1 Laboratoire de Biologie Moléculaire et Cellulaire, UMR 5665 CNRS/ENS, INRA 913, Ecole Normale Supérieure de Lyon, 46, allée d'Italie, 69364 Lyon CEDEX 7, France
2 Laboratoire de Biologie Moléculaire et Cellulaire de la Différenciation, INSERM U309, Institut Albert Bonniot, Faculté de Médecine, 38706 La Tronche Cedex, France
3 CRBM, CNRS FRE2593, 1919 route de Mende, 34293 Montpellier CEDEX 5, France
¶ Author for correspondence (e-mail: pjurdic{at}ens-lyon.fr)
Accepted 6 April 2005
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Summary |
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Key words: HDAC6, Rho GTPase, mDIA, Microtubule acetylation, Podosomes, Osteoclasts
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Introduction |
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Rho GTPases are known to promote F-actin and adhesion structure rearrangements and appear to be the probable signalling intermediates between microtubules and F-actin (Etienne-Manneville and Hall, 2003). Microtubule depolymerisation in fibroblasts promotes stress fibre formation and focal adhesion plaque assembly, which relies on RhoA activation (Enomoto, 1996
; Ren et al., 1999
). Although the function of Rho GTPases in podosome assembly or patterning is rather unclear, Rho GTPase activity needs to be tightly regulated to maintain podosome assembly (Linder and Aepfelbacher, 2003
) and as in fibroblasts, microtubule dynamics regulates Rho GTPase activity (Ory et al., 2002
). We recently showed that microtubule repolymerisation recapitulates the sequence of events that lead to podosome belt formation during osteoclast maturation. This process starts with podosome clustering at the early stage of microtubule repolymerisation and proceeds to the formation of podosome rings that eventually fuse together to generate the podosome belt at the cell periphery when microtubules are fully regrown (Destaing et al., 2003
). It should be noted that the kinetics of microtubule repolymerisation are faster than the reformation of the podosome belt, indicating that not only the dynamics of microtubules are crucial for early events in podosome patterning (clusters or rings) but also that the microtubule network needs to be in place or stabilised before the podosome belt can be formed. In cells, there are two pools of dynamic microtubules, those that exhibit dynamic instability and have half-lives of 5-10 minutes, and stabilised microtubules, which do not exhibit dynamic instability and persist for hours (Saxton et al., 1984
; Schulze and Kirschner, 1986
; Webster et al., 1987a
; Webster et al., 1987b
). Stable microtubules accumulate post-translational modifications including detyrosination or acetylation, and may contribute to specialised functions in cells (Bulinski and Gundersen, 1991
; Palazzo et al., 2001b
; Rosenbaum, 2000
).
Starting with these observations, we decided to investigate the molecular mechanisms that drive microtubule-dependent podosome belt stabilisation in osteoclasts and the extent to which Rho GTPase is involved in this process. Here, we report that Rho inhibition prevents podosome belt disruption following microtubule depolymerisation by nocodazole and, more surprisingly, that Rho inhibition increases the resistance of microtubules to nocodazole. Checking for microtubule post-translational modifications, we found that stable microtubules were acetylated and not detyrosinated. This led us to investigate whether Rho was involved in microtubule acetylation in osteoclasts. We used the fact that the histone acetylase HDAC6 has recently been described as a microtubule deacetylase (Hubbert et al., 2002; Matsuyama et al., 2002
) and that the Rho effectors of the mDia family are involved in the control of post-translational modification of microtubules (Palazzo et al., 2001b
) as well as the coordination of the microtubule and actin networks (Watanabe et al., 1999). This allowed us to reveal a pathway where activation of Rho promotes the deacetylation of microtubules through mDia2 and HDAC6 activation. Moreover, we present evidence that the level of microtubule acetylation is important for osteoclast function.
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Materials and Methods |
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Plasmids and constructs
GFP-mDIA2 and GFP-mDIA2-GBD were from Art Alberts (Van Andel Institute) and have been described previously (Palazzo et al., 2001a
). GFP-G14VRho and GFP-G12VRac were gifts from Philippe Fort (CRBM, Montpellier, France). pEGFP-Actin Vector® was from Clontech. TAT-C3 expression vector was a kind gift from Erik Sahai and was produced as described (Coleman et al., 2001
). Vectors expressing haemagglutinin (HA)-tagged mHDAC6 and deletion mutants have been described previously (Seigneurin-Berny et al., 2001
).
