Title: In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation
Abstract: Article16 December 2002free access In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation Akihisa Matsuyama Akihisa Matsuyama Chemical Genetics Laboratory, RIKEN, Wako, Saitama, 351-0198 Japan CREST Research Project, Japan Science and Technology Corporation, Saitama, 332-0012 Japan Search for more papers by this author Tadahiro Shimazu Tadahiro Shimazu Chemical Genetics Laboratory, RIKEN, Wako, Saitama, 351-0198 Japan Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657 Japan Search for more papers by this author Yuko Sumida Yuko Sumida Chemical Genetics Laboratory, RIKEN, Wako, Saitama, 351-0198 Japan CREST Research Project, Japan Science and Technology Corporation, Saitama, 332-0012 Japan Search for more papers by this author Akiko Saito Akiko Saito Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657 Japan Search for more papers by this author Yasuhiro Yoshimatsu Yasuhiro Yoshimatsu Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657 Japan Search for more papers by this author Daphné Seigneurin-Berny Daphné Seigneurin-Berny Laboratoire de Biologie Moléculaire et Cellulaire de la Différenciation-INSERM U309, Equipe, Chromatine et Expression des Gènes, Institut Albert Bonniot, Faculté de Médecine, Domaine de la Merci, France Search for more papers by this author Hiroyuki Osada Hiroyuki Osada Antibiotics Laboratory, RIKEN, Wako, Saitama, 351-0198 Japan Search for more papers by this author Yasuhiko Komatsu Yasuhiko Komatsu CREST Research Project, Japan Science and Technology Corporation, Saitama, 332-0012 Japan Search for more papers by this author Norikazu Nishino Norikazu Nishino CREST Research Project, Japan Science and Technology Corporation, Saitama, 332-0012 Japan Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Wakamatsu, Kitakyushu, 808-0196 Japan Search for more papers by this author Saadi Khochbin Saadi Khochbin Laboratoire de Biologie Moléculaire et Cellulaire de la Différenciation-INSERM U309, Equipe, Chromatine et Expression des Gènes, Institut Albert Bonniot, Faculté de Médecine, Domaine de la Merci, France Search for more papers by this author Sueharu Horinouchi Sueharu Horinouchi Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657 Japan Search for more papers by this author Minoru Yoshida Corresponding Author Minoru Yoshida Chemical Genetics Laboratory, RIKEN, Wako, Saitama, 351-0198 Japan CREST Research Project, Japan Science and Technology Corporation, Saitama, 332-0012 Japan Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657 Japan Search for more papers by this author Akihisa Matsuyama Akihisa Matsuyama Chemical Genetics Laboratory, RIKEN, Wako, Saitama, 351-0198 Japan CREST Research Project, Japan Science and Technology Corporation, Saitama, 332-0012 Japan Search for more papers by this author Tadahiro Shimazu Tadahiro Shimazu Chemical Genetics Laboratory, RIKEN, Wako, Saitama, 351-0198 Japan Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657 Japan Search for more papers by this author Yuko Sumida Yuko Sumida Chemical Genetics Laboratory, RIKEN, Wako, Saitama, 351-0198 Japan CREST Research Project, Japan Science and Technology Corporation, Saitama, 332-0012 Japan Search for more papers by this author Akiko Saito Akiko Saito Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657 Japan Search for more papers by this author Yasuhiro Yoshimatsu Yasuhiro Yoshimatsu Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657 Japan Search for more papers by this author Daphné Seigneurin-Berny Daphné Seigneurin-Berny Laboratoire de Biologie Moléculaire et Cellulaire de la Différenciation-INSERM U309, Equipe, Chromatine et Expression des Gènes, Institut Albert Bonniot, Faculté de Médecine, Domaine de la Merci, France Search for more papers by this author Hiroyuki Osada Hiroyuki Osada Antibiotics Laboratory, RIKEN, Wako, Saitama, 351-0198 Japan Search for more papers by this author Yasuhiko Komatsu Yasuhiko Komatsu CREST Research Project, Japan Science and Technology Corporation, Saitama, 332-0012 Japan Search for more papers by this author Norikazu Nishino Norikazu Nishino CREST Research