Myotubularin subcellular localization - Semantic Scholar

Research Article

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The PtdIns3P phosphatase myotubularin is a cytoplasmic protein that also localizes to Rac1inducible plasma membrane ruffles Jocelyn Laporte§, Francois Blondeau*, Anne Gansmuller, Yves Lutz‡, Jean-Luc Vonesch and Jean-Louis Mandel§ Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, 1 rue Laurent Fries, BP 10142, 67404 Illkirch Cedex, CU de Strasbourg, France *Present address: McGill Cancer Centre, McGill University, 3655 Promenade Sir William Osler, McIntyre Medical Sciences Building, Room 702, Montreal, Québec, Canada H3G 1Y6 ‡Present address: INSERM U 338, Centre de Neurochimie, 5 rue Blaise Pascal, 67084 Strasbourg, France §Authors for correspondence (e-mail: [email protected])

Accepted 1 May 2002 Journal of Cell Science 115, 3105-3117 (2002) © The Company of Biologists Ltd

Summary Myotubularin, the phosphatase mutated in X-linked myotubular myopathy, was shown to dephosphorylate phosphatidylinositol 3-monophosphate (PtdIns3P) and was also reported to interact with nuclear transcriptional regulators from the trithorax family. We have characterized a panel of specific antibodies and investigated the subcellular localization of myotubularin. Myotubularin is not detected in the nucleus, and localizes mostly as a dense cytoplasmic network. Overexpression of myotubularin does not detectably affect vesicle trafficking in the mammalian cells investigated, in contrast to previous observations in yeast models. Both mutation of a key

Introduction Myotubularin is a lipid phosphatase that was initially identified as the product of the human MTM1 gene, mutated in patients with X-linked myotubular myopathy (Laporte et al., 1996). X-linked recessive myotubular myopathy (XLMTM; OMIM310400) is a rare congenital muscle disorder characterized by severe hypotonia and generalized muscle weakness at birth in affected males (Fardeau, 1992; Wallgren-Pettersson et al., 1995). More than 130 different hMTM1 mutations have been found in patients, including 60 missense changes (Laporte et al., 2000; Mandel et al., 2002). The classic severe form usually leads to the death of patients within the first year of life, due to respiratory insufficiency, and is associated in most cases with a complete loss of protein function (truncating mutations). Missense mutations affecting non-catalytic regions are often associated with a milder phenotype, allowing prolonged survival (Laporte et al., 2000). In long-term survivors, tissues other than muscle may be affected (Herman et al., 1999) and, for example, several patients died from liver haemorrhagy. The characteristic muscle histopathology consists of small rounded muscle cells with centrally located nuclei resembling fetal myotubes, suggesting that the disorder results either from an arrest in the normal development of muscle fibers or from a defect in the structural organization of the fibers (Fardeau, 1992; Sewry, 1998). In these fibers, nuclei are surrounded by an area devoid of myofibrils but containing

aspartate residue of myotubularin and dominant activation of Rac1 GTPase lead to the recruitment of myotubularin to specific plasma membrane domains. Localization to Rac1induced ruffles is dependent on the presence of a domain highly conserved in the myotubularin family (that we named RID). We thus propose that myotubularin may dephosphorylate a subpool of PtdIns3P (or another related substrate) at the plasma membrane. Key words: Myotubularin, Myotubular myopathy, Phosphatidylinositol 3-monophosphate, Membrane trafficking, Rac GTPase, Phosphatase, RID domain

mitochondria and organelles, and myofibrils appear partially disorganized. Myotubularin defines a large family of proteins conserved through evolution, from yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe to mammals (Laporte et al., 1998). Myotubularin-related genes (MTMR) are also present in plants (Arabidopsis thaliana) but not in bacteria, and recent updates have shown the presence of at least ten expressed MTMR genes in the human genome (Laporte et al., 2001a; Wishart et al., 2001). Most of these genes present a ubiquitous expression, as tested on northern blot. Truncating mutations in hMTMR2, the closest homolog of hMTM1 or hMTMR2, are responsible for autosomal recessive demyelinating neuropathy, Charcot-Marie-Tooth type 4B [CMT4B (Bolino et al., 2000)]. Other forms of Charcot-Marie-Tooth neuropathy are caused by mutations in genes encoding myelin proteins or Schwann cellspecific proteins (Timmerman et al., 1998). Myotubularin contains the consensus signature of the tyrosine and dual-specificity phosphatase (PTP), His-Cys-X2Gly-X2-Arg, and was shown to dephosphorylate phosphoserineand phosphotyrosine-containing peptides in vitro (Cui et al., 1998). However, more recent work showed that myotubularin dephosphorylates phosphatidylinositol 3-monophosphate (PtdIns3P) much more efficiently, and in vivo experiments in yeast models showed that myotubularin modulates PtdIns3P levels (Blondeau et al., 2000; Taylor et al., 2000). Myotubularin may also directly downregulate phosphatidylinositol 3-kinase