Osteoclast differentiation
Spleen cells from six- to eight-week-old male OF1 mice were seeded at 2500 cells/mm2 and cultured for 8 days on coverslips in differentiation medium: -MEM medium (Life Technologies) containing 10% foetal calf serum (FCS, Hyclone) plus M-CSF and soluble recombinant RANK-L.
Microinjection
Mouse spleen cell-derived osteoclasts differentiated in vitro on Eppendorf CELLocate® coverslips for 7 days in differentiation medium were transferred to observation medium: -MEM without bicarbonate (Life Technologies) containing 10% foetal calf serum, M-CSF, 20 mM HEPES and soluble recombinant RANK-L. Intranuclear microinjections of cDNA (0.2 mg/ml in 0.05 M Tris-HCl, pH 7.4) were carried out at room temperature using Eclipse TE 200 inverted microscope (Nikon) with an InjectMan micromanipulator and an Eppendorf 5246 microinjector. After injection, cells were further maintained at 37°C and 5% CO2 for 6 hours in differentiation medium before imaging.
Immunoprecipitation and interaction site mapping
For co-immunoprecipitation and interaction site mapping, COS cells were lysed 24 hours after transfection with Fugene 6® following the manufacturer's recommendations (Roche). Lysis buffer consists of 100 mM HEPES pH 7.9, 6 mM MgCl2, 40% glycerol, 150 mM KCl, 0.1% Nonidet P40 and 1 mM dithiothreitol supplemented with a protease inhibitor cocktail. The lysate was incubated on ice for 20 minutes and cleared by centrifugation at 17,000 g for 10 minutes at 4°C. HA-tagged proteins were immunoprecipitated with anti-HA antibody and protein-G sepharose for 2 hours at 4°C. Immunoprecipitated proteins were washed three times in lysis buffer.
Confocal microscopy
For immunofluorescence, cells were fixed in Busson fixation solution at pH 6.9 (4% paraformaldehyde, 60 mM PIPES, 25 mM HEPES, 20 mM EGTA, 2 mM magnesium acetate, 0.05% glutaraldehyde w/v), processed as described (Ory et al., 2000) and imaged with a Zeiss LSM 510 microscope using a 63 x (NA 1.4) Plan NeoFluor objective. To prevent cross-contamination between fluorochromes, each channel was imaged sequentially using the multi-track recording module before merging.
Tubulin deacetylase assay
COS cells transfected with 1 µg HDAC6 and/or mDia plasmids were lysed at room temperature for 40 minutes in buffer A (15 mM Tris-HCl, pH 7.4, 15 mM NaCl, 60 mM KCl, 340 mM sucrose, 2 mM EDTA, 0.5 mM EGTA, 0.65 mM spermidine, 1 mM dithiothreitol, 0.5% Triton X-100, 50 ng/ml TSA) with a complete protease inhibitor cocktail (Roche Molecular Biochemicals). After centrifugation at 17,000 g at 4°C, the supernatant (cytoplasmic extract) was mixed with Laemmli buffer and the extent of tubulin acetylation monitored by western blotting, using an anti-acetylated tubulin antibody.
GTP-GTPase affinity precipitation assay
The GST-RBD construct used to evaluate the level of GTP-Rho in cell lysates was kindly provided by M. Schwartz (Scripps Research Institute, La Jolla, CA). The activity assay was performed as described (Ren et al., 1999) for GTP-Rho with slight modifications. Briefly, GST-fusion proteins containing the Rho-binding domain (RBD) from mouse Rhotekin (amino acids 7-89) were produced in Escherichia coli BL21 cells. After isopropylthiogalactoside (IPTG) induction, pellets of bacteria were resuspended in lysis buffer (50 mM Tris-HCl, pH 8, 2 mM MgCl2, 0.2 mM Na2S2O5, 10% glycerol, 20% sucrose, 2 mM DTT, 1 µ g/ml each aprotinin, leupeptin and pepstatin) and sonicated. Cell lysates were centrifuged for 20 minutes at 4°C, 45,000 g and the supernatants were incubated with glutathione-coupled sepharose 4B beads (Pharmacia Biotech) for 2 hours at 4°C. After three washes with lysis buffer, the amount of GST-RBD fusion proteins bound to the beads was estimated from Coomassie Blue-stained SDS gels.