Project, Japan Science and Technology Corporation, Saitama, 332-0012 Japan Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Wakamatsu, Kitakyushu, 808-0196 Japan Search for more papers by this author Saadi Khochbin Saadi Khochbin Laboratoire de Biologie Moléculaire et Cellulaire de la Différenciation-INSERM U309, Equipe, Chromatine et Expression des Gènes, Institut Albert Bonniot, Faculté de Médecine, Domaine de la Merci, France Search for more papers by this author Sueharu Horinouchi Sueharu Horinouchi Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657 Japan Search for more papers by this author Minoru Yoshida Corresponding Author Minoru Yoshida Chemical Genetics Laboratory, RIKEN, Wako, Saitama, 351-0198 Japan CREST Research Project, Japan Science and Technology Corporation, Saitama, 332-0012 Japan Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657 Japan Search for more papers by this author Author Information Akihisa Matsuyama1,2, Tadahiro Shimazu1,3, Yuko Sumida1,2, Akiko Saito3, Yasuhiro Yoshimatsu3, Daphné Seigneurin-Berny4, Hiroyuki Osada5, Yasuhiko Komatsu2, Norikazu Nishino2,6, Saadi Khochbin4, Sueharu Horinouchi3 and Minoru Yoshida 1,2,3 1Chemical Genetics Laboratory, RIKEN, Wako, Saitama, 351-0198 Japan 2CREST Research Project, Japan Science and Technology Corporation, Saitama, 332-0012 Japan 3Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657 Japan 4Laboratoire de Biologie Moléculaire et Cellulaire de la Différenciation-INSERM U309, Equipe, Chromatine et Expression des Gènes, Institut Albert Bonniot, Faculté de Médecine, Domaine de la Merci, France 5Antibiotics Laboratory, RIKEN, Wako, Saitama, 351-0198 Japan 6Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, Wakamatsu, Kitakyushu, 808-0196 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:6820-6831https://doi.org/10.1093/emboj/cdf682 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Trichostatin A (TSA) inhibits all histone deacetylases (HDACs) of both class I and II, whereas trapoxin (TPX) cannot inhibit HDAC6, a cytoplasmic member of class II HDACs. We took advantage of this differential sensitivity of HDAC6 to TSA and TPX to identify its substrates. Using this approach, α-tubulin was identified as an HDAC6 substrate. HDAC6 deacetylated α-tubulin both in vivo and in vitro. Our investigations suggest that HDAC6 controls the stability of a dynamic pool of microtubules. Indeed, we found that highly acetylated microtubules observed after TSA treatment exhibited delayed drug-induced depolymerization and that HDAC6 overexpression prompted their induced depolymerization. Depolymerized tubulin was rapidly deacetylated in vivo, whereas tubulin acetylation occurred only after polymerization. We therefore suggest that acetylation and deacetylation are coupled to the microtubule turnover and that HDAC6 plays a key regulatory role in the stability of the dynamic microtubules. Introduction The N-terminal part of histones is the target of various post-translational modifications, including phosphorylation, methylation and acetylation. Recently, it has been proposed that these histone modifications create a specific epigenetic code known as the ‘histone code’ (Cheung et al., 2000; Strahl and Allis, 2000; Turner, 2000). The ‘histone code’ hypothesis proposes that a specific combination of histone modifications provides information that is interpreted by machinery capable of locally modifying the structure and function of chromatin. Histone acetylation is believed to play a central role in this ‘histone code’ system (Turner, 1993). Many histone acetyltransferases (HATs) and histone deacetylases (HDACs) are, respectively, activators and repressors of transcription (Kuo and Allis, 1998). Besides chromatin-related functions, protein acetylation seems to be a signal directly controlling the activity of key cellular regulators (Kouzarides, 2000). HATs and HDACs have been shown to control the state of acetylation of many non-histone proteins, among which some are cytoplasmic (Kouzarides, 2000; Sterner and Berger, 2000). Although the significance of the nuclear protein acetylation is under intensive investigation, almost nothing is known