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(PtdIns 3-kinase), because a mutant where the putative catalytic aspartate has been replaced (D278A mutation, designed to have substrate-trap properties) localizes to the plasma membrane and can co-immunoprecipitate the VPS34 PtdIns 3-kinase activity in S. pombe (Blondeau et al., 2000). Some homologs of myotubularin contain a FYVE-finger domain (Laporte et al., 2001a; Wishart et al., 2001), known to bind specifically to PtdIns3P in proteins such as the early-endosomal antigen 1 [EEA1 (Gaullier et al., 1998)]. PtdIns3P localizes mainly onto the endosomes, where it interacts with FYVE-finger proteins that regulate the endocytic pathway (Gillooly et al., 2000). Myotubularin and the other MTMRs also contain a specific domain that was called SID (SET-interacting domain) as it was first reported in the inactive phosphatase hMTMR5/Sbf1 protein (Cui et al., 1998). Sbf1 is believed to protect the phosphorylation state of specific substrates, especially nuclear SET transcriptional regulators from the trithorax family (Firestein et al., 2000). To analyze the biological function of myotubularin and its potential role in membrane trafficking, we have developed a panel of specific antibodies and investigated the subcellular localization of myotubularin by subcellular fractionation and immunohistochemistry on transfected cells, and also under perturbed conditions either after mutation of myotubularin or by induction of plasma membrane changes. We conclude that myotubularin is not in the nucleus as first reported (Cui et al., 1998), but rather localizes as a dense cytoplasmic network which is not yet identified. Under perturbation by mutation or by Rac1 GTPase, it localizes to specific plasma membrane sites. We propose that myotubularin interacts at the plasma membrane with a subpool of PtdIns3P or with other phosphoinositides, and may be implicated in membrane trafficking. Materials and Methods Cell culture and antibodies COS cells were grown in Dulbecco+5% fetal calf serum (FCS) and HeLa cells in Dulbecco+10% FCS. Lymphoblastoid cell lines from control and from a patient (deleted for exons 1 to 13 of the hMTM1 gene) were grown in RPMI+10% FCS. C2C12 mouse myoblasts were maintained in DMEM+20% FCS and differentiated at confluence into myotubes in DMEM+5% FCS for at least 2 days. Media included 500 U/ml penicillin and 400 µg/ml gentamycin. Cells were tranfected with either the calcium phosphate method or Ex-Gen 500 (Euromedex, France), the precipitate was removed 12 hours after transfection and cells were allowed to grow for another 24 hours. We used monoclonal antibodies directed against vimentin (LN6), α-tubulin, vinculin (all from Sigma), phosphotyrosine (4G10, UBI), dynamin (Upstate Biotechnology). Rabbit anti-Rab5 and rabbit anticaveolin 1 were purchased from Santa Cruz Biotechnology and rabbit anti-actin from Sigma. Anti-myc and anti-flag monoclonal and polyclonal antibodies were produced in house (IGBMC). Secondary antibodies were goat anti-mouse Kappa-specific (Southern Biotechnology Associates) and goat anti-mouse or goat anti-rabbit (Jackson Immunoresearch Laboratories) all conjugated to peroxidase for western blotting detection, goat anti-mouse or anti-rabbit Cy3 and biotin-SP-conjugated donkey anti-rabbit from Jackson Immunoresearch Laboratories, and fluorescein (DTAF)-conjugated streptavidin (Immunotech). Wortmannin, LY294002 and Cytochalasin D were purchased from Sigma. Anti-myotubularin antibodies Monoclonal antibodies were raised against full-length human

myotubularin produced in Baculovirus (antibodies 1C7, 1F8, 1G1, 2D2, 2E12 and 2H6), or against peptides conjugated to ovalbumine: peptide SLENESIKRTSRDGVNRDLT corresponding to amino acids 13 to 32 (antibody 1G6) and peptide LANSAKLSDPPTSPSSPSQMM corresponding to amino acids 575 to 596 (antibody 1D10). Production of His-tagged human myotubularin in the Baculovirus system is described elsewhere (Laporte et al., 1998). Mice injections, myeloma fusions and ascite production were carried out as described (Devys et al., 1993). Polyclonal antibodies were raised in New Zealand White male rabbits either against full-length myotubularin (antibody R1208), against peptides described above (antibodies R929 and R1141) or against peptide SSGKSSVLVHCSDGWDRTAQL corresponding to the PTP active site (amino acids 365-385, antibody R1015). All the produced sera and ascite fluids were screened against the antigens on a differential ELISA test and against the full-length myotubularin overexpressed in COS cells by western blotting and immunofluorescence microscopy (Fig. 1A). They were also characterized by immunoprecipitation against the endogenous human myotubularin (Laporte et al., 2001b). Type and class of the monoclonal antibodies were determined by using an isotyping kit (Amersham). Epitope mapping was performed by immunocytochemistry in transfected cells with the full-length, Nterminal and C-terminal constructs described below. Plasmids and constructions The full-length open-reading-frame of human hMTM1 gene (GenBank U46024) was subcloned as described into pCS2 eukaryotic expression vector (Blondeau et al., 2000). A panel of deletions and amino acid changes were engineered by PCR-based mutagenesis from the wild-type myotubularin construct using Deep Vent DNA polymerase (Ozyme) and confirmed by sequencing. The 2XFYVE probe interacting with PtdIns3P provided by H. Stenmark, Institute for Cancer Research, Oslo, Norway (Gillooly et al., 2000) was recloned into pCMVTag3B (Stratagene) with an N-terminal myc-tag. The pCDNA3-desmin construct was provided by P. Vicart (CNRS VMR 7000, Paris, France), HA-tagged GTPase dominant and negative mutants cloned in pEF-BOS by Y. Imai (National Institute of Neuroscience, Tokyo, Japan) (Ohsawa et al., 2000), flag-tagged Rac1 V12 by Y. Takai and T. Takenawa (Institute fo Medical Science, Osaka and Tokyo, Japan) (Mochizuki et al., 1999) and PML expression construct by R. Losson (IGBMC, Illkirch-France). Immunofluorescence microscopy Cells were grown either onto a glass coverslip (COS) or onto glass Lab-Tek chamber slides (Nalge Nunc Int.), and transfected and fixed with 4% paraformaldehyde. They were subsequently permeabilised in PBS with 0.3% Triton X-100. Subcellular localization of myotubularin constructs was assessed using either monoclonal antibodies 1G6 (1:1000), or 1D10 (1:500) for C-terminal constructs, or with rabbit R929 (1:500). Cy3- or biotin-conjugated secondary antibodies and DTAF-streptavidin were used for single and colocalization experiments following manufacturers recommendations. Actin was labelled with phalloidin-TRITC. Fluorescence was examined under a DMLB microscope or a laser scanning TCS4D microscope for confocal analysis (Leica). Immunoprecipitation and immunoblotting The entire procedure was carried out at 4°C. Whole-cell extracts from cultured cells were obtained by homogenization in lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 1 mM Pefabloc and 1 mM sodium orthovanadate) and from mouse tissues by homogenization in TGEK buffer (50 mM Tris-HCl pH 7.8, 10% glycerol, 1% NP-40, 5 mM KCl, 1 mM EDTA, 1 mM Pefabloc and 1 mM orthovanadate). Extracts were passed five times through a 25G

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needle to disperse aggregates and insoluble material was removed by centrifugation at 7000 g for 10 minutes. The same amount of protein per sample (at least 3 mg) was mixed overnight with 5 µl of ascites fluid or rabbit sera or 30 µl of hybridoma supernatant. Immunocomplexes were collected by centrifugation after incubation with 40 µl of protein G-agarose beads for 1 hour. Beads were washed four times with lysis buffer (including 400 mM NaCl), resuspended in loading buffer (8% SDS, 40% glycerol, 240 mM Tris pH 6.8, 0.004% bromophenol blue), boiled for five minutes and loaded onto an 8% SDS-polyacrylamide gel. Proteins were electrotransferred onto nitrocellulose membranes that were blocked with 2% BSA in TBS (Tris buffer saline) plus 0.05% Tween-20 and then incubated with the different primary antibodies for 1 hour. Detection was achieved with secondary antibodies coupled to peroxidase with Supersignal Substrate (Pierce, IL).