Cells at different stages of the differentiation process were rapidly washed in ice-cold PBS and proteins were extracted with lysis buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 1% Triton-X100, 10% glycerol, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl and 1 µg/ml each leupeptin, pepstatin and aprotinin). Lysates were centrifuged for 5 minutes at 17,000 g and 4°C, and aliquots from the supernatant were used to determine total GTPase in the cell lysate. 20 µg of bacterially produced GST, GST-RBD fusion proteins bound to glutathione-coupled sepharose beads were added to cell lysates and incubated for 1 hour at 4°C. Beads were washed four times in lysis buffer and bound proteins were eluted in Laemmli sample buffer. Analyses for bound GTPases by western blotting were performed using monoclonal antibody 26C4 against RhoA (a generous gift from J. Bertoglio, Inserm U461, Paris, France).
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Results |
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Surprisingly, TAT-C3-treated osteoclasts showed a significant increase in nocodazole-resistant microtubules compared to TAT-GFP-treated cells (Fig. 1A,B). This observation contradicts previous published data showing that microtubule stabilisation is induced by Rho activation rather than Rho inhibition in NIH3T3 cells (Cook et al., 1998). To confirm that our results were not due to experimental deficiencies, we observed the microtubule content in NIH3T3 cells that were first stimulated by serum to activate Rho, and then treated with nocodazole in the presence of TAT-C3 or TAT-GFP. As described by others, and in contrast to osteoclasts, Rho activation by serum induced microtubule stabilisation that was otherwise blocked by TAT-C3 (Fig. 1C). We conclude that the effects of Rho inhibition on microtubule stability are cell type specific. It should be noted that microtubule stabilisation by Rho inhibition has also been found in astrocytes (S. Etienne-Manneville, personal communication).
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Osteoclasts were incubated for 5 hours with either TAT-GFP as a control or TAT-C3 to inhibit Rho, and processed for confocal microscopy analysis after staining of their actin and acetylated-microtubule (Ac-MT) cytoskeleton. Whereas the Ac-MT level was detectable but low in osteoclasts incubated with TAT-GFP fusion protein, it was increased in osteoclasts maintained in presence of TAT-C3 (Fig. 2A). On the other hand, we never observed changes in detyrosinated microtubules (data not shown), suggesting that Rho inhibition was promoting microtubule acetylation and also that Rho activation should trigger microtubule deacetylation. To test this hypothesis, one nucleus per osteoclast analysed was microinjected with vectors encoding GFP fused to a wild-type form or a constitutively activated form of RhoA (RhoA-WT, V14-RhoA respectively). Cells were then fixed and Ac-MT levels compared by immunostaining. GFP-RhoA WT did not significantly affect levels of Ac-MT whereas V14RhoA promoted a drastic decrease in the amount of Ac-MT together with disruption of the podosome belt (Fig. 2B). Thus, Rho appears to be a key player in regulating levels of Ac-MT in osteoclasts.
It has recently been shown that the histone deacetylase 6 (HDAC6) acts as a major microtubule deacetylase that can be inhibited by TSA (Matsuyama et al., 2002). To test whether HDAC6 could act on the Rho pathway, multinucleated osteoclasts were microinjected with V14RhoA cDNA and treated with TSA for 30 minutes. TSA treatment blocked the V14RhoA-mediated deacetylation of microtubules (Fig. 2B, lower panel), indicating that HDAC6 was downstream of Rho. To confirm that microtubule acetylation was increased by HDAC6 or Rho inhibition, we monitored levels of acetylated tubulin in osteoclast lysates treated with TAT-GFP, TAT-C3 or TSA (Fig. 2C). As expected, whereas levels of tubulin were comparable between samples, levels of acetylated tubulin increased an average of 2.5-fold in TAT-C3-treated osteoclasts when compared to TAT-GFP-treated osteoclasts (mean of five independent experiments). However, the relative increase of acetylated tubulin was much higher in TSA-treated compared to C3-treated cells indicating that Rho may partially control tubulin acetylation and/or only affect a subset of microtubules. In addition, in contrast to most HDAC proteins, TSA-sensitive HDAC6 has been shown to be insensitive to sodium butyrate. To assess whether microtubule deacetylation was dependent on other HDACs in osteoclasts, cells were treated with TSA or sodium butyrate and the amount of acetylated tubulin determined by western blotting. The amount of acetylated tubulin did not change in sodium butyrate-treated osteoclasts but did drastically increase when osteoclasts were treated with TSA (Fig. 2D). Altogether, these experiments indicate that Rho activation is able to stimulate microtubule deacetylation and that HDAC6 is a probable intermediate.