about the cytoplasmic protein acetylation. However, all members of class II HDACs show a regulated intracellular localization and could be found in both the cytoplasm and the nucleus (Khochbin et al., 2001). Class II HDACs are enzymes whose catalytic domain is related to that of yeast histone deacetylase HDA1 (Grozinger et al., 1999; Miska et al., 1999; Verdel and Khochbin, 1999; Wang et al., 1999; Kao et al., 2000). Among the members of this family, HDAC6 is a unique enzyme that is essentially cytoplasmic (Verdel et al., 2000). Moreover, it harbors a conserved zinc finger capable of directly interacting with ubiquitin in its C-terminal region (Seigneurin-Berny et al., 2001). The purification of an HDAC6-containing complex from mouse testis cytosolic extracts showed the association of HDAC6 with two proteins involved in the control of protein ubiquitylation, VCP/p97 and PLAP/UFD3 (Seigneurin-Berny et al., 2001). The presence of HDAC6, as well as other members of class II HDACs, in the cytoplasm strongly suggests that they participate in the control of the acetylation of cytoplasmic proteins (Khochbin et al., 2001). We took advantage of the difference in sensitivity of several HDACs to HDAC inhibitors to identify specific HDAC substrates, with a particular interest in HDAC6. Indeed, we have identified HDACs as the targets of trichostatin A (TSA) and trapoxin (TPX), both of which are microbial metabolites that induce cell differentiation, cell cycle arrest and reversal of transformed cells morphology (Yoshida et al., 1995). Recently, we showed that, although all class I and II HDACs were strongly inhibited by TSA, HDAC6 was specifically resistant to TPX (Furumai et al., 2001). We expected that this difference between TSA and TPX in the inhibition of HDAC activity would allow the identification of specific substrates for HDAC6, the acetylation of which would be enhanced by TSA but not by TPX. These investigations led to the identification of α-tubulin as a substrate for HDAC6. Tubulin acetylation occurs at the ϵ-amino group of a conserved lysine residue (Lys40) near the N-terminus (MacRae, 1997; Rosenbaum, 2000). However, the enzymes responsible for the α-tubulin acetylation and deacetylation are neither purified nor cloned. In this paper, after identifying α-tubulin as a substrate for HDAC6 in vitro and in vivo, we show that α-tubulin acetylation reduces the rate of depolymerization of the cytoplasmic microtubules by depolymerizing agents. We also show that tubulin deacetylation by HDAC6 promotes its disassembly. Our data suggest that the cycle of tubulin acetylation and deacetylation is linked to the microtubule dynamics and has a role in regulating the stability of the microtubules. These findings may provide a basis for understanding the biological function of microtubule acetylation. Results Identification of α-tubulin as a protein specifically acetylated in TSA-treated cells We looked for acetylated proteins accumulated in TSA-treated but not in TPX-treated cells by using an anti-acetylated lysine monoclonal antibody AKL5C1 (Figure 1A). Both TSA and TPX treatments caused marked enhancement of histone acetylation at low nanomolar concentrations. In contrast, the acetylation of a 54 kDa protein was specifically enhanced by TSA treatment but not by TPX. In this particular experiment, only this 54 kDa protein was detected as being specifically acetylated in the TSA-treated cells. Figure 1.Acetylation of α-tubulin induced by TSA. (A) A protein with an apparent molecular mass of 54 kDa was strongly acetylated in response to TSA. NIH 3T3 cells were treated with various concentrations of either TSA or TPX B for 6 h. Acetylated proteins were analyzed by immunoblotting using an anti-acetylated lysine antibody (clone AKL5C1). (B) Identification of p54 as α-tubulin. Cell lysates prepared from NIH 3T3 cells that had been treated with either TSA (upper) or EtOH (lower) were subjected to two-dimensional PAGE and immunoblotted with an anti-acetylated lysine antibody (AKL5C1; left). The same blots were reprobed with an anti-α-tubulin antibody (clone B-5-1-2; right). (C) Acetylation of Lys40 in α-tubulin in TSA-treated