Research Article 3105 The PtdIns3P phosphatase myotubularin is a cytoplasmic protein that also localizes to Rac1- inducible 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 aspartate residue of myotubularin and dominant activation myotubular myopathy, was shown to dephosphorylate of Rac1 GTPase lead to the recruitment of myotubularin to phosphatidylinositol 3-monophosphate (PtdIns3P) and was specific plasma membrane domains. Localization to Rac1- also reported to interact with nuclear transcriptional induced ruffles is dependent on the presence of a domain regulators from the trithorax family. We have highly conserved in the myotubularin family (that we characterized a panel of specific antibodies and named RID). We thus propose that myotubularin may investigated the subcellular localization of myotubularin. dephosphorylate a subpool of PtdIns3P (or another related Myotubularin is not detected in the nucleus, and localizes substrate) at the plasma membrane. mostly as a dense cytoplasmic network. Overexpression of myotubularin does not detectably affect vesicle trafficking Key words: Myotubularin, Myotubular myopathy, in the mammalian cells investigated, in contrast to previous Phosphatidylinositol 3-monophosphate, Membrane trafficking, Rac observations in yeast models. Both mutation of a key GTPase, Phosphatase, RID domain

Introduction mitochondria and organelles, and myofibrils appear partially Myotubularin is a lipid phosphatase that was initially disorganized. identified as the product of the human MTM1 gene, mutated Myotubularin defines a large family of proteins conserved in patients with X-linked myotubular myopathy (Laporte through evolution, from yeasts Saccharomyces cerevisiae and et al., 1996). X-linked recessive myotubular myopathy Schizosaccharomyces pombe to mammals (Laporte et al., (XLMTM; OMIM310400) is a rare congenital muscle 1998). Myotubularin-related genes (MTMR) are also present disorder characterized by severe hypotonia and generalized in plants (Arabidopsis thaliana) but not in bacteria, and recent muscle weakness at birth in affected males (Fardeau, 1992; updates have shown the presence of at least ten expressed Wallgren-Pettersson et al., 1995). More than 130 different MTMR genes in the human genome (Laporte et al., 2001a; hMTM1 mutations have been found in patients, including 60 Wishart et al., 2001). Most of these genes present a ubiquitous missense changes (Laporte et al., 2000; Mandel et al., 2002). expression, as tested on northern blot. Truncating mutations in The classic severe form usually leads to the death of patients hMTMR2, the closest homolog of hMTM1 or hMTMR2, are within the first year of life, due to respiratory insufficiency, responsible for autosomal recessive demyelinating neuropathy, and is associated in most cases with a complete loss of Charcot-Marie-Tooth type 4B [CMT4B (Bolino et al., 2000)]. protein function (truncating mutations). Missense mutations Other forms of Charcot-Marie-Tooth neuropathy are caused by affecting non-catalytic regions are often associated with a mutations in genes encoding myelin proteins or Schwann cell- milder phenotype, allowing prolonged survival (Laporte et specific proteins (Timmerman et al., 1998). al., 2000). In long-term survivors, tissues other than muscle Myotubularin contains the consensus signature of the may be affected (Herman et al., 1999) and, for example, tyrosine and dual-specificity phosphatase (PTP), His-Cys-X2- several patients died from liver haemorrhagy. The Gly-X2-Arg, and was shown to dephosphorylate phosphoserine- characteristic muscle histopathology consists of small and phosphotyrosine-containing peptides in vitro (Cui et al., rounded muscle cells with centrally located nuclei resembling 1998). However, more recent work showed that myotubularin fetal myotubes, suggesting that the disorder results either dephosphorylates phosphatidylinositol 3-monophosphate from an arrest in the normal development of muscle fibers or (PtdIns3P) much more efficiently, and in vivo experiments in from a defect in the structural organization of the fibers yeast models showed that myotubularin modulates PtdIns3P (Fardeau, 1992; Sewry, 1998). In these fibers, nuclei are levels (Blondeau et al., 2000; Taylor et al., 2000). Myotubularin surrounded by an area devoid of myofibrils but containing may also directly downregulate phosphatidylinositol 3-kinase 3106 Journal of Cell Science 115 (15)

(PtdIns 3-kinase), because a mutant where the putative catalytic myotubularin produced in Baculovirus (antibodies 1C7, 1F8, 1G1, aspartate has been replaced (D278A mutation, designed to have 2D2, 2E12 and 2H6), or against peptides conjugated to ovalbumine: substrate-trap properties) localizes to the plasma membrane and peptide SLENESIKRTSRDGVNRDLT corresponding to amino acids can co-immunoprecipitate the VPS34 PtdIns 3-kinase activity 13 to 32 (antibody 1G6) and peptide LANSAKLSDPPTSPSSPS- in S. pombe (Blondeau et al., 2000). Some homologs of QMM corresponding to amino acids 575 to 596 (antibody 1D10). myotubularin contain a FYVE-finger domain (Laporte et al., Production of His-tagged human myotubularin in the Baculovirus system is described elsewhere (Laporte et al., 1998). Mice injections, 2001a; Wishart et al., 2001), known to bind specifically to myeloma fusions and ascite production were carried out as described PtdIns3P in proteins such as the early-endosomal antigen 1 (Devys et al., 1993). Polyclonal antibodies were raised in New [EEA1 (Gaullier et al., 1998)]. PtdIns3P localizes mainly onto Zealand White male rabbits either against full-length myotubularin the endosomes, where it interacts with FYVE-finger proteins (antibody R1208), against peptides described above (antibodies R929 that regulate the endocytic pathway (Gillooly et al., 2000). and R1141) or against peptide SSGKSSVLVHCSDGWDRTAQL Myotubularin and the other MTMRs also contain a specific corresponding to the PTP active site (amino acids 365-385, antibody domain that was called SID (SET-interacting domain) as it was R1015). All the produced sera and ascite fluids were screened against first reported in the inactive phosphatase hMTMR5/Sbf1 the antigens on a differential ELISA test and against the full-length protein (Cui et al., 1998). Sbf1 is believed to protect the myotubularin overexpressed in COS cells by western blotting and phosphorylation state of specific substrates, especially nuclear immunofluorescence microscopy (Fig. 1A). They were also characterized by immunoprecipitation against the endogenous human SET transcriptional regulators from the trithorax family myotubularin (Laporte et al., 2001b). Type and class of the (Firestein et al., 2000). monoclonal antibodies were determined by using an isotyping kit To analyze the biological function of myotubularin and its (Amersham). Epitope mapping was performed by potential role in membrane trafficking, we have developed a immunocytochemistry in transfected cells