Rho activates HDAC6 via mDia2
To further evaluate the role of the Rho pathway in microtubule acetylation in osteoclasts, we decided to test whether the two best-characterised Rho effectors, namely ROCK and mDia proteins, modified levels of Ac-MT in osteoclasts. As the ROCK inhibitor Y27632 did not modify levels of Ac-MT in osteoclasts (data not shown), we focused on mDia2. Indeed, mDia2 was a good candidate as it has been shown to be involved in microtubule stabilisation and in coordinating microtubule and actin dynamics (Ishizaki et al., 2001; Palazzo et al., 2001a
). We thus microinjected plasmids encoding a constitutive active mutant of mDia2 fused to GFP (GFP-mDIA2-
GBD) into osteoclasts (Palazzo et al., 2001a
). Consistent with the effect of activated Rho, we found that activated mDia2 triggered a drastic decrease in levels of Ac-MT as well as disruption of the podosome belt without any new specific actin structures being formed (Fig. 3A). As our results suggest that both HDAC6 and mDia2 act downstream of Rho, we hypothesised that mDia2 and HDAC6 could interact together to regulate tubulin acetylation. To test this hypothesis, we cotransfected HDAC6 with either wild-type mDia2 or activated mDia2 fused to GFP (GFP-mDia2WT or GFP-mDIA2-
GBD) in COS cells. As a control, HDAC6 was transfected with GFP alone. HDAC6 proteins were then immunoprecipitated from cleared cell lysates and associated mDia2 revealed by immunoblotting with anti-GFP antibody. GFP-mDia2WT and GFP-mDIA2-
GBD were found in HDAC6 immunoprecipitates whereas GFP alone was not (Fig. 3B). To gain further insight into which HDAC6 domains were responsible for mDia2 binding, we cotransfected deletion mutants of HA-tagged HDAC6 together with GFP-mDia2WT. HDAC6 fragment proteins were immunoprecipitated with HA antibody and associated mDia2 was revealed by anti-GFP immunoblotting. We detected mDia2 in the DD1 (amino acids 85-428) and DD2 (aa 429-824) but not in the C-terminal (aa 825-1149) and N-terminal (aa 1-84) domain immunocomplexes (Fig. 3C). These results indicate that HDAC6 interacts with mDia2 in COS cells and that the two deacetylase domains, DD1 and DD2 are both able to interact with mDia2. Finally, to determine whether mDia2 was able to stimulate the deacetylase activity of HDAC6 in cells, we used an in vitro deacetylase assay (Zhang et al., 2003
). COS cells were transfected with either HDAC6 alone, GFP-mDia2 alone or HDAC6 and GFP-mDia2 together. We then analysed the level of acetylated tubulin by western blotting of COS cell lysates (Fig. 3D). HDAC6 or GFP-mDia2 alone was not able to promote deacetylation of tubulin. However, when both proteins were expressed in cells, the level of acetylated tubulin was clearly reduced despite comparable amounts of tubulin in the samples. Moreover, transfecting twice the amount of GFP-mDia2 still reduced the level of acetylated tubulin indicating that mDia2 is able to activate HDAC6 deacetylase activity.