cells. Cell lysates used in (A) were immunoblotted with anti-acetylated α-tubulin (clone 6-11B-1), anti-α-tubulin (B-5-1-2) or anti-acetylated lysine antibodies (AKL5C1). Note that TPX effectively enhanced the acetylated level of histones but not of α-tubulin. (D) Inhibitory potency of HDAC inhibitors against HDAC1 and HDAC6. The 50% inhibitory concentration (IC50) of each inhibitor was determined as described previously (Furumai et al., 2001). (E) Correlation between the ability of HDAC inhibitors to inhibit HDAC6 and to induce in vivo α-tubulin acetylation. NIH 3T3 cells were cultured for 6 h with each inhibitor at a concentration sufficient to induce histone acetylation, and then the levels of acetylation of the α-tubulin (Ac-α-tubulin), the total α-tubulin (α-tubulin) and acetylated proteins (Ac-Lys) were determined by immunoblotting. Download figure Download PowerPoint p53, importin-α and α-tubulin have been reported as potentially acetylated proteins with similar molecular sizes (L'Hernault and Rosenbaum, 1985; Gu and Roeder, 1997; Bannister et al., 2000). We therefore compared their mobility with that of the 54 kDa protein in a two-dimensional gel electrophoresis followed by immunoblotting. Only α-tubulin comigrated with the 54 kDa protein detected in the TSA-treated cells (Figure 1B). To confirm the identity of the 54 kDa protein, we used an anti-acetylated α-tubulin antibody 6-11B-1 (Piperno and Fuller, 1985) that recognizes a short region encompassing acetylated Lys40 in α-tubulin (Figure 1C). This antibody allowed us to show that the acetylation of α-tubulin was markedly enhanced by TSA treatment, whereas the total α-tubulin level determined with the pan-tubulin antibody B-5-1-2 was unchanged. In contrast, TPX, even at high concentrations, did not increase the acetylation level of the α-tubulin, although histone hyperacetylation was induced at a concentration 10-fold lower than that of TSA. Effect of other HDAC inhibitors on tubulin acetylation A number of natural and synthetic compounds have been described as HDAC inhibitors (Nakajima et al., 1998; Richon et al., 1998; Saito et al., 1999; Furumai et al., 2001; Komatsu et al., 2001). We next analyzed the correlation between the ability of these compounds to inhibit HDAC6 and to induce tubulin acetylation. First, the IC50 values of various HDAC inhibitors for HDAC6 inhibition were determined (Figure 1D). TSA inhibited both HDAC1 and HDAC6 to a similar extent. Although TPX strongly inhibited HDAC1 at sub-nanomolar concentrations, it failed to inhibit HDAC6 at these concentrations. Like TPX, all other inhibitors such as CHAPs, butyrate and FK228 were weaker inhibitors of HDAC6 activity than of HDAC1 activity. An immunoblot analysis showed that not only TPX but also FK228, MS-275, butyrate, CHAP31 and CHAP1 were very weak at inducing α-tubulin acetylation in cells, although they were capable of inducing histone hyperacetylation (Figure 1E). Thus, TSA is a unique compound in its abilities both to induce an α-tubulin acetylation in vivo and to inhibit HDAC6 activity in vitro. This suggested that HDAC6 is involved in α-tubulin deacetylation. In vivo deacetylation of α-tubulin by HDAC6 To see whether acetylated α-tubulin is the in vivo substrate of HDAC6, HDAC enzymes tagged with HA were overexpressed in NIH 3T3 cells, and the intracellular α-tubulin acetylation level of individual transfected cells was examined by immunofluorescent labeling using the antibody 6-11B-1 (Figure 2A). The acetylation of the tubulin network was greatly reduced in the HDAC6-transfected cells compared with that of the untransfected cells present in the same microscopic field. This decrease in tubulin acetylation was not observed with cells transfected with the enzymatically inactive HDAC6 mutant, which contained replacements of essential residues in both its deacetylase domains (HDAC6 m1/m2), indicating that the catalytic activity of HDAC6 is essential for the deacetylation of α-tubulin. All other HDACs, even HDAC10, the enzyme most related to HDAC6 (Guardiola and Yao, 2002), failed to deacetylate α-tubulin in vivo. Surprisingly, no increase in tubulin acetylation was observed in