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:
mtm{at}titus.u-strasbg.fr
)
Accepted 1 May 2002
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Summary |
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Key words: Myotubularin, Myotubular myopathy, Phosphatidylinositol 3-monophosphate, Membrane trafficking, Rac GTPase, Phosphatase, RID domain
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Introduction |
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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 cell-specific
proteins (Timmerman et al., 1998).
Myotubularin contains the consensus signature of the tyrosine and
dual-specificity phosphatase (PTP),
His-Cys-X2-Gly-X2-Arg, and was shown to dephosphorylate
phosphoserine-and 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
(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.
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Materials and Methods |
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We used monoclonal antibodies directed against vimentin (LN6),
-tubulin, vinculin (all from Sigma), phosphotyrosine (4G10, UBI),
dynamin (Upstate Biotechnology). Rabbit anti-Rab5 and rabbit anti-caveolin 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, N-terminal
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 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 and myotubes or in buffer B
(50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM Pefabloc, 1 mM
orthovanadate) for HeLa cells, and lysed by passing through a 25G syringe and
a Dounce homogenizer 20 times. P1 (nuclei) and S1 (organelles and cytoplasm)
were separated by centrifugation at 1,000 g for 15 minutes and
S1 supernatant was centrifuged at 100,000 g for 1 hour to
yield the P2 (big organelles) and S2 (microsomal fraction and cytoplasm)
fractions. Fractionation was monitored by phase-contrast microscopy and using
different antibodies.
For cytoskelatal fractionation, transfected HeLa cells or C2C12 myotubes
were lysed in the cytoskeleton stabilizing buffer (10 mM Pipes pH 6.8, 250 mM
sucrose, 3 mM MgCl2, 120 mM KCl, 1 mM EGTA, 0.15% Triton X-100, 1
mM Pefabloc), centrifuged at 4°C at 14,000 g for 10
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 myotubularin immunoprecipitation and western
blotting.
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.
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Results |
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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 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.
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 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).
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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 co-localization. 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 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 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 SET-interacting 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 C-terminal 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 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 (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). 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 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 (clathrin-coated 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.
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 membrane remodeling by overexpressing a Rac
1-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 myotubularin alone, a strong labeling of membrane ruffles was sometimes noted (see Fig. 7A, caveolin 1 co-staining).
In order to map the Rac1-induced localization domain (or RID), we
co-transfected Rac1 V12 with a panel of truncated and mutated myotubularin
constructs. Results are summarized in Fig.
5A. The N-terminal construct (aa 1-369) localized to Rac1-induced
ruffles (Fig. 8C), while the
C-terminal part (aa 336-603) did not. The smallest deletion that prevented
co-localization to Rac1-induced ruffles was del(233-237)
(Fig. 8C; see also
Fig. 5A). The region from amino
acids 179 to 248, which contains the RID domain, is highly conserved in the
myotubularin family (61% aa identity between human myotubularin and the
drosophila homolog) and is a hot spot for missense mutations in XLMTM patients
(Laporte et al., 2000). This
suggests that the property to localize to ruffles might be shared by
myotubularin homologous proteins. It has to be noted that other constructs did
not localize to ruffles: bigger deletions in the N-terminal region, but also
some missense mutations in the C-terminal part (e.g. H469P) that may render
myotubularin unstable.
These data show that myotubularin localization to plasma membrane ruffles is not dependent on its enzymatic activity, and suggest that myotubularin is recruited to the membrane rather than to the actin cytoskeleton. Moreover, overexpression of all the myotubularin constructs listed in Fig. 5A, including wild-type and phosphatase inactive mutants D278A and C375S, does not detectably affect the Rac1-induced ruffles at the edges and over the entire surface of the cell.
![]() |
Discussion |
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---|
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 ß 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 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
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 Rac1-induced 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 Rab-like 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 PtdIns5-phosphatases 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 Rac1-induced 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.
![]() |
Acknowledgments |
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