107: 881-890Crossref PubMed Google Scholar), resulting in 13 μm polymerized tubulin. With respect to Tau, Drubin et al. (40Drubin D.G. Feinstein S.C. Shooter E.M. Kirschner M.W. J. Cell Biol. 1985; 101: 1799-1807Crossref PubMed Scopus (419) Google Scholar) determined that the Tau:polymeric tubulin molar ratio in neuronally differentiated rat PC12 cells is ∼1:5, whereas it is ∼1:34 in undifferentiated PC12 cells. Thus, we concluded that a 1:10 Tau:polymeric tubulin molar ratio represents a reasonable approximation of in vivo neuronal conditions. Immunocytochemistry—Cells were rinsed once with PBS and fixed by the rapid addition of 100% methanol (4 °C). Fixed cells were incubated overnight in blocking buffer (3% bovine serum albumin, 0.1% Triton-X-100, and 1% horse serum in PBS). Cells were incubated first with mouse monoclonal Tau 5 antibody (1:100; BioSource International, Camarillo, CA), then Cy3-conjugated donkey anti-mouse secondary antibody (1:100; Jackson ImmunoResearch, West Grove, PA), followed by fluorescein isothiocyanate-conjugated mouse monoclonal tubulin antibody DM1α (1:50; Sigma). All incubations were for 1 h at room temperature, followed by four 15-min washes in blocking buffer. Coverslips were mounted on glass slides using Prolong (Molecular Probes, Eugene, OR). Images were obtained using a laser-scanning confocal microscope (MRC 1024; Bio-Rad). Images are 4 Kalman averages of the same plane in the z axis. Images were processed using Adobe Photoshop 6.0. Time-lapse Microscopy and Image Acquisition—Microinjected cells were mounted in a Rose chamber (41Rose G.G. Pomerat C.M. Shindler T. Trunnell J. J. Biophys. Biochem. Cytol. 1958; 4: 761-764Crossref PubMed Scopus (121) Google Scholar) in recording media supplemented with Oxyrase (1:25 dilution; Oxyrase, Mansfield, OH) to reduce photo-bleaching. Images were captured with an inverted fluorescence microscope (Nikon Eclipse E800) with a Nikon plan apochromat 1.4 N.A., ×100 objective lens, maintained at 36.5 ± 1 °C. Thirty images per cell were taken at 4-s intervals using a Hamamatsu Orca II (Middlesex, NJ) digital camera driven by MetaMorph software (Universal Imaging, Media, PA). Analysis of Microtubule Dynamic Instability—To determine the percentage of microtubules that displayed visually detectable growing and shortening dynamics, we analyzed the total change in length (growing plus shortening) of at least 50 randomly selected microtubules from four independent cells microinjected with a given Tau isoform. A region of the cell periphery was randomly boxed, and 10-20 microtubules within 1-2 boxes per cell were analyzed. A microtubule was considered to be not detectably dynamic if the total change in length during the 2-min time-lapse sequence was less than or equal to 1.0 μm. Analysis of microtubule dynamic instability behavior was performed as described previously (33Bunker J.M. Wilson L. Jordan M.A. Feinstein S.C. Mol. Biol. Cell. 2004; 15: 2720-2728Crossref PubMed Scopus (122) Google Scholar, 42Goncalves A. Braguer D. Kamath K. Martello L. Briand C. Horwitz S. Wilson L. Jordan M.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11737-11742Crossref PubMed Scopus (230) Google Scholar). The positions of the plus ends of individual microtubules over time were recorded using MetaMorph software, exported to Microsoft Excel, and analyzed using RTM software (43Walker R.A. O'Brien E.T. Pryer N.K. Soboeiro M.F. Voter W.A. Erickson H.P. Salmon E.D. J. Cell Biol. 1988; 107: 1437-1448Crossref PubMed Scopus (784) Google Scholar). The lengths of individual microtubules were plotted as a function of time. Changes in length greater than 0.5 μm were designated as growth or shortening events. Periods in which length changes were less than 0.5 μm were designated as phases of attenuated microtubule dynamics (or pause). A catastrophe was defined as a transition from either growth or attenuation to shortening. The catastrophe frequency