Research Article
353
BAD-LAMP defines a subset of early endocytic
organelles in subpopulations of cortical projection
neurons
Alexandre David1,2,3,*, Marie-Catherine Tiveron4,*, Axel Defays1,2,3, Christophe Beclin4,
Voahirana Camosseto1,2,3, Evelina Gatti1,2,3, Harold Cremer4,‡,§ and Philippe Pierre1,2,3,‡,§
1
Centre d’Immunologie de Marseille-Luminy, Université de la Méditerranée, Case 906, 13288 Marseille cedex 9, France
INSERM, U631 and 3CNRS, UMR6102,13288 Marseille, France
4
Institut de Biologie du Développement de Marseille-Luminy, CNRS UMR 6216, Université de la Méditerranée, Case 907, 13288 Marseille cedex 9,
France
2
*Both first authors contributed equally to this work
‡
Both last authors contributed equally to this work
§
Authors for correspondence (e-mail: cremer@ibdm.univ-mrs.fr; pierre@ciml.univ-mrs.fr)
Accepted 25 October 2006
Journal of Cell Science 120, 353-365 Published by The Company of Biologists 2007
doi:10.1242/jcs.03316
Journal of Cell Science
Summary
The brain-associated LAMP-like molecule (BAD-LAMP) is
a new member of the family of lysosome associated
membrane proteins (LAMPs). In contrast to other LAMPs,
which show a widespread expression, BAD-LAMP
expression in mice is confined to the postnatal brain and
therein to neuronal subpopulations in layers II/III and V
of the neocortex. Onset of expression strictly parallels
cortical synaptogenesis. In cortical neurons, the protein is
found in defined clustered vesicles, which accumulate along
neurites where it localizes with phosphorylated epitopes of
neurofilament H. In primary neurons, BAD-LAMP is
endocytosed, but is not found in classical lysosomal/
endosomal compartments. Modification of BAD-LAMP by
addition of GFP revealed a cryptic lysosomal retention
motif, suggesting that the cytoplasmic tail of BAD-LAMP
is actively interacting with, or modified by, molecules that
promote its sorting away from lysosomes. Analysis of BADLAMP endocytosis in transfected HeLa cells provided
evidence that the protein recycles to the plasma membrane
through a dynamin/AP2-dependent mechanism. Thus,
BAD-LAMP is an unconventional LAMP-like molecule and
defines a new endocytic compartment in specific subtypes
of cortical projection neurons. The striking correlation
between the appearance of BAD-LAMP and cortical
synatogenesis points towards a physiological role of this
vesicular determinant for neuronal function.
Introduction
Neurons are polarized cells specialized to carry out regulated
secretion of storage vesicles when an appropriate stimulus is
applied. Furthermore, synapse formation, stabilization and
maintenance require the delivery of transport vesicles to the
site of initial contact between axons and dendrites. These
vesicles, containing the different proteins necessary for proper
establishment and function of synapses, are the results of
complex interplay between the secretion and the endocytic
membrane transport pathways (Kennedy and Ehlers, 2006).
Another layer of complexity is introduced by the existence
of ordered lipid domains in the plasma membrane (Maxfield
and Tabas, 2005). In neurons, several types of microdomains
have been shown to be distinguishable by the partitioning of
different membrane-associated proteins such as thymus cell
antigen 1 (THY1) or the prion protein (PrP) (Sunyach et al.,
2003), which are found in different, albeit often closely
adjacent domains (Madore et al., 1999). These differences in
surface localization are reflected in the different trafficking and
functions of these proteins. THY1 is slowly internalized and
inhibits the activity of Src family kinases, whereas PrP is
rapidly endocytosed and induces axonal outgrowth via the
activation of fyn-related kinases (Santuccione et al., 2005).
Vesicular transport and lipid microdomain organization,
therefore, play key roles in neuronal development and function.
The LAMP family is composed of proteins bearing sequence
and structural homology with the canonical LAMP1 and
LAMP2 molecules. LAMP molecules harbor an endosomal
and lysosomal addressing signal within their short cytoplasmic
tail, and contain several conserved cysteine residues, which
allow the formation of particular structural loops known as
‘LAMP folds’. Although the structure, subcellular localization
and interaction partners of LAMP1 and LAMP2 have been
extensively characterized, their physiological function is still
elusive (Eskelinen et al., 2003). Lamp1-deficient mice are
viable and show a mild astrogliosis in the brain (Andrejewski
et al., 1999), whereas Lamp2 mutants show increased postnatal
lethality and massive accumulation of autophagic vesicles in
different tissues (Tanaka et al., 2000; Eskelinen et al., 2002).
Interestingly, LAMP2 deficiency in humans induces Danon
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/120/2/353/DC1
Key words: Corticogenesis, Endocytosis, Synaptogenesis, LAMP,
Lipid microdomains, Cortex
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disease, a lysosomal glycogen storage disorder characterized
by cardio- and skeletal myopathy and a variable degree of
mental retardation (Saftig et al., 2001).
We identified a new member of the LAMP protein family in
mice. Brain-associated LAMP-like molecule (BAD-LAMP) is
expressed after birth in cortical neurons of particular layers,
where it is enriched in defined zones along the neuronal
projections. BAD-LAMP mainly accumulates in distinct
intracellular vesicles, which do not contain any known markers
of classical intracellular transport pathways. BAD-LAMPcontaining vesicles have a remarkable clustered organization
mirroring at the neuronal surface the presence of THY1containing microdomains, but not of N-CAM and the
ganglioside GM1-enriched microdomains. Interestingly the
phosphoepitopes present on microtubule-associated protein 1B
and neurofilament H also define BAD-LAMP-containing
vesicle positioning in neurons. BAD-LAMP has the ability to
be endocytosed, but is not targeted to the late
endosomal/lysosome compartments (Gruenberg and Stenmark,
2004). The spatiotemporal specificity of BAD-LAMP
expression and its distribution reveal therefore a new level of
interplay involving unconventional endocytic compartments
and membrane microdomains in specific cortical neurons.