with the full-length, N- panel of specific antibodies and investigated the subcellular terminal and C-terminal constructs described below. localization of myotubularin by subcellular fractionation and immunohistochemistry on transfected cells, and also under perturbed conditions either after mutation of myotubularin or Plasmids and constructions by induction of plasma membrane changes. We conclude that The full-length open-reading-frame of human hMTM1 gene myotubularin is not in the nucleus as first reported (Cui et al., (GenBank U46024) was subcloned as described into pCS2 eukaryotic expression vector (Blondeau et al., 2000). A panel of deletions and 1998), but rather localizes as a dense cytoplasmic network amino acid changes were engineered by PCR-based mutagenesis which is not yet identified. Under perturbation by mutation or from the wild-type myotubularin construct using Deep Vent DNA by Rac1 GTPase, it localizes to specific plasma membrane sites. polymerase (Ozyme) and confirmed by sequencing. The 2XFYVE We propose that myotubularin interacts at the plasma membrane probe interacting with PtdIns3P provided by H. Stenmark, Institute with a subpool of PtdIns3P or with other phosphoinositides, and for Cancer Research, Oslo, Norway (Gillooly et al., 2000) was re- may be implicated in membrane trafficking. cloned 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 Materials and Methods mutants cloned in pEF-BOS by Y. Imai (National Institute of Cell culture and antibodies Neuroscience, Tokyo, Japan) (Ohsawa et al., 2000), flag-tagged Rac1 COS cells were grown in Dulbecco+5% fetal calf serum (FCS) and V12 by Y. Takai and T. Takenawa (Institute fo Medical Science, Osaka HeLa cells in Dulbecco+10% FCS. Lymphoblastoid cell lines from and Tokyo, Japan) (Mochizuki et al., 1999) and PML expression control and from a patient (deleted for exons 1 to 13 of the hMTM1 construct by R. Losson (IGBMC, Illkirch-France). 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 Immunofluorescence microscopy U/ml penicillin and 400 µg/ml gentamycin. Cells were tranfected with Cells were grown either onto a glass coverslip (COS) or onto glass either the calcium phosphate method or Ex-Gen 500 (Euromedex, Lab-Tek chamber slides (Nalge Nunc Int.), and transfected and fixed France), the precipitate was removed 12 hours after transfection and with 4% paraformaldehyde. They were subsequently permeabilised cells were allowed to grow for another 24 hours. in PBS with 0.3% Triton X-100. Subcellular localization of We used monoclonal antibodies directed against vimentin (LN6), myotubularin constructs was assessed using either monoclonal α-tubulin, vinculin (all from Sigma), phosphotyrosine (4G10, UBI), antibodies 1G6 (1:1000), or 1D10 (1:500) for C-terminal constructs, dynamin (Upstate Biotechnology). Rabbit anti-Rab5 and rabbit anti- or with rabbit R929 (1:500). Cy3- or biotin-conjugated secondary caveolin 1 were purchased from Santa Cruz Biotechnology and rabbit antibodies and DTAF-streptavidin were used for single and co- anti-actin from Sigma. Anti-myc and anti-flag monoclonal and localization experiments following manufacturers recommendations. polyclonal antibodies were produced in house (IGBMC). Secondary Actin was labelled with phalloidin-TRITC. Fluorescence was antibodies were goat anti-mouse Kappa-specific (Southern examined under a DMLB microscope or a laser scanning TCS4D Biotechnology Associates) and goat anti-mouse or goat anti-rabbit microscope for confocal analysis (Leica). (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 Immunoprecipitation and immunoblotting Immunoresearch Laboratories, and fluorescein (DTAF)-conjugated The entire procedure was carried out at 4°C. Whole-cell extracts from streptavidin (Immunotech). Wortmannin, LY294002 and Cytochalasin cultured cells were obtained by homogenization in lysis buffer (50 D were purchased from Sigma. 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% Anti-myotubularin antibodies glycerol, 1% NP-40, 5 mM KCl, 1 mM EDTA, 1 mM Pefabloc and Monoclonal antibodies were raised against full-length human 1 mM orthovanadate). Extracts were passed five times through a 25G Myotubularin subcellular localization 3107 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 inhibitor, 1 mM orthovanadate) for lymphoblasts Fig. 1. Characterization of myotubularin antibodies. (A) ‘Structural epitope’ means that and myotubes or in buffer B (50 mM Tris-HCl pH the antibodies recognized only the full-length myotubularin but not overlapping 7.5, 150 mM NaCl, 1% NP-40, 1 mM Pefabloc, 1 fragments. Western blotting and immunocytochemistry results are from transfected mM orthovanadate) for HeLa cells, and lysed by cells as detection of endogenous myotubularin was unsuccessful with these two passing through a 25G syringe and a Dounce methods. Immunoprecipitation data were obtained for the endogenous myotubularin homogenizer 20 times. P1 (nuclei) and S1 from muscle cells and are described elsewhere (Laporte et al., 2001b). +, the antibody (organelles and cytoplasm) were separated by is working with the corresponding technique; –, no signal has been detected. At least centrifugation at 1,000 g for 15 minutes and S1 2D2, 1G1 and R1208 crossreact with mouse myotubularin, while 1G6, 1D10, R929 and supernatant was centrifuged at 100,000 g for 1 hour R1141 do not. 1C7 does not immunoprecipitate the mouse myotubularin. R1208 to yield the P2 (big organelles) and S2 (microsomal crossreacts with MTMR1 while none of these antibodies crossreact with hMTMR2 and fraction and cytoplasm) fractions. Fractionation hMTMR3 proteins. (B) Example of immunoprecipitant antibodies crossreacting with was monitored by phase-contrast microscopy and the endogenous mouse mMTM1 myotubularin. Mouse C2C12 myotube protein extract using different antibodies. or buffer (/) were immunoprecipitated (IP) with the listed antibodies and the purified For cytoskelatal fractionation, transfected HeLa mouse myotubularin was detected by western blot with the 2D2 antibody (1/2000) cells or C2C12 myotubes were lysed in the followed by an anti-Kappa light chain (1/2500). Myotubularin has an estimated cytoskeleton stabilizing buffer (10 mM Pipes pH molecular weight of 70 kDa compared with size markers. Transfected COS cells with 6.8, 250 mM sucrose, 3 mM MgCl2, 120 mM KCl, human myotubularin serve as a size control on the left. R1203 is a serum from a 1 mM EGTA, 0.15% Triton X-100, 1 mM different rabbit immunized as for R1208. The dog myotubularin was also Pefabloc), centrifuged at 4°C at 14,000 g for 10 immunoprecipitated and detected with the 1G1 and 2D2 monoclonal antibodies minutes (Ogata et al., 1999). The pellet contained respectively (not shown). 