Subcellular fractionation Cells were resuspended at 4°C in buffer A (10 mM Tris-HCl pH 7.5, 0.3 M sucrose, 1.5 mM MgCl2, 5 mM KCl, 1 mM EDTA, 1 mM Pefabloc protease Fig. 1. Characterization of myotubularin antibodies. (A) ‘Structural epitope’ means that inhibitor, 1 mM orthovanadate) for lymphoblasts the antibodies recognized only the full-length myotubularin but not overlapping and myotubes or in buffer B (50 mM Tris-HCl pH fragments. Western blotting and immunocytochemistry results are from transfected 7.5, 150 mM NaCl, 1% NP-40, 1 mM Pefabloc, 1 cells as detection of endogenous myotubularin was unsuccessful with these two mM orthovanadate) for HeLa cells, and lysed by methods. Immunoprecipitation data were obtained for the endogenous myotubularin passing through a 25G syringe and a Dounce from muscle cells and are described elsewhere (Laporte et al., 2001b). +, the antibody homogenizer 20 times. P1 (nuclei) and S1 is working with the corresponding technique; –, no signal has been detected. At least (organelles and cytoplasm) were separated by 2D2, 1G1 and R1208 crossreact with mouse myotubularin, while 1G6, 1D10, R929 and centrifugation at 1,000 g for 15 minutes and S1 R1141 do not. 1C7 does not immunoprecipitate the mouse myotubularin. R1208 supernatant was centrifuged at 100,000 g for 1 hour crossreacts with MTMR1 while none of these antibodies crossreact with hMTMR2 and to yield the P2 (big organelles) and S2 (microsomal hMTMR3 proteins. (B) Example of immunoprecipitant antibodies crossreacting with fraction and cytoplasm) fractions. Fractionation the endogenous mouse mMTM1 myotubularin. Mouse C2C12 myotube protein extract was monitored by phase-contrast microscopy and or buffer (/) were immunoprecipitated (IP) with the listed antibodies and the purified using different antibodies. mouse myotubularin was detected by western blot with the 2D2 antibody (1/2000) For cytoskelatal fractionation, transfected HeLa followed by an anti-Kappa light chain (1/2500). Myotubularin has an estimated cells or C2C12 myotubes were lysed in the molecular weight of 70 kDa compared with size markers. Transfected COS cells with cytoskeleton stabilizing buffer (10 mM Pipes pH human myotubularin serve as a size control on the left. R1203 is a serum from a 6.8, 250 mM sucrose, 3 mM MgCl2, 120 mM KCl, different rabbit immunized as for R1208. The dog myotubularin was also 1 mM EGTA, 0.15% Triton X-100, 1 mM immunoprecipitated and detected with the 1G1 and 2D2 monoclonal antibodies Pefabloc), centrifuged at 4°C at 14,000 g for 10 respectively (not shown). minutes (Ogata et al., 1999). The pellet contained the polymerized actin and the intermediate filament, while the supernatant contained the depolymerized actin and tubulin. Fractions were further subjected to used were either peptides (see Materials and Methods) or fullmyotubularin immunoprecipitation and western blotting. length myotubularin produced in Baculovirus (Laporte et al., Pulse-chase COS cells transiently transfected with wild-type myotubularin were starved in DMEM methionine- and cysteine-free for 1.5 hours, and then pulsed with 300 µCi of [35S]methionine/cysteine at 37°C for 1 hour. Cells were washed in medium and chased with pre-warmed DMEM+10% FCS for the indicated time lapses. Proteins were prepared and immunoprecipitated as before.

Results Characterization of specific antibodies and myotubularin isoforms We raised eight monoclonal and four polyclonal antibodies directed against human myotubularin (hMTM1). Antigens

1998). Antibodies were characterized by western blotting and immunohistochemistry on transfected COS and HeLa cells and some of them can immunoprecipitate the endogenous myotubularin from muscle cells (Fig. 1A). Direct detection of endogenous myotubularin by immunohistochemistry was not successful and detection by western blot usually requires prior immunoprecipitation as a concentration step. Immunoprecipitation of myotubularin can be used as a complementary diagnostic test for XLMTM (Laporte et al., 2001b). This panel of antibodies, directed against different epitopes, allowed us to detect untagged full-length or truncated myotubularin in all subsequent experiments. Some antibodies crossreact with mouse myotubularin (Fig. 1B) and will thus be helpful for analysis in mouse. As the hMTM1 protein shares high sequence similarity with the hMTMR1 and hMTMR2

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proteins, we tested for possible crossreaction with these proteins. Only the R1208 polyclonal was found to crossreact with the hMTMR1 protein (not shown). The hMTM1 gene is ubiquitously expressed and ESTs from at least 24 different tissues can be found in databases. A muscle and testis mRNA isoform was found but differs from the ubiquitous 3.4 kb mRNA only by a different polyadenylation site (Laporte et al., 1996). In order to confirm the ubiquitous presence of myotubularin and to check if there are protein isoforms, which would give a clue to the tissue specificity of the disease, we immunoprecipitated myotubularin from ten different mouse tissues. This confirmed that myotubularin can be found in every tested tissues, although the amount was higher in heart and muscle and quite low in brain (Fig. 2A). This may be due to the difference of the stability of myotubularin in various tissues. Although the coding mRNA appears to be the same size in all tissues, a shorter protein isoform was detected in intestine and kidney, whereas the usual 70 kDa product was absent. We have not investigated further these two tissues. A longer separation on 8% acrylamide gels revealed a doublet specific to muscle and heart. The additional musclespecific isoform is absent in myoblasts and myotubes and is specific to adult muscle (Fig. 2B). Thus, it appears at a late differentiation step, believed to be primarily affected in XLMTM patients. We cannot exclude that it results from splice variants, but a search using a set of primers spanning the coding region did not reveal such variants (A. Buj-Bello, personal communication). This adult muscle-specific isoform may rather represent a post-translational modified form, such as a phosphorylated form.