Results
BAD-LAMP is a new member of the LAMP family
expressed specifically in the post-natal mouse brain
During a bioinformatics search to identify lysosomalassociated molecules, several overlapping nucleotide
sequences were identified. After PCR cloning of the full-length
cDNA from mouse cortex and extensive sequencing, we
identified a potential open reading frame coding for a new
putative member of the LAMP family. The new ORF codes for
a protein of 280 aa (PI 6.42 and molecular mass of 31.7 kDa)
predicted to contain a transmembrane domain (aa 236-256) and
a cytoplasmic tail of 24 residues (Fig. 1A). This cytoplasmic
domain contains a YKHM (aa 276) motif corresponding to a
classical Yxx⌽ internalization and endosomal targeting signal.
The sequence also contains four highly conserved cysteine
residues separated by a fixed number of amino acids and is
likely to form characteristic internal di-sulfide bonds required
for a classical ‘LAMP fold’. The protein was also predicted to
contain three consensus N-glycosylation sites. The nucleotide
sequence shares 45% identity with LAMP1 and LAMP2, the
founding members of the family, whereas alignments at the
protein level displayed 25% similarity (19% identity) (see
supplementary material Fig. S1). Thus, the protein was placed
on an evolutionary classification tree between LAMP1 and
DC-HIL sequences, clearly identifying it as a new member of
the LAMP family (Fig. 1B). The tree indicates that DC-HIL
(15.5% of similarity), a dendritic cell specific molecule
functioning as an integrin ligand (Shikano et al., 2001), shared
a common ancestor molecule after diverging away from the
LAMP1/CD68 evolutionary axis. The molecule is extremely
conserved, since it is found in worm, fly, fish, chicken, rodent
and human (see supplementary material Fig. S1). The degree
of identity at the amino acid level is close to 85% among
mammals and 45% between mouse and fugu. This very high
level of conservation across species suggests that the molecule
performs a conserved cellular function, not accommodating
many variations of its tertiary structure.
Northern blot analysis of the identified mRNA using
different mouse tissues indicated that it is expressed almost
exclusively in the adult brain, with a close to background signal
in the E14 embryo (Fig. 1C). The detected mRNA corresponds
to a unique transcript of around 1.8 kb with no apparent
Fig. 1. Characterization of a new LAMP molecule. (A) BAD-LAMP
protein sequence showing predicted glycosylation sites (bold and
blue), putative di-sulfide bridges between two pairs of cysteins (red
line), transmembrane domain (aa 236-256 in blue and underlined)
and tyrosine endocytic sorting motif (YKHM, aa 276 in red).
(B) Phylogram representation of all the LAMP family members in
human and mouse. (C) Tissue distribution of BAD-LAMP by
Northern blot. Among all mouse tissues tested BAD-LAMP appears
to be expressed specifically in brain as a 2 kb mRNA transcript.
Actin mRNA levels are shown as control.
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BAD-LAMP in cortical neurons
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Fig. 2. BAD-LAMP is heavily glycosylated and is expressed after birth. (A) Immunoblot performed with a polyclonal antibody raised against
the predicted peptide of the BAD-LAMP cytoplasmic tail. Lysates of mouse adult cortex and BAD-LAMP-transfected HeLa cells produced
several bands migrating at above 35 kDa, whereas no reactivity is observed in control untransfected cells. The lowest form in the transfected
cells is probably due to ER accumulation. (B) HeLa cells transfected with FLAG-BAD-LAMP cDNA were lysed and immunoprecipitated with
anti-FLAG antibody. Immunoprecipitated material was treated with endoglycosidase H (endo H) or N-glycosidase F (N-gly F) prior to
immunoblotting with anti-BAD-LAMP. In untransfected cells (NC), just the anti-FLAG IgG band (arrow) is observed, whereas several isoforms
of BAD-LAMP were detected in transfected cells (Wt). Endo H treatment shows that the major band is endo H sensitive (gH) thus probably
accumulating in the ER. The higher molecular mass bands were all N-gly F sensitive (gF). N-gly F treatment also demonstrated that all
isoforms of BAD-LAMP are glycosylated and that the native molecular mass of the molecule is around 32 kDa (g0). The anti-FLAG IgG bands
are also N-gly F sensitive, arrows. (C) After fractionation and isolation of cortical membranes, BAD-LAMP was found to be present
exclusively in the membrane pellet (MbP) and not in the supernatant (Sup). Control syntaxin 6 and syntaxin 13 were also found in the
membrane pellet, whereas RAB3, as expected, had a shared distribution due to its shuttling nature. (D) BAD-LAMP expression after birth.
Mouse cortex lysates of different ages were immunoblotted with BAD-LAMP polyclonal antibody. BAD-LAMP expression levels are increased
from birth to adulthood. (E) In situ hybridization for Bad-lamp on coronal post-natal brain sections from P2 to P12. Hemisections are
presented. Bad-lamp expression appears at P2 in the cingulate cortex (arrowhead) and extends ventrally during the first post-natal weeks as a
superficial and a deep band in the cortex. Subcortically, Bad-lamp is expressed transiently in the caudate putamen (cp). Bar, 500 ␮m.
alternative spliced forms. Based on its relationship to the
LAMP family and its restricted pattern of expression, the
molecule was named BAD-LAMP, for brain-associatedLAMP.
BAD-LAMP is a glycosylated membrane-associated
protein
To investigate further BAD-LAMP distribution and function,
we raised antibodies against peptide epitopes present in its
cytoplasmic tail. These antibodies were characterized by
immunoblot of HeLa cells transfected with the cDNA coding
for mouse BAD-LAMP (Fig. 2A). Several bands were detected
in extracts from transfected cells, whereas control extracts
remained non-reactive. Brain extracts also displayed several
bands, mostly corresponding to those observed in transfected
cells, confirming the existence of several isoforms of BADLAMP. The detected proteins had a significantly higher
molecular mass than the one predicted from the primary
sequence of BAD-LAMP (31.7 kDa). In order to define the
nature of these post-translational modifications and in absence
of immunoprecipitating antibodies, we transfected an Nterminally tagged form of BAD-LAMP (FLAG-BAD-LAMP),
allowing efficient immunoprecipitation and treatment with
endoglycosidase H (Endo H) and N-glycosidase F (N-gly F).
Immunoprecipitated FLAG-BAD-LAMP was shown to be
heavily glycosylated (Fig. 2B). The major form of the protein
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glycosylated on at least two of its three acceptor sites, a
situation likely to be shared with endogenous BAD-LAMP
detected in brain.