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 full- myotubularin immunoprecipitation and western blotting. length myotubularin produced in Baculovirus (Laporte et al., 1998). Antibodies were characterized by western blotting and Pulse-chase immunohistochemistry on transfected COS and HeLa cells COS cells transiently transfected with wild-type myotubularin were and some of them can immunoprecipitate the endogenous starved in DMEM methionine- and cysteine-free for 1.5 hours, and myotubularin from muscle cells (Fig. 1A). Direct detection of then pulsed with 300 µCi of [35S]methionine/cysteine at 37°C for 1 endogenous myotubularin by immunohistochemistry was not hour. Cells were washed in medium and chased with pre-warmed successful and detection by western blot usually requires DMEM+10% FCS for the indicated time lapses. Proteins were prior immunoprecipitation as a concentration step. prepared and immunoprecipitated as before. Immunoprecipitation of myotubularin can be used as a complementary diagnostic test for XLMTM (Laporte et al., 2001b). This panel of antibodies, directed against different Results epitopes, allowed us to detect untagged full-length or truncated Characterization of specific antibodies and myotubularin myotubularin in all subsequent experiments. Some antibodies isoforms crossreact with mouse myotubularin (Fig. 1B) and will thus be We raised eight monoclonal and four polyclonal antibodies helpful for analysis in mouse. As the hMTM1 protein shares directed against human myotubularin (hMTM1). Antigens high sequence similarity with the hMTMR1 and hMTMR2 3108 Journal of Cell Science 115 (15) proteins, we tested for possible crossreaction with these Myotubularin is cytoplasmic and at the plasma proteins. Only the R1208 polyclonal was found to crossreact membrane with the hMTMR1 protein (not shown). In order to document precisely the subcellular localization of The hMTM1 gene is ubiquitously expressed and ESTs from myotubularin, we performed numerous immunocytochemistry at least 24 different tissues can be found in databases. A muscle experiments on transfected COS cells, mouse myoblasts and and testis mRNA isoform was found but differs from the myotubes (Fig. 3A-C) and on HeLa cells, 3T3 fibroblasts and ubiquitous 3.4 kb mRNA only by a different polyadenylation human muscle cells (data not shown). Untagged full-length site (Laporte et al., 1996). In order to confirm the ubiquitous myotubularin was used and all the antibodies described in this 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 muscle- specific 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 Fig. 3. Myotubularin is cytoplasmic and associated with plasma mouse were immunoprecipitated with 1G1 antibody and loaded on a membrane. (A) Wild-type untagged myotubularin overexpressed in 8% SDS-PAGE gel. Myotubularin was detected after western COS cells was detected with the specific 1G6 monoclonal antibody blotting with the 2D2 antibody (1/2000). Below, a longer migration (1/1000) followed by Cy3-conjugated goat anti-mouse antibody reveals the presence of a doublet specific to muscle (and heart, not (1/300). Confocal microscopy analysis does not show any nuclear shown). Different percentages of acrylamide and gradients tested did signal. The inactive C375S mutant was also localized as a dense not resolve the two bands any better. The arrow indicates the cytoplasmic network in different cell types (COS, HeLa, 3T3 migration of the 603 amino acid myotubularin construct transfected fibroblasts, myoblasts and myotubes). Also note the labeling of into cells, representing the common isoform. (B) The muscle-specific plasma membrane. Preimmune serum or immunizing peptide isoform appears after myotube formation. C2C12 mouse myoblasts competition (100 µg, 30 minutes, room temperature) abolished the were differentiated at confluence in DMEM+5% FCS into myotubes, signal. (B,C) Localization in transfected C2C12 mouse myoblasts and myotubes were stimulated with insulin like growth factor (IGF1 and myotubes respectively. (D) Transfected COS cell showing from Calbiochem at 25 ng/ml) as indicated. Protein extracts were altered cell shape and presence of myotubularin in extended prepared at different time points during differentiation and compared filopodia. This pattern was also observed with inactive myotubularin with adult muscle and liver tissues. mutants. Myotubularin subcellular localization 3109 study showed the same pattern in all the cell lines tested. cells suggests that myotubularin is not associated to vesicles Myotubularin localizes as a dense cytoplasmic network with nor to internal membranes (not shown). In conclusion, no signal in the nucleus as shown by confocal microscopy (Fig. myotubularin localizes to a dense cytoplasmic network not 3A). We can thus rule out that myotubularin has a nuclear related to known cytoskeletons and labels plasma membrane, localization under normal growth conditions. Myotubularin including extended filopodia in highly overexpressing cells. also labeled the plasma membrane (Fig. 3; see also Figs 7, 8) The localization of endogenous myotubularin was also including plasma membrane extensions such as filopodia (Fig. assessed by subcellular fractionation followed by 3D; see also Fig. 8A) and ruffles (see example on Fig. 7A). immunoprecipitation from lymphoblasts, myoblasts and HeLa Myotubularin localization is not modified by fusion of cells (Fig. 4). Consistent with the previous results in myoblasts into myotubes (Fig. 3C). Similar localization data transfected cells, endogenous myotubularin from lymphoblasts were obtained with peroxidase labeling and optical microscope was enriched in the cytoplasmic fraction and nearly analysis (data not shown). In a small subset of transfected cells, undetectable in the nuclear fraction (Fig. 4A). In the same myotubularin overexpression altered the shape of the cell, experiment, myotubularin was not detected in an XLMTM producing numerous filopodias (Fig. 3D). This was especially patient cell line deleted for exons 1-13 of the hMTM1 gene. noted in highly overexpressing cells and confirmed in HeLa We fractionated further the first supernatant (S1) to separate cells, where the same morphology as in Fig. 3D could be big organelles from cytoplasm and small organelles (P2 and observed (not shown). As this phenotype could also be seen S2, respectively). This latter protocol confirmed in HeLa cells with enzymatically inactive myotubularin constructs (C375S that myotubularin was absent from the nuclear fraction and and D278A mutants), it is not dependent on the enzymatic from the big organelles, and was enriched in the most soluble activity (not shown). fraction containing cytoplasm and ribosomes (Fig. 4B). As a filamentous cytoplasmic localization was clearly Myotubularin co-purified with tubulin, a cytoplasmic observed in some cells, suggestive of cytoskeletal networks, protein, in each experiment. To check whether endogenous we performed co-localization experiments. Overexpressed myotubularin could be localized to other compartments upon desmin, the localization of which was reported to be modified differentiation of muscle cells, we also performed the same in some XLMTM patients (Sarnat, 1992), and endogenous fractionation experiment on C2C12 mouse myotubes extracts, cytoplasmic actin, tubulin and keratin showed no co- using antibodies crossreacting with mouse myotubularin (here, localization. Vimentin, an intermediate filament protein, 1G1 and 2D2). Again, myotubularin was absent in the nuclear showed partial co-localization but confocal microscopy fraction and enriched in the cytoplasmic fraction (Fig. 4C). analysis did not allow unambiguous conclusion, as both As the localization pattern of myotubularin, especially the myotubularin and vimentin appeared as dense networks (not association to the plasma membrane, suggests that shown). Electron microscopy analysis of transfected HeLa myotubularin could be linked to the actin microfilaments, we