Fig. 2. Tissue expression and isoforms of myotubularin. (A) Three mg of total protein extracts from different tissues of a 14-week-old mouse were immunoprecipitated with 1G1 antibody and loaded on a 8% SDS-PAGE gel. Myotubularin was detected after western blotting with the 2D2 antibody (1/2000). Below, a longer migration reveals the presence of a doublet specific to muscle (and heart, not shown). Different percentages of acrylamide and gradients tested did not resolve the two bands any better. The arrow indicates the migration of the 603 amino acid myotubularin construct transfected into cells, representing the common isoform. (B) The muscle-specific isoform appears after myotube formation. C2C12 mouse myoblasts were differentiated at confluence in DMEM+5% FCS into myotubes, and myotubes were stimulated with insulin like growth factor (IGF1 from Calbiochem at 25 ng/ml) as indicated. Protein extracts were prepared at different time points during differentiation and compared with adult muscle and liver tissues.

Myotubularin is cytoplasmic and at the plasma membrane In order to document precisely the subcellular localization of myotubularin, we performed numerous immunocytochemistry experiments on transfected COS cells, mouse myoblasts and myotubes (Fig. 3A-C) and on HeLa cells, 3T3 fibroblasts and human muscle cells (data not shown). Untagged full-length myotubularin was used and all the antibodies described in this

Fig. 3. Myotubularin is cytoplasmic and associated with plasma membrane. (A) Wild-type untagged myotubularin overexpressed in COS cells was detected with the specific 1G6 monoclonal antibody (1/1000) followed by Cy3-conjugated goat anti-mouse antibody (1/300). Confocal microscopy analysis does not show any nuclear signal. The inactive C375S mutant was also localized as a dense cytoplasmic network in different cell types (COS, HeLa, 3T3 fibroblasts, myoblasts and myotubes). Also note the labeling of plasma membrane. Preimmune serum or immunizing peptide competition (100 µg, 30 minutes, room temperature) abolished the signal. (B,C) Localization in transfected C2C12 mouse myoblasts and myotubes respectively. (D) Transfected COS cell showing altered cell shape and presence of myotubularin in extended filopodia. This pattern was also observed with inactive myotubularin mutants.

Myotubularin subcellular localization study showed the same pattern in all the cell lines tested. Myotubularin localizes as a dense cytoplasmic network with no signal in the nucleus as shown by confocal microscopy (Fig. 3A). We can thus rule out that myotubularin has a nuclear localization under normal growth conditions. Myotubularin also labeled the plasma membrane (Fig. 3; see also Figs 7, 8) including plasma membrane extensions such as filopodia (Fig. 3D; see also Fig. 8A) and ruffles (see example on Fig. 7A). Myotubularin localization is not modified by fusion of myoblasts into myotubes (Fig. 3C). Similar localization data were obtained with peroxidase labeling and optical microscope analysis (data not shown). In a small subset of transfected cells, myotubularin overexpression altered the shape of the cell, producing numerous filopodias (Fig. 3D). This was especially noted in highly overexpressing cells and confirmed in HeLa cells, where the same morphology as in Fig. 3D could be observed (not shown). As this phenotype could also be seen with enzymatically inactive myotubularin constructs (C375S and D278A mutants), it is not dependent on the enzymatic activity (not shown). As a filamentous cytoplasmic localization was clearly observed in some cells, suggestive of cytoskeletal networks, we performed co-localization experiments. Overexpressed desmin, the localization of which was reported to be modified in some XLMTM patients (Sarnat, 1992), and endogenous cytoplasmic actin, tubulin and keratin showed no colocalization. Vimentin, an intermediate filament protein, showed partial co-localization but confocal microscopy analysis did not allow unambiguous conclusion, as both myotubularin and vimentin appeared as dense networks (not shown). Electron microscopy analysis of transfected HeLa

Fig. 4. Subcellular distribution of endogenous myotubularin in (A) lymphoblasts from normal (L1421) or an XLMTM patient deleted for exons 1-13 of the hMTM1 gene (89-441), (B) HeLa cells and (C) mouse myotubes. Subcellular fractions were prepared as described in Materials and Methods and enrichment was confirmed under the microscope (example below the western blot in A) and with protein markers (tubulin is present in the same fraction as myotubularin). Myotubularin was immunoprecipitated from the different fractions with 1G1 antibody and detected on a western blot by 1G6 (1/10,000) for the human cell lines and 2D2 (1/2000) for the mouse myotubes. P1, nucleus; P2, big organelles; S1, cytoplasm and all organelles; S2, cytoplasm and small organelles; T, total extract; TR, myotubularin overexpressed in COS cells. (D) Cytoskeleton fractionation of HeLa cells transfected with wild-type (WT) and substrate-trap mutant (D278A) myotubularin. Actin-based microfilaments and intermediate filaments containing vimentin were recovered in the cytoskeletal (P) fraction, whereas actin monomers and tubulin were recovered in the cytosolic (S) fraction. (E) The same cytoskeleton fractionation applied to mouse C2C12 myotubes. Myotubularin was immunoprecipitated and detected as above.

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cells suggests that myotubularin is not associated to vesicles nor to internal membranes (not shown). In conclusion, myotubularin localizes to a dense cytoplasmic network not related to known cytoskeletons and labels plasma membrane, including extended filopodia in highly overexpressing cells. The localization of endogenous myotubularin was also assessed by subcellular fractionation followed by immunoprecipitation from lymphoblasts, myoblasts and HeLa cells (Fig. 4). Consistent with the previous results in transfected cells, endogenous myotubularin from lymphoblasts was enriched in the cytoplasmic fraction and nearly undetectable in the nuclear fraction (Fig. 4A). In the same experiment, myotubularin was not detected in an XLMTM patient cell line deleted for exons 1-13 of the hMTM1 gene. We fractionated further the first supernatant (S1) to separate big organelles from cytoplasm and small organelles (P2 and S2, respectively). This latter protocol confirmed in HeLa cells that myotubularin was absent from the nuclear fraction and from the big organelles, and was enriched in the most soluble fraction containing cytoplasm and ribosomes (Fig. 4B). Myotubularin co-purified with tubulin, a cytoplasmic protein, in each experiment. To check whether endogenous myotubularin could be localized to other compartments upon differentiation of muscle cells, we also performed the same fractionation experiment on C2C12 mouse myotubes extracts, using antibodies crossreacting with mouse myotubularin (here, 1G1 and 2D2). Again, myotubularin was absent in the nuclear fraction and enriched in the cytoplasmic fraction (Fig. 4C). As the localization pattern of myotubularin, especially the association to the plasma membrane, suggests that myotubularin could be linked to the actin microfilaments, we