Although glycosylation was in support of BAD-LAMP
membrane association, we demonstrated the membrane-bound
nature of BAD-LAMP by submitting mouse cortex postnuclear supernatants to high speed ultracentrifugation, in
which BAD-LAMP was found associated with the membranes
pellets similar to other membrane-associated molecules such
as RAB3a, syntaxin 6 and syntaxin 13 (Fig. 2C). Thus BADLAMP, is a glycosylated LAMP-like molecule associated with
cortical membranes.
Fig. 3. BAD-LAMP is specifically expressed in neurons of the
cortical layers II, III and V. (A) In situ hybridization for Bad-lamp
(top panel), Cux2 (middle) and ER81 (bottom) on adult mouse brain
coronal hemisections. (B) High magnification views of in situ
hybridization on wild-type cortex shown in A. Bad-lamp is expressed
in layers II, II and V, but excluded from layers IV and VI. In the
Scrambler cortex, the entire region appears disorganized. However,
the typical inversion of cortical layers is reflected by the altered
BAD-LAMP staining, demonstrating that projection neurons express
the protein. (C) Combined in situ hybridization for Bad-lamp (in
blue) with immunohistochemistry for the specific neuronal marker
NeuN (in brown). The left panel is a higher magnification of the
boxed area. Bad-lamp is co-expressed with this pan-neuronal marker
in many, although not all, neurons. (D) Immunohistochemistry of
BAD-LAMP in the indicated cortex layers. (E) Immunofluorescence
staining on adult cortex using anti-MAP2 (green) and anti-BADLAMP (red) antibodies; the merged image is on the left. Whereas
MAP2 is present along the entire dendrites, BAD-LAMP
accumulates in defined domains (arrows). Bars, 500 ␮m in A; 200
␮m in B; 100 ␮m (left panel) and 10 ␮m (right panel) in C; 10 ␮m
in D.
(38 kDa, gH) remained Endo H sensitive, thus reflecting
endoplasmic reticulum (ER) retention due to over-expression.
The two higher additional bands (47 and 53 kDa, gF) were
Endo H resistant but remained N-gly F sensitive, as indicated
by the accumulation of a fully trimmed 31 kDa protein (g0)
after treatment. Transfected BAD-LAMP is therefore heavily
BAD-LAMP is expressed in neurons of specific cortical
layers after birth
Analysis of mouse brain extracts by immunoblotting revealed
that levels of BAD-LAMP increased strongly after birth (P0)
reaching its maximum level at adulthood, but being already
strongly expressed at P10-P12 (Fig. 2D). We used in situ
hybridization to investigate in detail the expression pattern of
BAD-LAMP in the developing mouse forebrain. The first
expression of BAD-LAMP was found at P2 in the cingulate
cortex, in a thin band of intermediate cells (Fig. 2E). At P5,
expression extended ventrally into the cortical plate.
Furthermore, the caudate putamen showed a punctuate
expression of Bad-lamp transcripts. This expression pattern
was maintained at P7, when an additional broad band of large
and strongly Bad-lamp-positive cells appeared in superficial
parts of the cortical plate. Although the cortical expression
intensified until P9, no major regional changes in Bad-lamp
expression were obvious during this period. At P12, expression
of Bad-lamp in the striatum ceased, while expression further
intensified in the cortex. This expression pattern was stable
until adulthood. Altogether, this expression pattern indicates
that BAD-LAMP does not function in the early steps of brain
development, such as neurogenesis and cell migration, but
potentially during terminal steps of neuronal differentiation
and neuronal function.
Within the adult cortex, the homogenous staining in outer
regions of the cortical plate, as well as in a more restricted band
of cells localized centrally, was suggestive of an expression in
neurons of specific cortical layers. We used well-known
markers for cortical layers to further characterize the respective
populations. Comparison of the expression of Bad-lamp to that
of Cux2, a marker for layers II-IV, showed that the BADLAMP domain is included in the CUX2 domain and confined
to its outer part (Fig. 3A,B). Thus, BAD-LAMP is expressed
in the upper layers II and III of the neocortex, but is excluded
from layer IV. Furthermore, there was a perfect overlap with
the layer V marker ER81 (Fig. 3A,B) demonstrating that the
deeply positioned Bad-lamp-positive population is located in
layer V.
The size of the Bad-lamp-positive cells in the respective
cortical layers was suggestive of neuronal cells. To confirm this
observation we investigated the expression of Bad-lamp in
Scrambler mice. These animals show a well described
inversion of the layers of cortical projection neurons, with
upper layer neurons (layers II-IV) positioned deeply whereas
deep layer neurons (layers V and VI) are positioned
superficially (Rice and Curran, 1999). The organization of B
Bad-lamp-positive cells in the Scrambler cortex was strikingly
BAD-LAMP in cortical neurons
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Fig. 4. BAD-LAMP is present in
vesicles clustering in specific areas of
the neurons. Immunofluorescence
confocal microscopy of cortical
neurons. (A) Staining for BAD-LAMP
(red) and cholera toxin (GM1, green).
(B) Staining for BAD-LAMP (red) and
PrP (green). (C) Staining for BADLAMP (red) and N-CAM (green).
BAD-LAMP is expressed in small
vesicles clustered in neurites and
accumulates in areas lacking surface
semi-ordered lipid microdomain
resident proteins (arrowheads).
(D) THY1 labelling (green) defines the
zones in which BAD-LAMP vesicles
accumulate (red). However, THY1 (red)
is present at the cell surface and does
not co-localize with BAD-LAMP as
seen at higher magnification (bottom
panels, arrowheads). (E) Cholesterol
depletion disrupts cluster organization
and induces BAD-LAMP (red) and
cholera toxin (GM1, green) colocalization. Bars, 20 ␮m; 10 ␮m for
THY1 high magnification.
altered (Fig. 3B). Small, lighter stained cell bodies were
displaced towards the ventricular side, whereas larger and more
strongly labelled cells were merely found at the pial side of the
cortex. This pattern was in agreement with an inversion of the
position of Bad-lamp-positive cells and suggestive of a
projection neurone identity. Furthermore, all Bad-lamppositive cells in the cortex were co-expressing the neuronal
marker NeuN, again confirming the neuronal identity of
labelled cells (Fig. 3C), which were found also to express
BAD-LAMP protein (Fig. 3D).