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. 3110 Journal of Cell Science 115 (15) performed a cytoskeletal fractionation to separate the Fig. 5. Effect of protein domains and XLMTM mutations on the polymerized actin and vimentin network from the soluble actin subcellular localization of myotubularin. (A) Schematic and tubulin. HeLa cells transfected either with the wild-type representation of myotubularin showing protein domains. GRAM, myotubularin, or with the D278A mutant, which is solely glucosyltransferase, Rab-like GTPase activator and myotubularin found at the plasma membrane (Blondeau et al., 2000), were common domain (Doerks et al., 2000) (aa 29-97); RID, Rac1- extracted with a triton-based buffer. Wild-type myotubularin induced localization to membrane ruffles (around aa 233-237); PTP, tyrosine phosphatase-like signature implicated in the lipid was enriched 14-fold compared with actin in the soluble phosphatase activity (aa 371-385) with catalytic residues D278, fraction (Fig. 4D), suggesting that it is not tightly associated C375 and R381; SID, SET-interacting domain (aa 435-486); PEST, to polymerized actin and intermediate filaments, even at the (aa 581-598) with a significant PESTfind score of +8.23; PDZ-BS, plasma membrane. The enrichment ratio of the D278A mutant putative PDZ-binding site functional in the hMTMR1 homolog compared with actin in the soluble fraction is only twofold, but (Fabre et al., 2000) (aa 599-603). Highly conserved regions through this may be a bias as the pellet might contain some membranes. evolution correspond to a high frequency of missense mutations in Cytoskeletal fractionation also performed on C2C12 mouse XLMTM patients and are also indicated (aa 170-330, 45% identity myotubes confirmed that myotubularin is found essentially in with the S. cerevisiae protein; and aa 370-490, 55% identity with the the soluble fraction, even after myotube formation (Fig. 4E). 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 Effect of protein domains and XLMTM mutations constructs were obtained from immunofluorescence experiments with either the N-terminal 1G6 or the C-terminal 1D10 anti- We have investigated the protein domains involved in the myotubularin antibodies on transfected COS and HeLa cells. Other subcellular localization of myotubularin. For this purpose, we constructs produced unstable proteins (aggregates in the cytoplasm generated a collection of deleted myotubularin constructs, and near the nucleus probably in the Golgi and very low including deletion of known domains such as the SET- myotubularin levels on western blot): del(1-95), del(97-122), interacting domain [SID (Cui et al., 1998)] and the GRAM del(183-245), del(224-245), del(308-325), del(396-406), del(437- domain, found in glucosyltransferases, Rab-like GTPase 469), del(482-494) and amino acid changes G378R, D394A, G402A, activators and myotubularins (Doerks et al., 2000). The protein E404K, E410A, D443A, C444Y and H469P. Mutation of the domains are indicated on Fig. 5A, and in addition to the SID conserved aspartate at position 257 (D257A) did not affect the localization of myotubularin. Note that missense mutations affecting and GRAM domains, include the PTP signature responsible for R241 (mild phenotype) impaired the in vitro enzymatic activity the enzymatic activity, a PEST sequence with significant score, toward PtdIns3P (Taylor et al., 2000) and lead to a decrease in and a putative PDZ-binding site (PDZ-BS), which was shown protein level in a patient cell line (Laporte et al., 2001b), while to be functional in the homolog hMTMR1 (Fabre et al., 2000). mutations G378R (severe phenotype) impaired the in vitro enzymatic The vast majority of the deleted constructs produced unstable activity and G402A (probably severe) leads to a decrease in protein products, as the resulting protein localized as aggregates in the level in a patient cell line. (B) Confocal microscopy analysis of a cytoplasm and near the nucleus, probably in the Golgi, and a truncated myotubularin shows nuclear localization (C-ter construct). very low protein level was detected when some constructs were Deletion of the SET-interacting domain from this construct abolished tested on western blot (not shown). Strikingly, the C-terminal the nuclear localization. (C) Co-localization of the C-ter construct half of myotubularin (from amino acids 336 to the stop codon) with PML in more than 50% of co-transfected cells (confocal microscopy). In the same experiment, other co-transfected cells showed a punctuated nuclear signal, together with cytoplasmic showed no obvious co-localization. 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 aggregates and is trapped by PML bodies. The N-terminal (Cui et al., 1998), we checked whether this domain was construct encompassing amino acids 1-369 has a cytoplasmic responsible for the nuclear localization of truncated C-terminal localization more similar to that of wild-type myotubularin myotubularin. A C-terminal construct lacking the SID does not (Fig. 5A), and this region would thus be implicated in the localize to the nucleus any more (Fig. 5B), while deletion of a subcellular localization of full-length myotubularin. more terminal part (amino acids 482-494) did not affect the We tested the subcellular localization of mutants with an nuclear localization (Fig. 5A). This confirmed that the SID is amino acid change at key catalytic residues (C375S, D278A), indeed responsible for the punctuated nuclear localization of and mutants of conserved aspartate residues at position 377, the C-terminal construct. As shorter constructs (e.g. C-terminal 380, 394 or 443. As described previously, mutation D278A deleted for the SID) do not localize to the nucleus, the nuclear produces an enzymatically inactive myotubularin that behaves localization of the C-terminal construct is not due to passive as a substrate-trap and localizes solely to plasma membrane diffusion into the nucleus. The C-terminal construct does extensions (Blondeau et al., 2000). Indeed, mutation of the not co-localize in the nucleus with Hoechst-labeled catalytic aspartate in tyrosine phosphatases