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performed a cytoskeletal fractionation to separate the polymerized actin and vimentin network from the soluble actin and tubulin. HeLa cells transfected either with the wild-type myotubularin, or with the D278A mutant, which is solely found at the plasma membrane (Blondeau et al., 2000), were extracted with a triton-based buffer. Wild-type myotubularin was enriched 14-fold compared with actin in the soluble fraction (Fig. 4D), suggesting that it is not tightly associated to polymerized actin and intermediate filaments, even at the plasma membrane. The enrichment ratio of the D278A mutant compared with actin in the soluble fraction is only twofold, but this may be a bias as the pellet might contain some membranes. Cytoskeletal fractionation also performed on C2C12 mouse myotubes confirmed that myotubularin is found essentially in the soluble fraction, even after myotube formation (Fig. 4E). Effect of protein domains and XLMTM mutations We have investigated the protein domains involved in the subcellular localization of myotubularin. For this purpose, we generated a collection of deleted myotubularin constructs, including deletion of known domains such as the SETinteracting domain [SID (Cui et al., 1998)] and the GRAM domain, found in glucosyltransferases, Rab-like GTPase activators and myotubularins (Doerks et al., 2000). The protein domains are indicated on Fig. 5A, and in addition to the SID and GRAM domains, include the PTP signature responsible for the enzymatic activity, a PEST sequence with significant score, and a putative PDZ-binding site (PDZ-BS), which was shown to be functional in the homolog hMTMR1 (Fabre et al., 2000). The vast majority of the deleted constructs produced unstable products, as the resulting protein localized as aggregates in the cytoplasm and near the nucleus, probably in the Golgi, and a very low protein level was detected when some constructs were tested on western blot (not shown). Strikingly, the C-terminal half of myotubularin (from amino acids 336 to the stop codon) showed a punctuated nuclear signal, together with cytoplasmic dots and aggregates (Fig. 5A,B). As the SET-interacting domain shown to mediate interaction with heterochromatin proteins is localized in the C-terminal part of myotubularin (Cui et al., 1998), we checked whether this domain was responsible for the nuclear localization of truncated C-terminal myotubularin. A C-terminal construct lacking the SID does not localize to the nucleus any more (Fig. 5B), while deletion of a more terminal part (amino acids 482-494) did not affect the nuclear localization (Fig. 5A). This confirmed that the SID is indeed responsible for the punctuated nuclear localization of the C-terminal construct. As shorter constructs (e.g. C-terminal deleted for the SID) do not localize to the nucleus, the nuclear localization of the C-terminal construct is not due to passive diffusion into the nucleus. The C-terminal construct does not co-localize in the nucleus with Hoechst-labeled heterochromatin. PML (promyelocytic leukaemia), a nuclear protein that does not localize to heterochromatin, showed perfect co-localization in the majority of cells with the Cterminal construct, as assessed by confocal microscopy (Fig. 5C), and this was confirmed by 3D reconstruction (not shown). However, other transfected cells clearly showed smaller nuclear dots that do not co-localize with PML nor with Hoechst-stained heterochromatin (Fig. 5C). One explanation could be that the nuclear subset of the C-terminal construct

Fig. 5. Effect of protein domains and XLMTM mutations on the subcellular localization of myotubularin. (A) Schematic representation of myotubularin showing protein domains. GRAM, glucosyltransferase, Rab-like GTPase activator and myotubularin common domain (Doerks et al., 2000) (aa 29-97); RID, Rac1induced localization to membrane ruffles (around aa 233-237); PTP, tyrosine phosphatase-like signature implicated in the lipid phosphatase activity (aa 371-385) with catalytic residues D278, C375 and R381; SID, SET-interacting domain (aa 435-486); PEST, (aa 581-598) with a significant PESTfind score of +8.23; PDZ-BS, putative PDZ-binding site functional in the hMTMR1 homolog (Fabre et al., 2000) (aa 599-603). Highly conserved regions through evolution correspond to a high frequency of missense mutations in XLMTM patients and are also indicated (aa 170-330, 45% identity with the S. cerevisiae protein; and aa 370-490, 55% identity with the S. cerevisiae protein). Below are indicated the subcellular localization and the Rac1-induced localization to membrane ruffles for some constructs. The subcellular localization of the depicted constructs were obtained from immunofluorescence experiments with either the N-terminal 1G6 or the C-terminal 1D10 antimyotubularin antibodies on transfected COS and HeLa cells. Other constructs produced unstable proteins (aggregates in the cytoplasm and near the nucleus probably in the Golgi and very low myotubularin levels on western blot): del(1-95), del(97-122), del(183-245), del(224-245), del(308-325), del(396-406), del(437469), del(482-494) and amino acid changes G378R, D394A, G402A, E404K, E410A, D443A, C444Y and H469P. Mutation of the conserved aspartate at position 257 (D257A) did not affect the localization of myotubularin. Note that missense mutations affecting R241 (mild phenotype) impaired the in vitro enzymatic activity toward PtdIns3P (Taylor et al., 2000) and lead to a decrease in protein level in a patient cell line (Laporte et al., 2001b), while mutations G378R (severe phenotype) impaired the in vitro enzymatic activity and G402A (probably severe) leads to a decrease in protein level in a patient cell line. (B) Confocal microscopy analysis of a truncated myotubularin shows nuclear localization (C-ter construct). Deletion of the SET-interacting domain from this construct abolished the nuclear localization. (C) Co-localization of the C-ter construct with PML in more than 50% of co-transfected cells (confocal microscopy). In the same experiment, other co-transfected cells showed no obvious co-localization.