BAD-LAMP is distributed in specific domains of cortical
neurons
BAD-LAMP distribution in cortical brain sections was
monitored by confocal microscopy (Fig. 3E). BAD-LAMP was
found in vesicles mostly located in cell bodies as delineated by
the MAP2 staining. In addition, BAD-LAMP accumulated in
defined domains along cellular projections (Fig. 3E
arrowheads). It could be detected at the plasma membrane
and in vesicles present in the cell bodies, but was also
enriched in vesicles clustered in defined domains of
dendrites. BAD-LAMP mostly accumulated within the
boundaries of specific neuronal areas in vivo.
In order to confirm the relevance of these observations,
embryonic cortical neurons were explanted and BADLAMP sub-cellular distribution was investigated after 3
days in culture. Owing to the particular clustered
distribution of BAD-LAMP, we also investigated the
distribution of proteins known to partition in different
cellular domains, such as lipid microdomain-associated
proteins (Madore et al., 1999). Using confocal
microscopy we found that semi-ordered lipid
microdomain residents such as PrP, N-CAM, as well as
the ganglioside GM1 (stained with cholera toxin, CT)
were enriched in zones excluding BAD-LAMP vesicles (Fig.
4A-C). This observation was particularly striking with CT
staining and N-CAM, which accumulated almost exclusively
in areas negative for BAD-LAMP (Fig. 4A,C). By contrast, at
this low magnification, THY1, a molecule representing ordered
lipid microdomain-associated proteins, displayed an
overlapping distribution with BAD-LAMP (Fig. 4D). However,
at higher magnification, no direct co-localization of THY1 and
BAD-LAMP molecules could be observed. Instead,
accumulation of BAD-LAMP-containing vesicles was revealed
directly underneath THY1-enriched areas at the plasma
membrane (Fig. 4D, arrowheads). BAD-LAMP-containing
vesicles therefore accumulate in cellular zones, defined by the
presence of THY1 at the plasma membrane, whereas they are
segregated from the detergent-resistant microdomains
containing most of the PrP, GM1 and N-CAM (Madore et al.,
1999).
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Fig. 5. BAD-LAMP organization is
defined by microtubules.
Immunofluorescence confocal
microscopy of cortical neurons. (A)
BAD-LAMP vesicles (red)
accumulate in areas of the cell that
are strongly enriched in the phosphoepitope detected by the Smi31
antibody (blue), whereas the L1
molecule (green) is distributed
throughout the neuronal plasma
membrane. (B) (top) BAD-LAMP
vesicle accumulation (white)
coincides with microtubule bundling
(␤-tubulin, red) and weak GM1
staining (green). Higher
magnification (Z1) shows that BADLAMP vesicles align along
microtubules. (Bottom) Nocodazole
treatment induces microtubule
destabilization and disorganization of
BAD-LAMP vesicle clusters. Bars,
20 ␮m; 10 ␮m for Z1.
BAD-LAMP distribution and microdomain organization
seemed to be closely linked. Cholesterol depletion efficiently
affects lipid microdomains and was therefore tested for its
ability to influence BAD-LAMP distribution. Cortical neurons
were treated for 2 hours with cholesterol-esterase prior to
immunostaining and confocal microscopy visualization (Fig.
4E). As expected, cholesterol depletion had a potent effect on
GM1 distribution at the plasma membrane. Moreover, BADLAMP vesicular staining was also deeply affected, displaying
an extensive co-localization with GM1, which was never
observed in normal conditions. Thus, microdomain
organization
at
the
membrane
and
clustering
of BAD-LAMP-positive vesicles appeared to be directly
linked.
The cytoskeleton controls the distribution of BAD-LAMP
vesicles
This particular organization was likely to be maintained with
the active participation of the cytoskeleton and/or associated
proteins. In order to test this hypothesis, several candidate
molecules were followed by confocal microscopy in cortical
neurons. Surprisingly, BAD-LAMP containing-vesicles
clustered within punctate zones delimited by staining with the
Smi31 antibody (Fig. 5A). Smi31 detects phosphorylated
epitopes present in neurofilament H and mostly in the
microtubule-associated molecule 1B (MAP1B) (Fischer and
Romano-Clarke, 1990). Although the precise function of
MAP1B phosphorylation is still debated, experimental
evidence suggests a role in regulating microtubules and actin
dynamics as well as being necessary for axonal growth
(Dehmelt and Halpain, 2004; Del Rio et al., 2004). The perfect
overlapping distribution of BAD-LAMP and Smi31 strongly
suggested that microtubules or actin are likely to play in
important role in the organization and the clustering of BADLAMP-positive vesicles, however BAD-LAMP distribution is
not dependent on the neuronal polarity.
BAD-LAMP-positive vesicles were found in the close
vicinity of the microtubule network, mirroring, by their
accumulation, the intensity of the tubules bundling (Fig. 5B).
A treatment with the microtubule depolymerizing agent
nocodazole was thus carried out (Fig. 5B). Nocodazole induced
a strong redistribution of BAD-LAMP-containing vesicles and
a loss of BAD-LAMP staining intensity in cortical neurons.
Thus the microtubule network influences the positioning of
BAD-LAMP vesicles. Lipid microdomain organization and
BAD-LAMP distribution in cortical neurons are therefore
linked, use the microtubule network and possibly depend on
MAP1B phosphorylation for their regulation.
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BAD-LAMP in cortical neurons
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BAD-LAMP defines a specific subset of early
endosomes
The unusual distribution of BAD-LAMP vesicles led us to
investigate their relationship with other types of sub-cellular
compartments. We focused primarily on endocytic organelles,
likely to be relevant to a transmembrane molecule bearing a
Yxx⌽ motif in its cytoplasmic tail. BAD-LAMP could not be
detected in classical endosomal compartments as judged from
its lack of co-localization with LAMP2 (late endosomes and
lysosomes) and internalized transferrin-FITC (sorting and
recycling endosomes) (Fig. 6A,B) or syntaxin 13 (Hirling et
al., 2000). BAD-LAMP was not found in more specialized
endocytic compartment such as TI-VAMP-positive vesicles
(Coco et al., 1999) (Fig. 6C). Co-labeling with synaptic vesicle
proteins such as synaptotagmin 1, RAB3a, VAMP2 revealed
some level of co-localization with BAD-LAMP in the growth
cones (Fig. 6D-F). Interestingly, co-localization was not
observed in other cellular areas and a similar overlapping
distribution in the growth cone was observed with TI-VAMP,
which is not found enriched in synaptic vesicles (Coco et al.,
1999). Thus, this co-distribution in the growth cone probably
reflects the difficulty of segregating, at this optical resolution,
individual carrier vesicles congregating in the same area of the
cone, rather than a true co-localization in the same vesicles.