the HDAC6-overexpressing cells in the presence of 100 nM TSA, whereas microtubules in the untransfected cells or cells transfected with other HDACs were highly acetylated upon TSA treatment (Figure 2B). More than 1 μM TSA was required to enhance tubulin acetylation in the HDAC6-overexpressing cells (Figure 2C). These results clearly indicate that HDAC6 deacetylates microtubules in vivo and, moreover, that an elevated cellular concentration of HDAC6 confers a resistance to TSA at 100 nM, a concentration at which the endogenous HDAC6 is efficiently inhibited. Figure 2.In vivo deacetylation of α-tubulin by HDAC6. (A) Effects of HDAC expression on tubulin acetylation in individual cells. NIH 3T3 cells grown on coverslips were transfected with HA-HDACs. After fixation, cells were immunostained with anti-acetylated α-tubulin and anti-HA antibodies. The cells transfected with HDAC6 are indicated by arrowheads. (B) Effects of HDAC expression on tubulin acetylation in TSA-treated cells. NIH 3T3 cells grown on coverslips were transfected with HA-HDACs and then treated with 100 nM TSA for 6 h. The cells transfected with HDAC6 are indicated by arrowheads. (C) Inhibition of the HDAC6-mediated tubulin deacetylation by a high concentration of TSA. NIH 3T3 cells grown on coverslips were transfected with HA-HDAC6 and then treated with 3 μM TSA for 6 h. The cells transfected with HDAC6 are indicated by arrowheads. Download figure Download PowerPoint In vitro deacetylation of α-tubulin by HDAC6 In order to confirm the capability of HDAC6 to deacetylate α-tubulin, an endogenous HDAC6-containing complex was purified from mouse testis by immunoprecipitation, taking advantage of the overexpression of HDAC6 in the testis (Grozinger et al., 1999; Verdel and Khochbin, 1999). The substrate was prepared by purifying paclitaxel-polymerized tubulin from TSA-treated cells (Vallee and Collins, 1986). When the HDAC6-containing complex was incubated with the acetylation-enriched tubulin, the amount of acetylated α-tubulin was reduced (Figure 3A, lane 3). The deacetylation of α-tubulin was not observed when the acetylated tubulin was incubated with material immunoprecipitated with an anti-HDAC6 antibody blocked by an excess of the peptide antigen (Figure 3A, lane 2). This in vitro deacetylation by HDAC6 was inhibited by TSA but not by TPX (Figure 3A, lanes 6 and 7, respectively). To further examine the substrate specificity of HDACs, His-tagged human HDAC enzymes were produced using the baculoviral expression system and affinity purified (Figure 3B). These enzymes were catalytically active, and their activity was inhibited by TSA (Figure 3C; data not shown). The recombinant HDAC6 was resistant to TPX, as was the enzyme prepared from mammalian cells (Figure 3C). Their ability to deacetylate α-tubulin was examined with the fluorescence-labeled 20 amino acid peptide containing acetylated Lys40 of α-tubulin (Dansylated 30-IQPDGQMPSDK (Ac)TIGGGDDSF-49). Since the specific activities of the tested HDACs to deacetylate 3H-labeled histones were different, enzyme concentrations were adjusted to equivalent total activities of the enzymes. As shown in Figure 3D, the HPLC analysis showed that HDAC6 could deacetylate the acetylated peptide. About 60% of the acetylated peptide was converted to a non-acetylated form within 3 h. In contrast, other HDACs had no deacetylating activity on the peptide (Figure 3D–F). The time-course experiments showed that the reaction proceeded linearly during the initial 2 h (Figure 3D). To compare the specific activities between histone and α-tubulin, their abilities to deacetylate the fluorescent-labeled 20 amino acid peptide containing acetylated Lys16 of histone H4 (Dansylated 1-SGRGKGGKGLGKGGAK(Ac)RHRK-20) were also examined (Figure 3F). Only HDAC6 could deacetylate both substrates to a similar extent. These results indicate that HDAC6 is specifically responsible for α-tubulin deacetylation both in vivo and in vitro. Figure 3.In vitro deacetylation of α-tubulin by HDAC6. (A) Deacetylation of acetylated microtubules by HDAC6 from mouse testis. HDAC6 was isolated from mouse testis by immunoprecipitation using an anti-mouse