leads to the heterochromatin. PML (promyelocytic leukaemia), a nuclear localization of the phosphatase to the substrate’s subcellular protein that does not localize to heterochromatin, showed sites, or the reverse (Flint et al., 1997). Additional sequences perfect co-localization in the majority of cells with the C- from homologs highlighted another conserved aspartate terminal construct, as assessed by confocal microscopy (Fig. (D257), but subcellular localization of a D257A construct was 5C), and this was confirmed by 3D reconstruction (not shown). similar to wild-type myotubularin in transfected COS cells (not However, other transfected cells clearly showed smaller shown). Mutation of the catalytic cysteine does not affect nuclear dots that do not co-localize with PML nor with localization, while, in some tyrosine phosphatases, it also Hoechst-stained heterochromatin (Fig. 5C). One explanation induced a different localization of the protein (Liu and could be that the nuclear subset of the C-terminal construct Chernoff, 1997). Positively charged aspartate residues at Myotubularin subcellular localization 3111

position 377 and 380 in the phosphatase signature are believed (Fig. 5A), but abrogated their lipid phosphatase activity to contribute to the substrate interaction and would explain the (Wishart et al., 2001). specificity towards phosphatidylinositol monophosphate Lastly, we also tested the subcellular localization of mutants (Laporte et al., 2001a; Wishart et al., 2001). Mutation of these found in XLMTM patients (R241C and G378R in the PTP, residues did not affect the localization of the resulting proteins C444Y and H469P in the SID, G402A, E404K and R421Q ). 3112 Journal of Cell Science 115 (15) Most of these mutants showed a cytoplamic signal with S. pombe impaired vesicle trafficking (Blondeau et al., 2000). cytoplasmic aggregates. Aggregation suggests that these We first tested the effect of overexpression of wild-type mutants are unstable and this is consistent with the fact that myotubularin on different endocytosis pathways. In COS cells, immunoprecipitation from XLMTM patient cell lines showed we observed no effect on the distribution of dynamin (clathrin- a decrease in myotubularin level in 87% of the cases including coated vesicles), caveolin 1 (uncoated vesicles) and Rab5 some missense mutations (Laporte et al., 2001b). Thus, (endosomes) compared with untransfected cells (Fig. 7A). absence or instability of mutated myotubularin appears as the However, we cannot exclude that a fraction of myotubularin main cause of the disease. However, the R421Q mutant found may associate with these structures. in severe cases of XLMTM localized as wild-type We then compared the distribution of myotubularin and myotubularin, and labeled filopodia were consistently present PtdIns3P in cells after cotransfection with constructs expressing in all transfected cells (J.L., unpublished). 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., Turn-over of myotubularin 2000). Myotubularin did not co-localize with the typical Sequence analysis using the PESTfind algorithm predicted in vesicular staining pattern of PtdIns3P-coated endosomes (Fig. the C-terminal portion of the protein a PEST sequence with a 7B). In cells overexpressing wild-type myotubularin, there was significant score of +8.23 (Rechsteiner and Rogers, 1996). The no obvious change in the level or localization of the 2XFYVE presence of the PEST sequence and the fact that we cannot probe. Moreover, overexpression of the 2XFYVE probe detect the endogenous protein by immunohistochemistry or by generated expanded vacuolar structures in about 30% of the direct western blot suggested that myotubularin is rapidly cells, probably due to displacement of FYVE-proteins (such as degraded, especially as the amount of myotubularin mRNA EEA1) from PtdIns3P, resulting in deregulation of the detected by northern blot and the rather high number of endocytic pathway (Gillooly et al., 2000). Overexpression of corresponding ESTs indicate that the level of transcript is myotubularin did not cause similar changes (Fig. 7A), and not very low (Laporte et al., 1998). Moreover, stability of deregulation of the endocytic pathway by the 2XFYVE probe myotubularin seems very sensitive to sequence changes did not modify the localization of transfected myotubularin (Laporte et al., 2001b). Despite the presence of this PEST (data not shown). Thus myotubularin does not seem to motif, a pulse-chase labelling experiment indicated that detectably regulate PtdIns3P content in this system. It also does myotubularin has a slow turn-over in transfected cells, with an not co-localize with endosomal PtdIns3P in most co-transfected approximate half-life of 4-5 hours (Fig. 6). Thus, the PEST cells, although it cannot be excluded that a fraction of it may sequence in myotubularin does not, under these conditions, associate. Inactive mutants C375S and D278A behaved direct very rapid degradation. 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). Myotubularin, vesicle trafficking and PtdIns3P As shown above, some truncated myotubularin constructs distribution containing the phosphatase active site localized as cytoplasmic Myotubularin was recently found to be able to dephosphorylate dots (e.g. C-ter del SID in Fig. 5C). We show in Fig. 7B that PtdIns3P in vitro and in yeast systems (Blondeau et al., 2000; these cytoplasmic dots are not PtdIns3P-containing Taylor et al., 2000). PtdIns3P is a second messenger localized endosomes. The C-ter del SID protein appears membrane mainly on endosomal vesicles (Gillooly et al., 2000) and associated and in some cells produces structures similar to interacts with FYVE-domain-containing proteins that regulate vacuoles (Fig. 7B) that have not yet been identified. vesicle trafficking in yeast and mammalian cells (Stenmark and Aasland, 1999). Overexpression of myotubularin in the yeast 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 Fig. 6. Turn-over of myotubularin. COS cells transfected with wild- 35 myotubularin both underlined the shape of the untreated cells. type myotubularin were labeled with [ S]methionine/cysteine and Collapse of the actin network and stress fibers altered the cold chased for 0, 15 or 30 minutes and 1, 2, 3 or 5 hours. general distribution of myotubularin constructs. However, Myotubularin was immunoprecipitated with the 1G1 monoclonal antibody. The period of cold chase is indicated above the lanes. wild-type and D278A mutant were still present at the plasma Myotubularin is indicated by an arrow. Above, an additional protein membrane (Fig. 8A). In fact, wild-type myotubularin clearly is trapped probably by the beads as it is also present in the first lane labeled longer filopodias. This suggests that myotubularin is without immunoprecipitant antibody. The estimated half-life is about bound directly to plasma membrane components rather than to 4 hours. the underlying