Pre-and post-synaptic transport carriers are derived from transGolgi network (TGN) vesicles, which aggregate at initial
contacts between axons and dendrites (Sytnyk et al., 2002).
We, therefore, examined the possible association of BADLAMP with other known vesicular markers of these pathways,
such as syntaxin 6 or N-CAM (Sytnyk et al., 2004) (Fig. 6E).
We failed to detect co-localization of BAD-LAMP with any of
these markers (see supplementary material Fig. S2), suggesting
that the molecule is sorted in an uncharacterized type of
vesicles, which can accumulate in the growth cone of
developing axon, as well as in defined and organized domains
along the cellular processes.
BAD-LAMP distribution at the plasma membrane as well as
in localized intracellular vesicles suggested a possible shuttling
of the molecule between the cell surface and the vesicles. The
co-localization of GM1 and BAD-LAMP upon cholesterol
depletion suggests that BAD-LAMP vesicles are accessible to
plasma membrane constituents under specific conditions. To
address this issue, cortical neurons were surface biotinylated at
4°C prior to incubation at 37°C. Biotinylated surface proteins
could either diffuse, or be internalized, and their intermixing
with BAD-LAMP-positive compartments was evaluated at
different time points by confocal microscopy (Fig. 7).
Biotinylated proteins were detected rapidly co-localizing with
BAD-LAMP after 5 minutes of internalization. This significant
overlapping distribution decreased after 45 minutes, suggesting
that BAD-LAMP-containing organelles could represent a
subset of early endocytic vesicles, rapidly accessible from the
neuronal surface and serving as an intermediate step for the
intracellular sorting of specific surface molecules present in
developing neurons.
Fig. 6. Confocal immunofluorescence microscopy analysis of BADLAMP transport in cortical neurons. (A) Staining for LAMP2
(green) and BAD-LAMP (red). (B) Internalized transferrin-FITC
(green) in early and recycling endosomes and BAD-LAMP (red).
(C) Staining for Ti-VAMP (green) and BAD-LAMP (red).
(D) Staining of a growth cone for synaptotagmin 1 (SYT1, green)
and BAD-LAMP (red). (E) Staining of a growth cone for VAMP2
(green) and BAD-LAMP (red). (F) Staining for syntaxin 6 (green)
and BAD-LAMP (red). BAD-LAMP does not display any significant
co-localization with LAMP1 and internalized transferrin. Bars, 20
␮m in A,B,C,F, 10 ␮m in D,E.
BAD-LAMP sorting in transfected neurons
To further investigate the distribution of BAD-LAMP, we
generated N- terminally FLAG-tagged and C-terminally GFPtagged BAD-LAMP constructs and monitored their behavior
by microscopy in co-transfection experiments of cortical
neurons (Fig. 8). Surprisingly, endogenous BAD-LAMP
expression and domain organization were strongly inhibited in
electroporated neurons. Nevertheless, transfected FLAGtagged BAD-LAMP was found enriched in vesicles clustered
in specific zones along the neurites. Clearly, the tagged protein
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Fig. 7. Surface biotinylation reveals
the endocytic nature of BADLAMP-containing vesicles.
Cortical neurons were surface
biotinylated for 15 minutes at 4°C,
prior to warming at 37°C for
different times, fixation and
visualization by confocal
microscopy. (A) Prior to warming,
no significant co-localization of
biotinylated proteins with BADLAMP was observed. Colocalization was evaluated using
the Image J image analysis
software. A low Pearson’s
coefficient and strong negative
pixel shift are both indicative of the
absence of staining overlap (right).
(B) After 5 minutes of endocytosis
at 37°C, extensive co-localization
of biotinylated proteins (green) was
observed with BAD-LAMP (red) in
neurites (arrowheads), as also
shown by a higher Pearson’s
coefficient and the absence of pixel
shift (right). (C) After 45 minutes
of endocytosis co-localization of
BAD-LAMP with biotin is
decreased as shown by a decreased
Pearson’s coefficient and negative
pixel shift (right). Bar, 10 ␮m.
is not addressed in conventional endo/lysosomes as judged by
its lack of co-localization with LAMP2 (Fig. 8A), internalized
transferrin (not shown) or cholera toxin (see supplementary
material Fig. S3). The exact location of FLAG-BAD-LAMP in
the cell body was difficult to establish since its over-expression
induced an accumulation of the molecule in the ER and Golgi
network. Surprisingly, the C-terminally GFP-tagged, BADLAMP (BAD-GFP) was found accumulating in LAMP2positive lysosomal compartments (Fig. 8A, arrowheads in Z1).
Therefore, the BAD-LAMP cytoplasmic tail contains a cryptic
lysosomal retention motif, which is revealed by the addition of
the GFP moiety. This observation also suggests that the
cytoplasmic tail of BAD-LAMP is actively interacting with, or
modified by, molecules that promote its sorting away from
traditional endocytic compartments. Co-localization of BADGFP and FLAG-tagged BAD-LAMP was observed in discrete
vesicles in neurites (Fig. 8A, Z2 arrowheads), despite the fact
that BAD-GFP was found mostly accumulating in large
lysosomes in the cell body. This demonstrates that a small
fraction of BAD-GFP can be sorted normally.
We next evaluated the internalization dynamics of BADLAMP by using the N-terminally FLAG-tagged construct and
by monitoring FLAG antibody uptake after cold binding (Fig.
8B). The antibody was rapidly endocytosed after 5 minutes at
37°C. Inside the cell, it was detected in a different compartment
from conventional endo/lysosomes as shown by the absence of
co-localization with co-transfected BAD-GFP, LAMP2 (Z3
and arrows) and internalized cholera toxin (supplementary
material Fig. S3A). After 30 minutes of synchronous uptake
(Z4 and arrowheads), co-localization of the antibodies with
BAD-LAMP-GFP and LAMP 2 indicated that BAD-LAMP
can reach conventional endocytic compartments, after being
internalized from the surface. Surprisingly, this co-localization
was more evident in the more discrete LAMP2-positive
organelles present in the neurite (late endosomes, arrowheads)
than in the large lysosomes observed in the cell body (Fig. 8B).