aggregates and is trapped by PML bodies. The N-terminal construct encompassing amino acids 1-369 has a cytoplasmic localization more similar to that of wild-type myotubularin (Fig. 5A), and this region would thus be implicated in the subcellular localization of full-length myotubularin. We tested the subcellular localization of mutants with an amino acid change at key catalytic residues (C375S, D278A), and mutants of conserved aspartate residues at position 377, 380, 394 or 443. As described previously, mutation D278A produces an enzymatically inactive myotubularin that behaves as a substrate-trap and localizes solely to plasma membrane extensions (Blondeau et al., 2000). Indeed, mutation of the catalytic aspartate in tyrosine phosphatases leads to the localization of the phosphatase to the substrate’s subcellular sites, or the reverse (Flint et al., 1997). Additional sequences from homologs highlighted another conserved aspartate (D257), but subcellular localization of a D257A construct was similar to wild-type myotubularin in transfected COS cells (not shown). Mutation of the catalytic cysteine does not affect localization, while, in some tyrosine phosphatases, it also induced a different localization of the protein (Liu and Chernoff, 1997). Positively charged aspartate residues at


position 377 and 380 in the phosphatase signature are believed to contribute to the substrate interaction and would explain the specificity towards phosphatidylinositol monophosphate (Laporte et al., 2001a; Wishart et al., 2001). Mutation of these residues did not affect the localization of the resulting proteins

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(Fig. 5A), but abrogated their lipid phosphatase activity (Wishart et al., 2001). Lastly, we also tested the subcellular localization of mutants found in XLMTM patients (R241C and G378R in the PTP, C444Y and H469P in the SID, G402A, E404K and R421Q ).

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Most of these mutants showed a cytoplamic signal with cytoplasmic aggregates. Aggregation suggests that these mutants are unstable and this is consistent with the fact that immunoprecipitation from XLMTM patient cell lines showed a decrease in myotubularin level in 87% of the cases including some missense mutations (Laporte et al., 2001b). Thus, absence or instability of mutated myotubularin appears as the main cause of the disease. However, the R421Q mutant found in severe cases of XLMTM localized as wild-type myotubularin, and labeled filopodia were consistently present in all transfected cells (J.L., unpublished). Turn-over of myotubularin Sequence analysis using the PESTfind algorithm predicted in the C-terminal portion of the protein a PEST sequence with a significant score of +8.23 (Rechsteiner and Rogers, 1996). The presence of the PEST sequence and the fact that we cannot detect the endogenous protein by immunohistochemistry or by direct western blot suggested that myotubularin is rapidly degraded, especially as the amount of myotubularin mRNA detected by northern blot and the rather high number of corresponding ESTs indicate that the level of transcript is not very low (Laporte et al., 1998). Moreover, stability of myotubularin seems very sensitive to sequence changes (Laporte et al., 2001b). Despite the presence of this PEST motif, a pulse-chase labelling experiment indicated that myotubularin has a slow turn-over in transfected cells, with an approximate half-life of 4-5 hours (Fig. 6). Thus, the PEST sequence in myotubularin does not, under these conditions, direct very rapid degradation. Myotubularin, vesicle trafficking and PtdIns3P distribution Myotubularin was recently found to be able to dephosphorylate PtdIns3P in vitro and in yeast systems (Blondeau et al., 2000; Taylor et al., 2000). PtdIns3P is a second messenger localized mainly on endosomal vesicles (Gillooly et al., 2000) and interacts with FYVE-domain-containing proteins that regulate vesicle trafficking in yeast and mammalian cells (Stenmark and Aasland, 1999). Overexpression of myotubularin in the yeast

Fig. 6. Turn-over of myotubularin. COS cells transfected with wildtype myotubularin were labeled with [35S]methionine/cysteine and cold chased for 0, 15 or 30 minutes and 1, 2, 3 or 5 hours. Myotubularin was immunoprecipitated with the 1G1 monoclonal antibody. The period of cold chase is indicated above the lanes. Myotubularin is indicated by an arrow. Above, an additional protein is trapped probably by the beads as it is also present in the first lane without immunoprecipitant antibody. The estimated half-life is about 4 hours.

S. pombe impaired vesicle trafficking (Blondeau et al., 2000). We first tested the effect of overexpression of wild-type myotubularin on different endocytosis pathways. In COS cells, we observed no effect on the distribution of dynamin (clathrincoated vesicles), caveolin 1 (uncoated vesicles) and Rab5 (endosomes) compared with untransfected cells (Fig. 7A). However, we cannot exclude that a fraction of myotubularin may associate with these structures. We then compared the distribution of myotubularin and PtdIns3P in cells after cotransfection with constructs expressing myotubularin and the PtdIns3P-specific probe 2XFYVE. The latter contains two FYVE domains, from the receptor tyrosine kinase substrate Hrs, fused to a myc epitope (Gillooly et al., 2000). Myotubularin did not co-localize with the typical vesicular staining pattern of PtdIns3P-coated endosomes (Fig. 7B). In cells overexpressing wild-type myotubularin, there was no obvious change in the level or localization of the 2XFYVE probe. Moreover, overexpression of the 2XFYVE probe generated expanded vacuolar structures in about 30% of the cells, probably due to displacement of FYVE-proteins (such as EEA1) from PtdIns3P, resulting in deregulation of the endocytic pathway (Gillooly et al., 2000). Overexpression of myotubularin did not cause similar changes (Fig. 7A), and deregulation of the endocytic pathway by the 2XFYVE probe did not modify the localization of transfected myotubularin (data not shown). Thus myotubularin does not seem to detectably regulate PtdIns3P content in this system. It also does not co-localize with endosomal PtdIns3P in most co-transfected cells, although it cannot be excluded that a fraction of it may associate. Inactive mutants C375S and D278A behaved similarly, and treatment with the PtdIns 3-kinase inhibitors wortmannin or LY294002 did not change the localization of myotubularin wild-type and D278A mutant (not shown). As shown above, some truncated myotubularin constructs containing the phosphatase active site localized as cytoplasmic dots (e.g. C-ter del SID in Fig. 5C). We show in Fig. 7B that these cytoplasmic dots are not PtdIns3P-containing endosomes. The C-ter del SID protein appears membrane associated and in some cells produces structures similar to vacuoles (Fig. 7B) that have not yet been identified. Myotubularin and plasma membrane remodeling A subset of wild-type myotubularin localizes to the plasma membrane and filopodia (this study), and the D278A mutant, which gains substrate-trapping properties, solely localizes to plasma membrane extensions (Blondeau et al., 2000). This suggested that myotubularin could have a role in plasma membrane remodeling. We first checked whether the plasma membrane localization of wild-type or D278A myotubularin was dependent on the actin cytoskeleton by treatment of transfected cells with the actin microfilament-disrupting agent cytochalasin D. Actin and myotubularin both underlined the shape of the untreated cells. Collapse of the actin network and stress fibers altered the general distribution of myotubularin constructs. However, wild-type and D278A mutant were still present at the plasma membrane (Fig. 8A). In fact, wild-type myotubularin clearly labeled longer filopodias. This suggests that myotubularin is bound directly to plasma membrane components rather than to the underlying actin fibers.