actin fibers. Myotubularin subcellular localization 3113 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 over- expressing myotubularin constructs in order to monitor cell spreading. Next, we induced plasma mem- brane 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 3- kinase, 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 Rac- induced 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 myotubularin alone, a strong labeling transfected with wild-type myotubularin do not show changes in endocytic markers such as of membrane ruffles was sometimes dynamin (clathrin-coated vesicles), caveolin 1 (uncoated vesicles), and Rab5 (endosomes). Note noted (see Fig. 7A, caveolin 1 co- the strong labeling of a membrane ruffle with anti-myotubularin antibody in the caveolin 1 co- staining). staining. (B) COS cells co-transfected with wild-type or C-ter delSID myotubularin constructs In order to map the Rac1-induced and a myc-tagged 2XFYVE expression construct show no co-localization and no effect of localization domain (or RID), we co- myotubularin with the endosomal staining by 2XFYVE. transfected Rac1 V12 with a panel of truncated and mutated myotubularin constructs. Results are summarized in Fig. 5A. The N-terminal other constructs did not localize to ruffles: bigger deletions in construct (aa 1-369) localized to Rac1-induced ruffles (Fig. the N-terminal region, but also some missense mutations in 8C), while the C-terminal part (aa 336-603) did not. The the C-terminal part (e.g. H469P) that may render smallest deletion that prevented co-localization to Rac1- myotubularin unstable. induced ruffles was del(233-237) (Fig. 8C; see also Fig. 5A). These data show that myotubularin localization to plasma The region from amino acids 179 to 248, which contains the membrane ruffles is not dependent on its enzymatic activity, RID domain, is highly conserved in the myotubularin family and suggest that myotubularin is recruited to the membrane (61% aa identity between human myotubularin and the rather than to the actin cytoskeleton. Moreover, overexpression drosophila homolog) and is a hot spot for missense mutations of all the myotubularin constructs listed in Fig. 5A, including in XLMTM patients (Laporte et al., 2000). This suggests that wild-type and phosphatase inactive mutants D278A and the property to localize to ruffles might be shared by C375S, does not detectably affect the Rac1-induced ruffles at myotubularin homologous proteins. It has to be noted that the edges and over the entire surface of the cell. 3114 Journal of Cell Science 115 (15)

Discussion sheets. The SID is unlikely to mediate nuclear localization in Myotubularin defines a large subgroup of phosphatases within the context of full-length myotubularin. After submission of the family of tyrosine and dual-specificity phosphatases (PTP), the present work, another group reported that full-length and has been recently shown to dephosphorylate PtdIns3P Sbf1/MTMR5 protein and myotubularin are indeed (Blondeau et al., 2000; Taylor et al., 2000). We characterized cytoplasmic (Firestein and Cleary, 2001). specific antibodies against myotubularin. Direct western blot Myotubularin is cytoplasmic in various transfected cells and or immunocytochemistry did not allow detection of the its localization is not changed after differentiation of myoblasts endogenous myotubularin, suggesting that it is present at a very into myotubes. Endogenous myotubularin is also present in low level. This contrasts with the fact that it is not difficult to cytoplasmic fractions, as tested by immunoprecipitation and detect the RNA transcript by northern blot and with the high western blotting. Under normal culture conditions, number of cDNAs represented as ESTs in the databases. This myotubularin localization appears as a dense cytoplasmic suggests either a high turnover rate of the protein or a tight network with plasma membrane staining of occasional ruffles translational control. However, myotubularin does not have a and of filopodia in highly overexpressing cells. However, there very short half-life in transfected cells, in spite of the presence is no obvious co-localization with known cytoskeletal of a predicted PEST sequence at the C-terminus. networks. While myotubularin actively dephosphorylates In contrast with an initial report that myotubularin (like the PtdIns3P, it is striking to note that it does not co-localize phosphatase-inactive Sbf1/MTMR5 protein) is located in the extensively with PtdIns3P-containing endosomes and its nucleus and interacts with nuclear SET proteins via its SID overexpression does not affect localization of proteins domain (Cui et al., 1998), our results do not show any evidence implicated in endocytosis. A punctate and reticular for the presence of full length myotubularin in the nucleus. cytoplasmic staining was also recently reported for a tagged However, we have noted that the SID can drive the localization MTMR3 protein, with no colocalization with endosomal of a truncated portion of myotubularin into nuclear dots. No markers (Walker et al., 2001). nuclear localization signal-like sequence is present in the SID, In our experiments, overexpression of wild-type which is rich in hydrophobic residues presumably forming β myotubularin did not detectably affect PtdIns3P level and Myotubularin subcellular localization 3115

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 PtdIns3P labelling (Kim et al., 2002). The localization at the specific probe (2XFYVE). Although PtdIns3P was shown to plasma membrane of the enzymatically inactive, substrate-trap be a most effective substrate of myotubularin in yeast and in D278A mutant also suggests that the physiological substrate of in vitro experiments, other phosphoinositides might also serve myotubularin might not be endosomal PtdIns3P, but rather a as substrates in higher eukaryotic cells. After submission of the plasma membrane subpool of PtdIns3P (or of another present work, it was reported that the MTMR3 protein phosphoinositide). Monitoring levels of PtdIns3P and other dephosphorylates PtdIns(3,5)P2, to yield PtdIns5P (Walker et phosphoinositide in tissues from myotubularin knockout mice al., 2001). However, it is also possible that, in our experiments, may provide a more definitive answer on the nature of the concomitant overexpression of the 2XFYVE probe blocked myotubularin physiological substrate. the transient interaction of myotubularin with PtdIns3P. This The presence of myotubularin at the plasma membrane could explain the apparent discrepancy with the work of Kim (enhanced in the D278A mutant) may also be the result of an et al., published after initial submission of our manuscript, interaction with a PtdIns 3-kinase. The latter enzymes are where the authors used a biotinylated 2XFYVE probe for known to localize to plasma membrane under growth factor 3116 Journal of Cell Science 115 (15) stimulation and membrane remodeling (Tsakiridis