We next investigated the contribution of tyrosine 276 to
BAD-LAMP trafficking by introducing a mutational change to
alanine at this position (Tyr276Ala). The FLAG-tagged mutant
was also found accumulating in the ER and Golgi network of
transfected neurons. However, the fraction of the mutant that
exited these organelles accumulated at the surface of the
neurites in a manner very distinct from the normal molecule
(wild type), which was mostly found in intracellular vesicles
(supplementary material Fig. S3B). Similar results were
obtained with a construct lacking the entire cytoplasmic tail of
BAD-LAMP (not shown). Thus, tyrosine 276 is directly
involved in intracellular addressing of BAD-LAMP and allows
BAD-LAMP in cortical neurons
361
Journal of Cell Science
its internalization from the surface. FLAG
antibody uptake after binding in the cold
was performed in neurons expressing
FLAG-BAD-LAMP
Tyr276Ala.
Transfected cells remained mostly
antibody-decorated at the surface 30
minutes after warming at 37°C
(supplementary material Fig. S3C). Thus,
BAD-LAMP is probably cycling between
the plasma membrane and a subset of
endocytic vesicles.
BAD-LAMP recycles in HeLa cells
In order to further dissect the molecular
mechanisms
governing
BAD-LAMP
endocytosis, we studied the distribution
and transport of transfected BAD-LAMP in
a cell type easy to manipulate, such as
HeLa and mouse NIH 3T3 cells. In HeLa
cells, FLAG-BAD-LAMP was found at the
cell surface and in internalized transferrincontaining vesicles distributed in the
vicinity of the plasma membrane, whereas
the Tyr276Ala mutant accumulated only at
the cell surface (Fig. 9A). No colocalization was found in LAMP1-positive
late endosomes or lysosomes, nor with cotransfected DC-LAMP tagged with GFP
(Fig. 9B), another lysosomal resident of the
LAMP family (de Saint-Vis et al., 1998).
These observations were confirmed after
Percoll density gradient subcellular
fractionation of transfected HeLa cells
(supplementary material Fig. S4). BADLAMP was mostly detected in the low
density fractions of the gradient containing
plasma membrane, ER and early
endosomes, but it was absent from the high
density fractions containing lysosomes, as
indicated by ␤-hexosaminidase activity.
Thus, most of transfected BAD-LAMP was
found on the cell surface contrasting with
transfected neurons in which BAD-LAMP
mostly
accumulated
intracellularly,
underlining the specificity of its sorting
even when over-expressed.
Fig. 8. Localization of FLAG-tagged BAD-LAMP in transfected cortical neurons.
Anti-FLAG
antibody
uptake
in
Cortical neurons co-transfected with BAD-GFP and FLAG-BAD-LAMP were visualized
transfected cells saturated with FITCby confocal microscopy. (A) Staining for FLAG antibody (red), BAD-GFP (green) and
transferrin (FITC-TF), confirmed that
LAMP2 (white). FLAG-BAD-LAMP does not co-localize with LAMP 2. BAD-GFP is
BAD-LAMP could be internalized rapidly
targeted to lysosomes upon addition of the GFP moiety at the C-terminal end of BADin sorting endosomes (Fig. 9C).
LAMP. Bar, 20 ␮m; 10 ␮m for high magnification of Z1 and 5 ␮m for Z2. (B)
Interestingly, 15 minutes after uptake
Internalization of FLAG antibody (red) in transfected neurons for indicated times and
BAD-LAMP was found present in
staining for LAMP2 (white). High magnifications reveal a late accessibility of BADLAMP into LAMP2 positive compartments in neurites. Bars, 20 ␮m; 5 ␮m for high
transferrin-positive recycling endosomes
magnification.
clustered
around
the
microtubule
organizing center, suggesting that BADLAMP could recycle to the plasma
again a difference with neurons, in which the antibodies could
membrane after internalization. This hypothesis was supported
be detected in discrete LAMP1-positive compartment 45
by the poor co-localization of the antibody with LAMP1 after
minutes after uptake.
45 minutes of uptake, indicating that the molecule does not
We next investigated the molecular mechanisms involved in
efficiently reach late endocytic compartments. This underlines
362
Journal of Cell Science 120 (2)
Journal of Cell Science
Fig. 9. BAD-LAMP is targeted to early
endosomes and recycles in HeLa cells.
HeLa cells transfected with FLAG-BADLAMP were submitted to
immunofluorescent staining and confocal
microscopy visualization. (A) FLAG-tagged
BAD-LAMP (anti-flag antibody, red) was
found at the cell surface and in internalized
transferrin-FITC-containing endosomes.
Cytoplasmic tail tyrosine 276 mutant
(Tyr276-Ala) was found accumulating at the
surface of transfected cells (anti-flag
antibody, red) with little intracellular
distribution (transferrin-FITC, green). (B)
Transfected FLAG-BAD-LAMP is not
detected in LAMP1- (blue) and DC-LAMP
(green)-positive late endosomes and
lysososomes. (C) Kinetics of FLAG
antibody uptake after cold binding on the
surface of transfected HeLa cells. Only
transfected cells accumulate the antibody
(red) on their surface, which upon warming
reaches rapidly sorting (5 minutes) and
recycling (15 minutes) endosomes
containing transferrin-FITC (green). No colocalization with LAMP1 (white, 45
minutes) could be observed, suggesting that
BAD-LAMP and associated antibodies do
not access the late endocytic pathway. (D)
Co-expression of dynamin dominant
negative mutant A44K (right panel, green)
in FLAG-BAD-LAMP-transfected HeLa
cells prevents the internalization of
associated flag antibodies (right panel, red),
whereas expression of wild-type dynamin
has no effect (green, left panel). (E) Cotransfection of HeLa cells with FLAGBAD-LAMP (anti-BAD-LAMP, red) and
pSuper control plasmid (left) has no effect
on the internalization of associated flag
antibodies (green). Conversely RNAi
inhibition of the clathrin adaptor AP2 blocks
flag antibodies uptake (green). Bars, 20 ␮m.