HDAC6 antibody raised against an HDAC6 peptide (Verdel et al., 2000). As a control, the antigen peptide was added in excess to block the antibody binding to HDAC6 (Block). HDAC6 was incubated with acetylated microtubules isolated from TSA-treated cells in 20 μl for 3 h at 37°C in the presence or absence of TSA (100 nM) or TPX (100 nM). The success of the immunoprecipitation and the deacetylation of the microtubules were visualized by immunoblotting using the anti-mouse HDAC6 and anti-acetylated tubulin antibodies, respectively. (B) Purification of HDAC enzymes produced in insect cells via a baculovirus system. Asterisks denote the produced enzymes in the CBB stained gel of SDS–PAGE. HDAC4, HDAC6 and HDAC8 were efficiently expressed and highly purified. Although the production of HDAC1 was not efficient, the preparation had a sufficient activity to deacetylate 3H-histone. (C) Effects of TSA and TPX on the recombinant enzymes. The enzyme activities of HDAC1 and HDAC6 were determined with 3H-histone in the presence of various concentrations of TSA and TPX. (D) In vitro deacetylation of an acetylated tubulin peptide by recombinant human HDAC6. Recombinant human HDAC6 was incubated for 3 h with 0.5 mM Dns-tubulin peptide containing acetylated Lys40, and the reaction mixtures were analyzed with HPLC using a fluorescent detector. The retention time of the new peak (deAc) was identical to that of Dns-tubulin peptide without acetylation. (E) Enzyme specificity of deacetylation. The deacetylation of the Dns-acetylated tubulin peptide was analyzed over time with recombinant HDAC1, HDAC4, HDAC6 and HDAC8. The enzyme preparations used for the deacetylation assay were normalized based on their specific activities obtained with 3H-histone. The amounts of the deacetylated peptide after incubation with HDAC1 (open circles), HDAC4 (filled circles), HDAC6 (open squares) and HDAC8 (filled squares) for various lengths of time were plotted. (F) Specific activities of the recombinant enzymes for deacetylation of tubulin and histone H4 peptides. N.D., not detected. Download figure Download PowerPoint TSA enhances acetylation of entire microtubules We next investigated whether TSA affects the morphology of the cytoplasmic microtubule network by immunofluorescent labeling of formaldehyde-fixed NIH 3T3 cells (Figure 4A and B). Immunofluorescent staining using the anti-acetylated α-tubulin antibody showed that acetylated α-tubulin was mostly localized in segments of a number of microtubules in untreated 3T3 cells (Figure 4A, first row). In TSA-treated cells, the microtubules were entirely labeled with the anti-acetylated α-tubulin antibody, even in the cell periphery region (Figure 4A and B, second row). Cell fractionation experiments demonstrated that α-tubulin in both the polymer and free subunits became highly acetylated by TSA treatment in a time-dependent manner (Figure 4C). Observation at a higher magnification revealed that the microtubules appeared to be partially bundled in the TSA-treated cells (Figure 4B, second row). In contrast, TPX did not cause any change in the intensity of the tubulin acetylation (Figure 4A and B, third row). Figure 4.Effect of TSA on microtubule morphology. (A) Immunostaining of cells treated with TSA, TPX, paclitaxel and demecolcin. NIH 3T3 cells were treated with the various drugs for 6 h. The cells were fixed and stained for tyrosinated α-tubulin (α-tubulin) and acetylated α-tubulin (Ac-α-tubulin). (B) Microtubule morphology in the cell edge region. The boxed regions in (A) were viewed at higher magnification. (C) NIH 3T3 cells were treated with either TSA, paclitaxel or demecolcin for the indicated time and lysed. Total cell lysates from the drug-treated cultures were separated into the precipitates and supernatants by 16 000 g centrifugation. The fractions were immunoblotted with anti-acetylated α-tubulin (upper two panels) and anti-α-tubulin (middle two panels) antibodies. The band intensities were measured using densitometry, and the precipitate/supernatant ratios were determined (lower two panels). Download figure Download PowerPoint Tubulin deacetylation and destabilization by HDAC6 It has