et al., 1999). membrane ruffles, and with its PtdIns3P phosphatase site as a Indeed, the D278A mutant was able to co-immunoprecipitate potential regulation domain for endosomal trafficking. For the only known PtdIns 3-kinase activity in S. pombe (Blondeau instance, recycling of endosomes back to the plasma et al., 2000). membrane at sites of membrane remodeling may depend upon Rac1-induced remodeling of the plasma membrane leads to the release of FYVE-finger regulatory proteins following the localization of myotubularin to membrane ruffles, and PtdIns3P dephosphorylation by myotubularin. this is independent of the phosphatase activity. High We have recently constructed a mouse knockout model of overexpression of myotubularin also induces filopodia myotubular myopathy, that indicates that MTM1 deficiency extension and affects cell shape, although it does not modify does not cause a defect in muscle maturation, but rather impairs the Rac1-induced membrane ruffles. Localization to Rac1- the function or organisation of muscle fibers (A. Buj-Bello induced ruffles is dependent on a myotubularin domain that we and J.-L.M., unpublished). The study of phosphoinositol named RID, which is located within a highly conserved region metabolism and membrane trafficking in this model should in the myotubularin family, but that shows no resemblance with allow a better understanding of the role of myotubularin, protein domains of known function. especially in muscle. The presence of a GRAM domain in myotubularin (different from the Rac1-induced localization domain), which is shared We thank Christine Kretz and Laurent Weiss for excellent technical with proteins implicated in membrane trafficking such as Rab- assistance, Isabelle Kolb-Cheynel for production of recombinant like GTPases activators (Doerks et al., 2000), also suggests a proteins, Yoshinori Imai for Rac1, Rho and Cdc42 dominant active role at the plasma membrane. A putative PDZ-binding site is and inactive HA-tagged constructs, Yoshimi Takai for the Rac1 V12- flag construct, Patrick Vicart for the desmin construct, Harald also present in myotubularin, and was shown to be active in its Stenmark for the PtdIns3P 2XFYVE probe and Mustapha Oulad- close homolog hMTMR1 (Fabre et al., 2000). It could thus Abdelghani and Anna Buj-Bello for useful discussions. This study mediate interaction of myotubularin with PDZ-containing was supported by funds from the Institut National de la Santé et de la proteins that are known to be implicated in the organization of Recherche Médicale, the Centre National de la Recherche specific plasma membrane domains (Fanning and Anderson, Scientifique, the Hôpital Universitaire de Strasbourg (HUS), the Louis 1999) and in membrane trafficking (Cao et al., 1999). It is also Jeantet Foundation and by grants from the Association Française worth noting the striking resemblance between the phosphatase contre les Myopathies (AFM). active site of myotubularin and that of the PtdIns5- phosphatases Sac1p and synaptojanin (they share a DCXD motif not present in other phosphatases); the latter protein is References known to play an essential role in synaptic vesicle recycling Al-Awar, O., Radhakrishna, H., Powell, N. N. and Donaldson, J. G. (2000). (Cremona et al., 1999; Harris et al., 2000). Separation of membrane trafficking and actin remodeling functions of ARF6 with an effector domain mutant. Mol. Cell. Biol. 20, 5998-6007. In summary, we propose a model where cytoplasmic Blondeau, F., Laporte, J., Bodin, S., Superti-Furga, G., Payrastre, B. and myotubularin may be localized to the plasma membrane upon Mandel, J.-L. (2000). Myotubularin, a phosphatase deficient in Myotubular Rac and/or PtdIns 3-kinase activation through interaction with Myopathy, acts on PI3-kinase and phosphatidylinositol 3 phosphate one of the many proteins present at these sites, including PtdIns pathway. Hum. Mol. Genet. 9, 2223-2229. 3-kinases and GTPase exchange factors, but not with actin as Bolino, A., Muglia, M., Conforti, F. L., LeGuern, E., Salih, M. A., Georgiou, D. M., Christodoulou, K., Hausmanowa-Petrusewicz, I., we showed that myotubularin still labels plasma membrane in Mandich, P., Schenone, A. et al. (2000). Charcot-marie-tooth type 4B is cells where actin fibers are disrupted. At the plasma membrane, caused by mutations in the gene encoding myotubularin-related protein-2. myotubularin would be in contact with its substrate, either a Nat. Genet. 25, 17-19. subpool of PtdIns3P or another phosphoinositide [such as Cao, T. T., Deacon, H. W., Reczek, D., Bretscher, A. and von Zastrow, M. (1999). A kinase-regulated PDZ-domain interaction controls endocytic PtdIns(3,5)P2 (Walker et al., 2001)]. PtdIns(4,5)P2 has been sorting of the beta2-adrenergic receptor. Nature 401, 286-290. shown to be synthesised at membrane ruffles and is important Carpenter, G. (2000). The EGF receptor: a nexus for trafficking and signaling. for vesicle trafficking (Honda et al., 1999; Carpenter et al., Bioessays 22, 697-707. 2000), but to date it does not appear as an effective substrate Cremona, O., di Paolo, G., Wenk, M. R., Luthi, A., Kim, W. T., Takei, K., for myotubularin (Taylor et al., 2000). Daniell, L., Nemoto, Y., Shears, S. B., Flavell, R. A. et al. (1999). Essential role of phosphoinositide metabolism in synaptic vesicle recycling. Cell 99, As GTPases link plasma membrane remodeling to endocytic 179-188. trafficking (Ellis and Mellor, 2000), myotubularin may act as Cui, X., de Vivo, I., Slany, R., Miyamoto, A., Firestein, R. and Cleary, M. a modulator of GTPase activity or action. Rho and Rac L. (1998). Association of SET domain and myotubularin-related proteins GTPases have been shown to regulate inositol lipid kinases modulates growth control. Nat. Genet. 18, 331-337. Devys, D., Lutz, Y., Rouyer, N., Bellocq, J.-P. and Mandel, J.-L. (1993). and phosphoinositides levels (Ren and Schwartz, 1998); The FMR-1 protein is cytoplasmic, most abundant in neurons and appears myotubularin lipid phosphatase may play a role at the plasma normal in carriers of a fragile X premutation. Nat. Genet. 4, 335-340. membrane to regulate the phosphoinositides pool created by Di Cesare, A., Paris, S., Albertinazzi, C., Dariozzi, S., Andersen, J., Mann, Rac-induced activation of PtdIns 3-kinases. The localization of M., Longhi, R. and de Curtis, I. (2000). P95-APP1 links membrane the D278A mutant to plasma membrane protrusions suggests transport to Rac-mediated reorganization of actin. Nat. Cell. Biol. 2, 521- 530. also a link with the ADP-ribosylation factor 6 (Arf6) GTPase Doerks, T., Strauss, M., Brendel, M. and Bork, P. (2000). GRAM, a novel (Radhakrishna et al., 1999; Di Cesare et al., 2000). Arf6 domain in glucosyltransferases, myotubularins and other putative activation is required for the recycling of the endosomal membrane-associated proteins. Trends Biochem. Sci. 25, 483-485. membrane back to plasma membrane and also for plasma Ellis, S. and Mellor, H. (2000). Regulation of endocytic traffic by rho family GTPases. Trends Cell. Biol. 10, 85-88. membrane ruffling induced by Rac (Al-Awar et al., 2000). Fabre, S., Reynaud, C. and Jalinot, P. (2000). Identification of functional Myotubularin may play a similar dual role with its Rac1- PDZ domain binding sites in several human proteins. Mol. Biol. Rep. 27, induced localization domain allowing localization to 217-224 Myotubularin subcellular localization 3117