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As Rho inhibition is crucial for maintaining the podosome belt at the cell periphery, we expected that Rho inhibition in immature osteoclasts (D6) should accelerate the formation of podosome belts. Macrophages were differentiated into osteoclasts and at day 6, TAT-GFP or TAT-C3 was added to the differentiation medium for 6 hours. The number of osteoclasts with podosome rings (Fig. 4E, arrowhead) or podosome belts (Fig. 4E, arrows) was then quantified and compared to cells maintained in differentiation medium alone. At day 6, only 40% of osteoclasts in untreated or TAT-GFP-treated osteoclasts exhibited podosome belts versus 70% in TAT-C3-treated osteoclasts (Fig. 4F). In an attempt to confirm the relation between the level of Rho activation, microtubule deacetylation and the podosome belt formation we performed GST-pull down experiments to evaluate the RhoGTP levels along osteoclastogenesis. Unfortunately, we could not detect any significant variation of the ratio of RhoGTP versus total Rho (Fig. 4G). This may indicate that the localisation of activated Rho is more important to mediate its effect than the overall Rho activity. Nevertheless, altogether our results in osteoclasts indicate that Rho is involved in podosome belt formation as well as in cytoskeleton maturation process by promoting a switch from podosome rings to podosome belts, although its precise mode of action remains to be elucidated.
Bone resorbing osteoclasts exhibit high levels of acetylated microtubules within the sealing zone
As microtubule acetylation is an essential process in osteoclast maturation, we reasoned that this could be important in osteoclast function, namely bone resorption. When resorbing bone, osteoclasts rearrange their actin cytoskeleton into a sealing zone, a large band of actin that delineates the resorption pit. We have recently shown that osteoclasts alternate between resorbing phases with a sealing zone and migrating phases without any specific actin structures (Saltel et al., 2004). To determine whether microtubule acetylation could be correlated with osteoclast function, we cultured osteoclasts on coverslips coated with an apatite/collagen matrix known to mimic the bone surface (Shibutani et al., 2000
; Saltel et al., 2004
). Ac-MTs and detyrosinated microtubules were analysed with F-actin by immunostaining and confocal microscopy. Ac-MTs were barely detectable in migrating osteoclasts (Fig. 5B), but increased drastically in resorbing osteoclasts (Fig. 5A). Interestingly, resorbing or non-resorbing osteoclasts did not show any accumulation of detyrosinated microtubules (Fig. 5A,B), suggesting that microtubule acetylation may reflect a specific post-translational modification of microtubules in active osteoclasts.
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Discussion |
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The fact that, in our study, TAT-C3-mediated Rho inhibition promoted podosome belt assembly during osteoclast maturation is rather surprising. Indeed, exoenzyme C3 has been reported to disrupt the sealing zone and to block osteoclast resorption on bone (Zhang et al., 1995; Saltel et al., 2004
) as well as disrupting isolated podosomes in macrophage polykaryons (Chellaiah et al., 2000
; Ory et al., 2000
). In src-transformed cells, Rho inhibition also disrupted the podosome whereas activated Rho has been found localised in podosomes (Berdeaux et al., 2004
). However, inconsistent with a function of Rho in podosome formation, and in contrast to Cdc42 (Dutartre et al., 1996
; Moreau et al., 2003
), activated Rho or, interestingly activated mDia, do not lead to typical podosome formation but rather to their disruption (Burgstaller and Gimona, 2004
; Chellaiah et al., 2000
; Ory et al., 2000
) indicating that the Rho activation pathway by itself is not sufficient for podosome formation. However, as revealed by our recent study (Saltel et al., 2004
), Rho inhibition in osteoclasts cultured on bone-like substrate led to the loss of both their resorptive function and their apico-basal polarity. Interestingly, under these conditions, F-actin reorganised into a podosome belt, mimicking the actin organisation found in osteoclasts seeded on glass, an organisation never seen in osteoclasts seeded on bone-like substrate in normal conditions. Together with the present study, it indicates that Rho activity is not required for the podosome belt formation. However, despite what we were expecting, we could not observe any RhoGTP level variation along osteoclastogenesis indicating that to get more insight into the function of Rho in such large multinucleated cells, the subcellular localisation of activated Rho should be investigated. It would be more informative than the overall activity measured by pull-down assay. Indeed, we should expect Rho to be only locally inhibited as its full inhibition by exoenzyme C3 triggered not only the podosome belt stabilisation, but also excessive spreading and loss of cell polarity (Ory et al., 2000
; Saltel et al., 2004
). This may engage local regulation of Rho activity, variations of which may be insufficient to discriminate it from the overall Rho activity in cells. This idea is also supported by the fact that acetylation of microtubules was mainly observed on a subset of microtubules localised in the vicinity of the podosome belt in differentiated osteoclasts. This suggests that a local change in microtubule properties is occurring, and consequently, a local change in signalling events leading to microtubule acetylation.