been reported that Taxol, an agent that stabilizes microtubules, causes an increased acetylation of α-tubulin (Piperno et al., 1987). Indeed, paclitaxel enhanced α-tubulin acetylation of polymer microtubules but not unpolymerized tubulins in 3T3 cells, whereas demecolcin (colcemid), a depolymerizing agent, reduced the amount of acetylation (Figure 4C). We therefore compared the effect of paclitaxel and TSA on the microtubule morphology and acetylation (Figure 4A and B). Staining of total microtubules with the anti-α-tubulin antibody revealed that paclitaxel stabilizes the cytoplasmic microtubules, which normally disassemble and assemble at high rates, thereby forming bundled microtubules. As reported previously, immunostaining with the antibody 6-11B-1 showed that the microtubules are highly acetylated in the paclitaxel-treated cells. However, the microtubule morphology in paclitaxel-treated cells was totally different from that in the presence of TSA (Figure 4A and B, fourth row). We further determined the relative polymer levels in drug-treated cells. The paclitaxel treatment greatly increased the polymer content (Figure 4C, α-tubulin ppt/sup) as well as the polymer acetylation relative to the free tubulin acetylation level (Figure 4C, Ac-α-tubulin ppt/sup), whereas demecolcin reduced the polymer level in cells. In the TSA-treated cells, both the polymer level and the polymer acetylation level were slightly but reproducibly increased. These results suggest that TSA-induced acetylation may affect the stability of microtubules. Effect of acetylation on the drug-induced depolymerization We next examined whether the TSA-induced α-tubulin acetylation affects the sensitivity of the interphase arrays of cytoplasmic microtubules to a microtubule-depolymerizing agent. Cells pretreated with ethanol (control), TSA or TPX for 6 h were exposed to 1 μM demecolcin for various lengths of time up to 60 min (Figure 5A), and the cellular levels of acetylated and total α-tubulin were monitored by immunoblotting (Figure 5B). Immunofluorescent staining of the cells with the α-tubulin antibody showed that, in the control culture, a large part of the cytoplasmic microtubule networks was disrupted by demecolcin within 15 min (Figure 5C, first row) and only a subset of stable microtubules was observed 30 min after demecolcin addition. TPX pretreatment did not affect this pattern of changes in the microtubule morphology by demecolcin (Figure 5C, third row). In contrast, the entirely acetylated microtubules in the TSA-treated cells showed a slow depolymerization, with most of the cytoplasmic microtubule network being intact after 15 min. Some of the microtubules remained intact even 30 min after demecolcin treatment. The microtubules were finally disrupted by 60 min (Figure 5C, second row). Since colchicine inhibits the assembly of tubulins by sub-stoichiometric binding of tubulin dimers rather than by direct dissociation of formed microtubules (Wilson, 1975), these results indicate that tubulin acetylation does not block the microtubule depolymerization itself but reduces its rate. The amount of acetylated α-tubulin in the TSA-treated cells did not change during the demecolcin treatment (Figure 5B), showing that the sequential treatment with TSA and demecolcin eventually led to the accumulation of acetylated, unpolymerized tubulin in the cytoplasm. Additionally, the removal of demecolcin resulted in the microtubule assembly of acetylated tubulin in TSA-treated cells without delay, suggesting that acetylated, unpolymerized tubulin has essentially the same ability to be assembled into microtubules as unmodified tubulin (data not shown). Figure 5.Delayed depolymerization of microtubules in TSA-treated cells. (A) Schematic representation of experimental procedures. Bars indicate the periods during which the cells were treated with drugs. Arrows indicate the time-points at which cells were taken for immunoblot analysis (B) and immunofluorescent microscopy (C). (B) Cellular levels of tubulin acetylation in the time-course experiments. The amounts of acetylated and total tubulin in the cells treated with various drugs in the time-course e