Fanning, A. S. and Anderson, J. M. (1999). PDZ domains: fundamental Laporte, J., Kress, W. and Mandel, J.-L. (2001b). Diagnosis of X-linked building blocks in the organization of protein complexes at the plasma Myotubular Myopathy by detection of myotubularin. Ann. Neurol. 50, 42- membrane. J. Clin. Invest. 103, 767-772. 46 Fardeau, M. (1992). Congenital myopathies. In Skeletal Muscle Pathology Liu, F. and Chernoff, J. (1997). Protein tyrosine phosphatase 1B interacts (ed. F. L. Mastaglia and Lord Walton of Detchant), pp. 237-281. Edinburgh: with and is tyrosine phosphorylated by the epidermal growth factor receptor. Churchill Livingstone. Biochem. J. 327, 139-145. Firestein, R., Cui, X., Huie, P. and Cleary, M. L. (2000). Set domain- Mandel, J. L., Laporte, J., Buj-Bello, A., Sewry, C. and Wallgren- dependent regulation of transcriptional silencing and growth control by Pettersson, C. (2002). X-linked myotubular myopathy. In Structural and SUV39H1, a mammalian ortholog of Drosophila Su(var)3-9. Mol. Cell. Molecular Basis of Skeletal Muscle Disease (ed. G. Karpati), pp. 124-129. Biol. 20, 4900-4909. Basel: International Society of Neuropathology Press. Firestein, R. and Cleary, M. L. (2001). Pseudo-phosphatase Sbf1 contains Mochizuki, Y. and Takenawa, T. (1999). Novel inositol polyphosphate 5- an N-terminal GEF homology domain that modulates its growth regulatory phosphatase localizes at membrane ruffles. J. Biol. Chem. 274, 36790- properties. J. Cell Sci. 114, 2921-2927. 36795. Flint, A. J., Tiganis, T., Barford, D. and Tonks, N. K. (1997). Development Ogata, M., Takada, T., Mori, Y., Oh-hora, M., Uchida, Y., Kosugi, A., of “substrate-trapping” mutants to identify physiological substrates Miyake, K. and Hamaoka, T. (1999). Effects of overexpression of PTP36, of protein tyrosine phosphatases. Proc. Natl. Acad. Sci. USA 94, 1680- a putative protein tyrosine phosphatase, on cell adhesion, cell growth, and 1685. cytoskeletons in HeLa cells. J. Biol. Chem. 274, 12905-12909. Gaullier, J. M., Simonsen, A., D’Arrigo, A., Bremnes, B., Stenmark, H. Ohsawa, K., Imai, Y., Kanazawa, H., Sasaki, Y. and Kohsaka, S. (2000). and Aasland, R. (1998). FYVE fingers bind PtdIns(3)P. Nature 394, 432- Involvement of Iba1 in membrane ruffling and phagocytosis of 433. macrophages/microglia. J. Cell. Sci. 113, 3073-3084. Gillooly, D. J., Morrow, I. C., Lindsay, M., Gould, R., Bryant, N. J., Radhakrishna, H., Al-Awar, O., Khachikian, Z. and Donaldson, J. G. Gaullier, J. M., Parton, R. G. and Stenmark, H. (2000). Localization of (1999). ARF6 requirement for Rac ruffling suggests a role for membrane phosphatidylinositol 3-phosphate in yeast and mammalian cells. EMBO J. trafficking in cortical actin rearrangements. J. Cell. Sci. 112, 855-866. 19, 4577-4588. Rechsteiner, M. and Rogers, S. W. (1996). PEST sequences and regulation Harris, T. W., Hartwieg, E., Horvitz, H. R. and Jorgensen, E. M. (2000). by proteolysis. Trends Biochem. Sci. 21, 267-271. Mutations in synaptojanin disrupt synaptic vesicle recycling. J. Cell Biol. Ren, X. D. and Schwartz, M. A. (1998). Regulation of inositol lipid kinases 150, 589-600. by Rho and Rac. Curr. Opin. Genet. Dev. 8, 63-67. Herman, G. E., Finegold, M., Zhao, W., de Gouyon, B. and Metzenberg, Sarnat, H. B. (1992). Vimentin and desmin in maturing skeletal muscle and A. (1999). Medical complications in long-term survivors with X-linked developmental myopathies. Neurology 42, 1616-1624. myotubular myopathy. J. Pediatrics 134, 206-214. Sewry, C. A. (1998). The role of immunohistochemistry in congenital Honda, A., Nogami, M., Yokozeki, T., Yamazaki, M., Nakamura, H., myopathies. Neuromuscul. Disord. 8, 394-400. Watanabe, H., Kawamoto, K., Nakayama, K., Morris, A. J., Frohman, Stenmark, H. and Aasland, R. (1999). FYVE-finger proteins – effectors of M. A. and Kanaho, Y. (1999). Phosphatidylinositol 4-phosphate 5-kinase an inositol lipid. J. Cell Sci. 112, 4175-4183. alpha is a downstream effector of the small G protein ARF6 in membrane Taylor, G. S., Maehama, T. and Dixon, J. E. (2000). Myotubularin, a protein ruffle formation. Cell 99, 521-532. tyrosine phosphatase mutated in myotubular myopathy, dephosphorylates Kim, S. A., Taylor, G. S., Torgersen, K. M. and Dixon, J. E. (2002). the lipid second messenger, phosphatidylinositol 3-phosphate. Proc. Natl. Myotubularin and MTMR2, phosphatidylinositol 3-phosphatases mutated in Acad. Sci. USA 97, 8910-8915. myotubular myopathy and type 4B Charcot-Marie-Tooth disease. J. Biol. Timmerman, V. (1998). Charcot-Marie-Tooth neuropathies. In Chem. 8, 4526-4531. Neuromuscular Disorders: Clinical and Molecular Genetics (ed. A. E. H. Laporte, J., Hu, L. J., Kretz, C., Mandel, J.-L., Kioschis, P., Coy, J. F., Emery). Wiley & Sons. Klauck, S. M., Poustka, A. and Dahl, N. (1996). A gene mutated in X- Tsakiridis, T., Tong, P., Matthews, B., Tsiani, E., Bilan, P. J., Klip, A. and linked myotubular myopathy defines a new putative tyrosine phosphatase Downey, G. P. (1999). Role of the actin cytoskeleton in insulin action. family conserved in yeast. Nat. Genet. 13, 175-182. Microsc. Res. Tech. 47, 79-92. Laporte, J., Blondeau, F., Buj-Bello, A., Tentler, D., Kretz, C., Dahl, N. Wallgren-Pettersson, C., Clarke, A., Samson, F., Fardeau, M., Dubowitz, and Mandel, J.-L. (1998). Characterization of the myotubularin tyrosine V., Moser, H., Grimm, T., Barohn, R. J. and Barth, P. G. (1995). The phosphatase gene family, from yeast to human. Hum. Mol. Genet. 7, 1703- myotubular myopathies: differential diagnosis of the X-linked recessive, 1712. autosomal dominant, and autosomal recessive forms and present state of Laporte, J., Biancalana, V., Tanner, S. M., Kress, W., Schneider, V., DNA studies. J. Med. Genet. 32, 673-679. Wallgren-Pettersson, C., Herger, F., Buj-Bello, A., Blondeau, F., Liechti- Walker, D. M., Urbé, S., Dove, S. K., Tenza, D., Raposo, G. and Clague, Gallati, S. and Mandel, J.-L. (2000). MTM1 mutations in X-linked M. J. (2001). Characterization of MTMR3: an inositol lipid 3-phosphatase myotubular myopathy. Hum. Mutat. 15, 393-409. with novel substrate specificity. Curr. Biol 11, 1600-1605. Laporte, J., Blondeau, F., Buj-Bello, A. and Mandel, J.-L. (2001a). The Wishart, M. J., Taylor, G. S., Slama, J. T. and Dixon, J. E. (2001). PTEN myotubularin family: from genetic diseases to phosphoinositide metabolism. and myotubularin phosphoinositide phosphatases: bringing bioinformatics Trends Genet. 17, 221-229. to the lab bench. Curr. Opin. Cell Biol. 13, 172-181.