was calculated either as the total number of catastrophes divided by the total time spent growing and attenuated or as the total number of catastrophes divided by the total length grown. A rescue was defined as a transition from shortening to either growth or attenuation. Frequencies of rescue were calculated either as the total number of rescues divided by the total amount of time spent shortening or as the total number of rescues divided by the total length shortened. Dynamicity was calculated as the total length grown and shortened divided by the total time measured. On-line Supplemental Material—Time-lapse movies of enhanced GFP-microtubules undergoing dynamic instability in cells are available as Supplemental Material. Each movie consists of 30 frames taken at 4-s intervals and is played 30 times faster than real time. Movie 1 shows microtubule dynamics in a control cell injected with buffer; Movie 2 shows microtubule dynamics in a cell injected with wild-type 4-repeat Tau; and Movie 3 shows microtubule dynamics in a cell injected with 4-repeat Tau containing the ΔK280 mutation. Localization of Microinjected Wild-type and FTDP-17 Mutated Tau in Cells—Prior to assessing the effects of FTDP-17 mutations on dynamic instability, we first sought to assess the subcellular distribution of injected wild-type and FTDP-17-mutated Tau. We injected MCF7 cells, which do not express any endogenous Tau, with either wild-type or FTDP-17 mutant Tau and then returned the cells to the 37 °C incubator for 2 h to allow sufficient time for the Tau to equilibrate throughout the cells. We then fixed the cells and double-stained for Tau and tubulin. We found that wild-type and the various FTDP-17-mutated Tau proteins associated with microtubules in injected cells, whereas no Tau was detected in uninjected cells, as expected (Fig. 2). Tau staining was evenly distributed along the microtubules, with little or no Tau staining elsewhere in the cells. Additionally, there was no detectable difference in the intensity of Tau staining on microtubules between wild-type Tau and Tau containing the various FTDP-17 mutations. Thus, differences in the abilities of various Tau molecules to regulate microtubule dynamics most likely reflect intrinsic mechanistic differences in the Tau molecules being tested rather than differential binding of particular Tau molecules to the microtubules. Microinjection of Exogenous Wild-type or FTDP-17 Mutant Tau Does Not Markedly Affect the Fraction of Microtubules That Display Dynamic Instability—To visualize dynamic instability behavior, we captured time-lapse images of microtubules in the flat peripheral region of living cells stably transfected with GFP-tubulin. As shown in Fig. 3, and the online supplemental movies, the microtubules were well resolved, and their ends were clearly visible. Initial analyses revealed that all cells possess two populations of microtubules, a stable population and a population that exhibits visually detectable dynamic behavior. To determine whether the different Tau isoforms used in this study differentially affected the percentage of dynamic versus nondynamic microtubules in cells, we operationally defined dynamic microtubules as those exhibiting total length changes (growth plus shortening lengths) of 1.0 μm or more during 2 min of observation (dynamicity ≥0.5 μm/min); stable microtubules were defined as those that grew and/or shortened less than 1.0 μm during the same period. Using these criteria, there were no significant differences in the percentage of dynamic microtubules in cells injected with control buffer versus any of the Tau-injected cells (Table 1). Most cells had ∼80% dynamic microtubules. Cells injected with either 4-repeat wild-type Tau or ΔK280 had slightly fewer dynamic microtubules than the control cells, but these differences were not statistically significant (p = 0.20 and p = 0.39, respectively, using χ2 test). These data are