BAD-LAMP endocytosis. Experiments performed in cells cotransfected with wild-type GTPase dynamin II or dominantnegative mutant A44K, indicated that BAD-LAMP
internalization is mediated in a dynamin-dependent manner,
since antibody internalization was abolished in cells
expressing dynamin A44K (Fig. 9D and control,
supplementary material Fig. S4C). In order to further define
the endocytic pathway used by BAD-LAMP to enter the cell,
we used an RNA inhibition approach to reduce the expression
of molecules involved in protein triage from the surface, such
as the clathrin adaptor AP2 (Dugast et al., 2005; McCormick
et al., 2005). Antibody uptake was monitored by
immunostaining and FACS detection after binding at 4°C and
internalization at 37°C. Cells co-transfected with FLAGBAD-LAMP and control RNAi plasmid showed rapid
internalization of the antibody (Fig. 9E), whereas RNAi
depletion of AP2 clearly inhibited BAD-LAMP
internalization as well as transferin uptake (supplementary
material Fig. S4C). In cells depleted for AP2, higher surface
levels of BAD-LAMP were also consistently detected (not
shown), suggesting that BAD-LAMP is internalized
constantly through a dynamin/AP2-dependent endocytic
pathway.
Interestingly, monitoring of surface anti-FLAG antibody by
FACS also indicated that the molecule was rapidly internalized
between 5 and 7.5 minutes after warming (supplementary
material Fig. S4B). Surface levels of antibodies then reincreased after 10 minutes, to be diminished again but with a
relatively slower internalization rate. These observations
confirm that BAD-LAMP and associated antibodies constantly
recycle to the plasma membrane with a relatively high
efficiency.
Discussion
BAD-LAMP sequence analysis clearly indicates that it
represents a new member of the LAMP family. However, its
expression pattern and intracellular distribution are
unconventional compared to other LAMP family members,
which show a widespread expression and specifically
accumulate in the lysosomes.
Journal of Cell Science
BAD-LAMP in cortical neurons
Our observations on BAD-LAMP intracellular distribution
are clearly indicative of a strong regulation of its trafficking in
a subset of early endosomes. Although we have not been able
to identify molecular markers able to identify these organelles,
the absence of transferrin or synaptotagmin 1, as well as late
endosomal markers such as LAMP1 suggests that these
vesicles represent a distinct class of neuronal endosomes. The
kinetics of biotinylated proteins and antibody uptake indicate
that they can serve as sorting platforms, prior to transport to
other organelles, which are positive for LAMP1, but only
represent a minor fraction of the neuronal organelles
containing LAMP1.
We have shown that BAD-LAMP, through an interaction
with its YKHM domain, requires dynamin and AP2 to be
internalized and sorted towards the early endocytic recycling
pathway of transfected HeLa cells. LAMP1 has also been
shown to require the AP2 adaptor, but its sorting is directed
towards lysosomes (Janvier and Bonifacino, 2005).
Interestingly, modification of the BAD-LAMP C-terminal
domain by GFP deeply affects its transport in neurons and
demonstrates the existence of an active sorting pathway in
these cells, which normally prevents the accumulation of BADLAMP in the lysosomes. The YKHM domain is a relatively
weak consensus endosomal/lysosomal addressing signal
(Bonifacino and Traub, 2003), although it is also found in
CTLA-4, a molecule known to recycle upon activation of T
cells (Linsley et al., 1996). As suggested by its early
endosomes distribution, we could show that BAD-LAMP also
recycles in transfected HeLa cells. Whether this is the case in
neurons remains to be further investigated, although it clearly
indicates that the ‘YKHM’ domain is not normally used as a
lysosomal addressing signal.
One of the features of BAD-LAMP-containing organelles is
their clustered distribution. This distribution mirrors the
organization of the different microdomains at the cell surface.
Whether BAD-LAMP-containing organelles participate in the
maintenance of this organization within the neuritic plasma
membrane remains to be proved. Nevertheless their sensitivity
to cholesterol-depleting drugs suggests that microdomains and
BAD-LAMP-containing vesicles are functionally linked.
Strikingly, the clustering the BAD-LAMP-containing vesicles
is also defined by the distribution of the phosphorylated
epitopes (SMI31) found on the microtubule-associated protein
MAP1B or neurofilament H (Fischer and Romano-Clarke,
1990). MAP1B in the cortex has been strongly implicated in
synapse formation and function (Kawakami et al., 2003). Such
a role has been recently functionally demonstrated through the
observation that mice lacking the phosphorylated form of
MAP1B specifically in the hippocampus, show deficits in longterm potentiation in the Schaeffer collaterals pathway (Zervas
et al., 2005). Therefore, it is conceivable that MAP1B is
implicated in the positioning and transport of BAD-LAMP
vesicles at sites of postsynaptic densities on the dendrites of
cortical neurons, and that this process could be essential for
stabilization, function and plasticity of cortical synapses.
Indeed, BAD-LAMP expression is temporally and spatially
restricted in cortical neurons of layers II, III and V. Whereas
the generation and migration of cortical neurons in rodents is
an embryonic process, synaptogenesis in the cortex occurs in
the postnatal animal with a peak between P10 and P15 to
approach adult values (Micheva and Beaulieu, 1996). This
363
increase in functional synapses in the cortex is strikingly
mirrored by the expression of BAD-LAMP during
corticogenesis. Thus, it appears very possible that BADLAMP, together with MAP1B, is involved in the terminal
maturation steps and/or function of defined cortical neurone
populations.
Most of our observations point towards a link between BADLAMP and endocytosis. The transformation of a transient
contact between two neurons into a stable and functional
synapse requires major changes in the membrane composition
of the respective neuronal surface areas. Endocytic processes
have been implicated in the regulation of synaptic function and
plasticity in vertebrates (Vissel et al., 2001) and in Drosophila
(Dickman et al., 2006). For example, NMDA receptors are
subject to constitutive (Roche et al., 2001) as well as agonistinduced (Vissel et al., 2001) internalization through clathrinmediated endocytosis. Interestingly, in situ hybridization for
NMDAR1 resulted in strong cellular labeling in neurons of
layers II/III, V and VI (Rudolf et al., 1996), resembling the
pattern we found for BAD-LAMP in the postnatal cortex. The
BAD-LAMP-containing endocytic compartment could
therefore play a regulatory role in these events by maintaining
specific zones in the neuronal projections.
Materials and Methods
Bioinformatics
The BAD-LAMP protein sequence ID in Ensembl database is
ENSMUSP00000061180. All LAMPs sequences were aligned using CLUSTALW
package (EBI) and results were treated with TreeView for phylogeny. Image
analysis was performed with the Image J software and the plugin JacoP.