Microtubule acetylation during osteoclast differentiation
Our results have shown that podosome belt and sealing zone formation during osteoclast maturation and bone resorption, respectively, are associated with an increase in tubulin acetylation corresponding to microtubule stabilisation. Interestingly, observations of increases in stable microtubules have been made in other differentiated cell types (Bulinski and Gundersen, 1991). For example, in muscle cell differentiation, which involves the fusion of myoblasts to form multinucleated myotubes, detyrosinated microtubules accumulate in myogenic precursors shortly before the fusion events. Detyrosinated microtubules are maintained in myotubes but acetylation is only observed at a later stage (Gundersen et al., 1989
). Unlike myogenesis (Chang et al., 2002
), osteoclastogenesis is not associated with detyrosination of microtubules, as we did not detect any significant changes in the levels of detyrosinated microtubules. However, as in myotubes, acetylated microtubules increased at a later stage in mature osteoclasts. These observations indicate that, although PTMs take place on stable microtubules, they are not necessarily occurring at the same time during the differentiation process, suggesting that they are dependent on their subcellular micro-environment and do not have the same biological functions. Differences in the function of post-translational tubulin modifications have been well exemplified in Tetrahymena thermophila in which the endogenous ß-tubulin gene has been replaced by mutated forms preventing either acetylation or polyglycylation PTMs. Although there were no detectable abnormalities when non-acetylatable tubulin was expressed (Gaertig et al., 1995
), preventing the polyglycylation of ß-tubulin, in contrast, had an affect on cytokinesis and was lethal (Thazhath et al., 2002
).
Although both the underlying molecular mechanisms of PTM and the function of microtubule acetylation remain to be elucidated, recent reports have highlighted an intriguing property of detyrosinated microtubules. In mammalian cells, kinesins have a stronger affinity for detyrosinated tubulin and may be preferentially recruited on stable detyrosinated microtubules (Gurland and Gundersen, 1995; Kreitzer et al., 1999
). Whether a similar function can be attributed to acetylation remains to be seen, but microtubule acetylation is highly regulated in osteoclast resorption function and podosome patterning.
Rho activates HDAC6 through mDia2 and controls microtubule quality
HDAC6 has been recently characterised as a cytoplasmic tubulin deacetylase (Hubbert et al., 2002; Matsuyama et al., 2002
; Zhang et al., 2003
). We found that Rho inhibition increased microtubule acetylation. Conversely, Rho activation as well as microinjection of activated mDia2, promoted microtubule deacetylation whereas no changes in the levels of microtubule acetylation were observed with the ROCK inhibitor Y27632 (data not shown). These results indicate that mDia2 is the specific Rho effector involved in microtubule deacetylation. This hypothesis is reinforced by the fact that mDia2 coprecipitated with and activated HDAC6 in COS cells. Thus, we propose that Rho activates mDia2, which in turn stimulates HDAC6. However, the precise mechanism by which HDAC6 is activated will require more experiments. Indeed, we noticed that deacetylation of microtubules was not significantly different when HDAC6 was cotransfected with either the constitutively activated form of mDia2 (GFP-mDIA2-
GBD) or its wild-type counterpart. Together with the fact that mDia2 partially colocalised with HDAC6 on microtubules in osteoclasts (data not shown), we propose that an mDia2/HDAC6 complex is constitutively formed and that Rho activation localises that complex on microtubules to promote its in vivo deacetylation. Knowing where Rho is activated in this context is of major importance. Finally, mDia2 binding requires the cooperation of the deacetylase domains, DD1 and DD2 of HDAC6, which are also required for HDAC6 to bind tubulin (Zhang et al., 2003
). Whether that complex is dependent upon tubulin binding to be assembled and/or active remains to be answered. Nonetheless, our results highlight the requirement for a fine control of Rho-dependent microtubule acetylation in osteoclasts. It would be of interest to analyse osteoclast podosome patterning in an HDAC6/ background.
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Acknowledgments |
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Footnotes |
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Present address: Boyer Center for Molecular Medicine, Yale School of Medicine, 295 Congress Avenue, 06510 New Haven CT, USA
Present address: CMU-Dpt physiologie cellulaire et métabolisme, 1 rue Michel Servet, 1211 Geneve 4, Switzerland
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