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As high overexpression of myotubularin had an effect on cell shape and as the D278A mutant localizes to a punctuated pattern at the plasma membrane, we investigated whether myotubularin could take part in focal adhesion. Endogenous vinculin at the focal adhesions does not co-localize with the D278A myotubularin mutant in the moving cell of Fig. 8B. This is in agreement with preliminary results showing that this mutant localizes to membrane not in contact with the substratum (Blondeau et al., 2000). This suggests that myotubularin is not implicated in cell movement, although we failed to establish stable cell lines overexpressing myotubularin constructs in order to monitor cell spreading. Next, we induced plasma membrane remodeling by overexpressing a Rac1-dominant-activated construct (Rac1 V12). Rac1 is part of a GTPase subfamily that includes Rho and Cdc42, and is known to play a role in membrane ruffling and pinocytosis through actin remodeling (Ellis and Mellor, 2000). Moreover, Rac1 is a downstream mediator of PtdIns 3kinase, and myotubularin was shown to be implicated in the PtdIns 3-kinase pathway (Blondeau et al., 2000). Induction of membrane ruffles by Rac1 V12 was evident in transfected cells and actin was concomitantly co-localized (not shown). Wild-type myotubularin localized to these Racinduced ruffles (Fig. 8C), as did the inactive mutants C375S and D278A (not shown). In cells transfected by Fig. 7. Effect of myotubularin on endocytic markers and PtdIns3P distribution. (A) COS cells transfected with wild-type myotubularin do not show changes in endocytic markers such as myotubularin alone, a strong labeling dynamin (clathrin-coated vesicles), caveolin 1 (uncoated vesicles), and Rab5 (endosomes). Note of membrane ruffles was sometimes the strong labeling of a membrane ruffle with anti-myotubularin antibody in the caveolin 1 conoted (see Fig. 7A, caveolin 1 costaining. (B) COS cells co-transfected with wild-type or C-ter delSID myotubularin constructs staining). and a myc-tagged 2XFYVE expression construct show no co-localization and no effect of In order to map the Rac1-induced myotubularin with the endosomal staining by 2XFYVE. localization domain (or RID), we cotransfected Rac1 V12 with a panel of truncated and mutated myotubularin other constructs did not localize to ruffles: bigger deletions in constructs. Results are summarized in Fig. 5A. The N-terminal the N-terminal region, but also some missense mutations in construct (aa 1-369) localized to Rac1-induced ruffles (Fig. the C-terminal part (e.g. H469P) that may render 8C), while the C-terminal part (aa 336-603) did not. The myotubularin unstable. smallest deletion that prevented co-localization to Rac1These data show that myotubularin localization to plasma induced ruffles was del(233-237) (Fig. 8C; see also Fig. 5A). membrane ruffles is not dependent on its enzymatic activity, The region from amino acids 179 to 248, which contains the and suggest that myotubularin is recruited to the membrane RID domain, is highly conserved in the myotubularin family rather than to the actin cytoskeleton. Moreover, overexpression (61% aa identity between human myotubularin and the of all the myotubularin constructs listed in Fig. 5A, including drosophila homolog) and is a hot spot for missense mutations wild-type and phosphatase inactive mutants D278A and in XLMTM patients (Laporte et al., 2000). This suggests that C375S, does not detectably affect the Rac1-induced ruffles at the property to localize to ruffles might be shared by the edges and over the entire surface of the cell. myotubularin homologous proteins. It has to be noted that

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Discussion Myotubularin defines a large subgroup of phosphatases within the family of tyrosine and dual-specificity phosphatases (PTP), and has been recently shown to dephosphorylate PtdIns3P (Blondeau et al., 2000; Taylor et al., 2000). We characterized specific antibodies against myotubularin. Direct western blot or immunocytochemistry did not allow detection of the endogenous myotubularin, suggesting that it is present at a very low level. This contrasts with the fact that it is not difficult to detect the RNA transcript by northern blot and with the high number of cDNAs represented as ESTs in the databases. This suggests either a high turnover rate of the protein or a tight translational control. However, myotubularin does not have a very short half-life in transfected cells, in spite of the presence of a predicted PEST sequence at the C-terminus. In contrast with an initial report that myotubularin (like the phosphatase-inactive Sbf1/MTMR5 protein) is located in the nucleus and interacts with nuclear SET proteins via its SID domain (Cui et al., 1998), our results do not show any evidence for the presence of full length myotubularin in the nucleus. However, we have noted that the SID can drive the localization of a truncated portion of myotubularin into nuclear dots. No nuclear localization signal-like sequence is present in the SID, which is rich in hydrophobic residues presumably forming β

sheets. The SID is unlikely to mediate nuclear localization in the context of full-length myotubularin. After submission of the present work, another group reported that full-length Sbf1/MTMR5 protein and myotubularin are indeed cytoplasmic (Firestein and Cleary, 2001). Myotubularin is cytoplasmic in various transfected cells and its localization is not changed after differentiation of myoblasts into myotubes. Endogenous myotubularin is also present in cytoplasmic fractions, as tested by immunoprecipitation and western blotting. Under normal culture conditions, myotubularin localization appears as a dense cytoplasmic network with plasma membrane staining of occasional ruffles and of filopodia in highly overexpressing cells. However, there is no obvious co-localization with known cytoskeletal networks. While myotubularin actively dephosphorylates PtdIns3P, it is striking to note that it does not co-localize extensively with PtdIns3P-containing endosomes and its overexpression does not affect localization of proteins implicated in endocytosis. A punctate and reticular cytoplasmic staining was also recently reported for a tagged MTMR3 protein, with no colocalization with endosomal markers (Walker et al., 2001). In our experiments, overexpression of wild-type myotubularin did not detectably affect PtdIns3P level and