in contrast to our earlier assertion, based on nonquantitative assessments, suggesting that Tau might increase the percentage of nondynamic microtubules in the cells (33Bunker J.M. Wilson L. Jordan M.A. Feinstein S.C. Mol. Biol. Cell. 2004; 15: 2720-2728Crossref PubMed Scopus (122) Google Scholar). In retrospect, it is likely that our earlier subjective observation was based upon the fact that Tau markedly increases the percentage of time microtubules spend in the attenuated state (33Bunker J.M. Wilson L. Jordan M.A. Feinstein S.C. Mol. Biol. Cell. 2004; 15: 2720-2728Crossref PubMed Scopus (122) Google Scholar).TABLE 1Percentage of dynamic microtubules in Tau-injected cells 4R indicates 4-repeat Tau; 3R indicates 3-repeat Tau.No. microtubulesDynamic% of totalBuffer control60804R WT63684R G272V70804R ΔK28060724R P301L78784R R406W50843R WT60833R G272V65773R R406W7882 Open table in a new tab Most FTDP-17 Missense Mutations Reduce the Ability of Tau to Regulate the Percentage of Time Dynamic Microtubules Spend Growing, Shortening, or Attenuated—The FTDP-17 mutations examined here all map to regions of Tau known to be important for normal Tau function (see Fig. 1). G272V is present in both 3-repeat and 4-repeat Tau, residing in the first repeat. ΔK280 and P301L are both encoded by the alternatively spliced exon 10 and therefore are present only in 4-repeat Tau; these mutations reside in the R1-R2 inter-repeat and at the end of repeat 2, respectively. Each of these mutations map to the region of Tau believed to interact directly with microtubules (44Brandt R. Lee G. J. Biol. Chem. 1993; 268: 3414-3419Abstract Full Text PDF PubMed Google Scholar, 45Butner K.A. Kirschner M.W. J. Cell Biol. 1991; 115: 717-730Crossref PubMed Scopus (452) Google Scholar, 46Goode B.L. Feinstein S.C. J. Cell Biol. 1994; 124: 769-782Crossref PubMed Scopus (344) Google Scholar, 47Goode B.L. Chau M. Denis P.E. Feinstein S.C. J. Biol. Chem. 2000; 275: 38182-38189Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). An additional mutation, R406W, is present in both 3-repeat and 4-repeat Tau and resides in the flanking sequence on the carboxyl side of the repeat-inter-repeat region; these flanking sequences are believed to influence Tau action indirectly via protein folding and/or phosphorylation effects (47Goode B.L. Chau M. Denis P.E. Feinstein S.C. J. Biol. Chem. 2000; 275: 38182-38189Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 48Delobel P. Flament S. Hamdane M. Jakes R. Rousseau A. Delacourte A. Vilain J.P. Goedert M. Buee L. J. Biol. Chem. 2002; 277: 9199-9205Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). To assess the effects of the various Tau molecules on the regulation of microtubule dynamic instability, we injected cells either with buffer, wild-type Tau, or FTDP-17 mutated Tau and then measured the changes in length of individual microtubules over time by tracking the positions of microtubule ends (see "Materials and Methods"). Typical microtubule life history plots for buffer-injected, wild-type 4-repeat Tau-injected and ΔK280-injected cells are presented in Fig. 4. From these plots, we determined the dynamic instability parameters (as described under "Materials and Methods"). Dynamic microtubules transition among three phases: growth, shortening, and attenuation (Fig. 4). To begin our analyses, we first assessed the ability of each wild-type and FTDP-17 mutant Tau isoform to influence the fraction of time that dynamic microtubules spent in each phase relative to the total time tracked. Wild-type 3-repeat and 4-repeat Tau increased the fraction of time microtubules spent attenuated while reducing the fraction of time spent growing, relative to microtubules in buffer-injected control cells (33Bunker J.M. Wilson L. Jordan M.A. Feinstein S.C. Mol. Biol. Cell. 2004; 15: 2720-2728Crossref PubMed Scopus (122) Google Scholar) (see also Tab
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