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Fig. 8. Myotubularin and plasma membrane remodeling. (A) HeLa cells transfected with either wild-type or D278A substrate-trap mutant were left untreated (–) or were treated with cytochalasin D at 400 µM for 2 hours (+). Co-localization with endogenous actin showed disruption of the actin filaments, while both myotubularin constructs still showed labeling of the plasma membrane. (B) D278A mutant (in green) transfected in HeLa cells showed no co-localization with focal adhesion labeled by an anti-vinculin antibody (in red). (C) Co-localization of different myotubularin constructs with Rac1-induced plasma membrane ruffles in COS cells co-transfected with constitutively activated Rac1 V12 (flag-tagged). Wild-type myotubularin and its N-terminal part localized to the ruffles while the del(233-237) construct did not.

localization, as tested by cotransfection with a PtdIns3P specific probe (2XFYVE). Although PtdIns3P was shown to be a most effective substrate of myotubularin in yeast and in in vitro experiments, other phosphoinositides might also serve as substrates in higher eukaryotic cells. After submission of the present work, it was reported that the MTMR3 protein dephosphorylates PtdIns(3,5)P2, to yield PtdIns5P (Walker et al., 2001). However, it is also possible that, in our experiments, the concomitant overexpression of the 2XFYVE probe blocked the transient interaction of myotubularin with PtdIns3P. This could explain the apparent discrepancy with the work of Kim et al., published after initial submission of our manuscript, where the authors used a biotinylated 2XFYVE probe for

PtdIns3P labelling (Kim et al., 2002). The localization at the plasma membrane of the enzymatically inactive, substrate-trap D278A mutant also suggests that the physiological substrate of myotubularin might not be endosomal PtdIns3P, but rather a plasma membrane subpool of PtdIns3P (or of another phosphoinositide). Monitoring levels of PtdIns3P and other phosphoinositide in tissues from myotubularin knockout mice may provide a more definitive answer on the nature of myotubularin physiological substrate. The presence of myotubularin at the plasma membrane (enhanced in the D278A mutant) may also be the result of an interaction with a PtdIns 3-kinase. The latter enzymes are known to localize to plasma membrane under growth factor

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stimulation and membrane remodeling (Tsakiridis et al., 1999). Indeed, the D278A mutant was able to co-immunoprecipitate the only known PtdIns 3-kinase activity in S. pombe (Blondeau et al., 2000). Rac1-induced remodeling of the plasma membrane leads to the localization of myotubularin to membrane ruffles, and this is independent of the phosphatase activity. High overexpression of myotubularin also induces filopodia extension and affects cell shape, although it does not modify the Rac1-induced membrane ruffles. Localization to Rac1induced ruffles is dependent on a myotubularin domain that we named RID, which is located within a highly conserved region in the myotubularin family, but that shows no resemblance with protein domains of known function. The presence of a GRAM domain in myotubularin (different from the Rac1-induced localization domain), which is shared with proteins implicated in membrane trafficking such as Rablike GTPases activators (Doerks et al., 2000), also suggests a role at the plasma membrane. A putative PDZ-binding site is also present in myotubularin, and was shown to be active in its close homolog hMTMR1 (Fabre et al., 2000). It could thus mediate interaction of myotubularin with PDZ-containing proteins that are known to be implicated in the organization of specific plasma membrane domains (Fanning and Anderson, 1999) and in membrane trafficking (Cao et al., 1999). It is also worth noting the striking resemblance between the phosphatase active site of myotubularin and that of the PtdIns5phosphatases Sac1p and synaptojanin (they share a DCXD motif not present in other phosphatases); the latter protein is known to play an essential role in synaptic vesicle recycling (Cremona et al., 1999; Harris et al., 2000). In summary, we propose a model where cytoplasmic myotubularin may be localized to the plasma membrane upon Rac and/or PtdIns 3-kinase activation through interaction with one of the many proteins present at these sites, including PtdIns 3-kinases and GTPase exchange factors, but not with actin as we showed that myotubularin still labels plasma membrane in cells where actin fibers are disrupted. At the plasma membrane, myotubularin would be in contact with its substrate, either a subpool of PtdIns3P or another phosphoinositide [such as PtdIns(3,5)P2 (Walker et al., 2001)]. PtdIns(4,5)P2 has been shown to be synthesised at membrane ruffles and is important for vesicle trafficking (Honda et al., 1999; Carpenter et al., 2000), but to date it does not appear as an effective substrate for myotubularin (Taylor et al., 2000). As GTPases link plasma membrane remodeling to endocytic trafficking (Ellis and Mellor, 2000), myotubularin may act as a modulator of GTPase activity or action. Rho and Rac GTPases have been shown to regulate inositol lipid kinases and phosphoinositides levels (Ren and Schwartz, 1998); myotubularin lipid phosphatase may play a role at the plasma membrane to regulate the phosphoinositides pool created by Rac-induced activation of PtdIns 3-kinases. The localization of the D278A mutant to plasma membrane protrusions suggests also a link with the ADP-ribosylation factor 6 (Arf6) GTPase (Radhakrishna et al., 1999; Di Cesare et al., 2000). Arf6 activation is required for the recycling of the endosomal membrane back to plasma membrane and also for plasma membrane ruffling induced by Rac (Al-Awar et al., 2000). Myotubularin may play a similar dual role with its Rac1induced localization domain allowing localization to

membrane ruffles, and with its PtdIns3P phosphatase site as a potential regulation domain for endosomal trafficking. For instance, recycling of endosomes back to the plasma membrane at sites of membrane remodeling may depend upon the release of FYVE-finger regulatory proteins following PtdIns3P dephosphorylation by myotubularin. We have recently constructed a mouse knockout model of myotubular myopathy, that indicates that MTM1 deficiency does not cause a defect in muscle maturation, but rather impairs the function or organisation of muscle fibers (A. Buj-Bello and J.-L.M., unpublished). The study of phosphoinositol metabolism and membrane trafficking in this model should allow a better understanding of the role of myotubularin, especially in muscle. We thank Christine Kretz and Laurent Weiss for excellent technical assistance, Isabelle Kolb-Cheynel for production of recombinant proteins, Yoshinori Imai for Rac1, Rho and Cdc42 dominant active and inactive HA-tagged constructs, Yoshimi Takai for the Rac1 V12flag construct, Patrick Vicart for the desmin construct, Harald Stenmark for the PtdIns3P 2XFYVE probe and Mustapha OuladAbdelghani and Anna Buj-Bello for useful discussions. This study was supported by funds from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Hôpital Universitaire de Strasbourg (HUS), the Louis Jeantet Foundation and by grants from the Association Française contre les Myopathies (AFM).

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