Animals and tissues
All animals were treated according to protocols approved by the French Ethical
Committee. CD1 mice (Iffa-Credo, Town?, France) were used to determine the Badlamp expression pattern. Disabled 1 deficient Scrambler mice were purchased from
Jackson Laboratories. The day of the vaginal plug appearance was considered as
embryonic day (E)0.5 and the day of the birth as postnatal day (P)0. For in situ
hybridization and immunohistochemistry, postnatal and adult brains were collected
after the animals were anaesthetized with a lethal dose of Rompun/Imalgen 500 and
intracardially perfused with 4% paraformaldehyde (PFA). Brains were further fixed
in 4% PFA overnight. Adult brains were sectioned at 80 ␮m on a vibratome whereas
P2-P12 brains were cryoprotected in 20% sucrose/PBS, frozen in OCT compound
and sectioned at 16 ␮m on a cryostat. Sections collected on Superfrost slides were
treated as described below.
Molecular biology
Northern blot analysis was done with FirstChoice Northern Blot Mouse Blot I
(Ambion) using a probe corresponding to exons 4, 5 and 6 of BAD-LAMP (clone
IMAGE 2588577). 2 mg of Trizol extracted total mouse cortex RNA was used for
reverse transcription with oligo(dT) primers. The cDNAs coding for BAD-LAMP
were amplified after 30 cycles of PCR using Taq polymerase. Sense primer was
ACC GGC CAC TTT GAG GGA and antisense GGG GCG GCC TTT GCA GCA
(1.5 kb). PCR products were cloned into pGEM-Teasy plasmid (Promega). BADLAMP-GFP fusion construct was constructed using pEGFP-NI vector (Clontech).
FLAG-BAD-LAMP was constructed using pTEJ-8-HA- FLAG plasmid (Didier
Marguet, Marseille, France). A tyrosine mutant of BAD, FLAG -BAD-Tyr-276-Ala
was produced by targeted PCR mutagenesis. FLAG-BAD-LAMP cDNA were
transferred into pCX-MCS2 plasmid, a pCAAGS derived plasmid with an extended
cloning site (a kind gift from Xavier Morin, Marseille, France). Dynamin-GFP wt
plasmid and dynamin-GFP A44K were kindly given by M. McNiven, Rochester,
MN. RNAi constructs pSUPER AP2 ␮2 and pSUPER control were a gift from
Philippe Benaroch, Paris, France.
In situ hybridization and immunohistochemistry
IMAGE clone 2588577 was used to make an antisense RNA probe. Antisense RNA
probes for Bad-lamp, Cux2 (Zimmer et al., 2004) and ER81 (Lin et al., 1998) were
generated using the Dig-RNA labelling kit (Roche). Single in situ hybridization and
combined in situ hybridization with immunohistochemistry were described
previously (Tiveron et al., 1996; Zimmer et al., 2004) for all probes and the NeuN
monoclonal mouse IgG (MAB377; Chemicon).
364
Journal of Cell Science 120 (2)
Antibodies and immunocytochemistry
A polyclonal rabbit anti-BAD-LAMP was raised in rabbit against two peptides
of the BAD-LAMP cytoplasmic tail, KMTANQVQIPRDRSQC and
KQIPRDRSQYKHMC. Anti-synaptotagmin 1 and anti-RAB3a/b antibodies were
obtained from P. Di Camilli, New Haven, CT, anti-FLAG M2 antibody and anti-␤tubulin-Cy3 were obtained from Sigma, anti-VAMP2 from SYSY, anti-syntaxin 6
from BD Transduction Laboratories; Aanti-PrP (6H4) was from Prionics (Schlieren,
Switzerland), anti-syntaxin 13 from Stressgen (Ann Arbor, MI); anti-Thy1 from
Michel Pierres, Marseille, France; human Alexa Fluor 568-Tf from Molecular
Probes; mouse FITC-Tf from Rockland (Gilbertsville, PA), Cy3-␤-tubulin from
Sigma, FITC-cholera toxin B subunit (GM1 staining) from Sigma; anti-NCAM H28
from C. Goridis, Paris, France; Anti-Ti-VAMP from T. Galli, Paris, France; Rat antimouse LAMP2 from I. Mellman (New Haven, CT) and anti-human LAMP1 from
Abcam. All FITC and Cy3-5 secondary antibodies were from Jackson
ImmunoResearch. All Alexa secondary antibodies were from Molecular Probes.
Immunofluorescence and confocal microscopy was performed with a Zeiss LSM
510 microscope as described previously (Cappello et al., 2004). Vibratome adult
brain sections were immunostained with rabbit anti-BAD-LAMP and mouse antiMAP2.
Cell culture
Journal of Cell Science
HeLa cells were grown in DMEM containing 10% FCS. Cortical neurons were
prepared from E15.5 embryonic cortices. Cortices were dissected out in HBSS,
treated for 15 minutes at 37°C in Trypsin/EDTA-HBSS (Invitrogen), washed once
in NeuroBasal medium (NB; Invitrogen) complemented with 10% horse serum to
block trypsin activity and washed once more in NB alone. Cortical neurons were
dissociated, plated on glass coverslips in NB with B27 complement, 2 mM Lglutamine and 50 ␮g/ml penicillin/streptomycin (Invitrogen) and cultured for 3 days
at 37°C, 5% CO2. Coverslips were coated overnight with poly-L-lysine (10 ␮g/ml).
Transfection and internalization experiments
Neurons were electroporated using Amaxa Nucleofactor Kit according to the
manufacturer’s instructions. HeLa cells were grown on coverslips and transfected
using Lipofectamine 2000 (Invitrogen) using the manufacturer’s protocol. After 824 hours of transfection the HeLa cells were processed to study internalization
kinetics or fixed using 3% paraformaldehyde. Internalization assays were performed
using FITC-conjugated transferrin or unconjugated antibodies. The cells were first
incubated for 20 minutes at 37°C in DMEM/100 mM HEPES to eliminate
endogenous transferrin. Cells were incubated for 15 minutes at 4°C with ligand
and/or antibody and washed twice in ice-cold PBS before incubation with DMEM,
1% BSA, 100 mM Hepes at 37°C, for different times prior to fixation and
immunocytochemistry. Neurons were processed identically in NB medium. Cortical
neurone biotinylation was performed using EZ-Link Sulfo-NHS-Biotin kit (Pierce)
with a 15-minute reaction time at 4°C, followed by three washes with ice-cold PBS
containing 10 mM glycine. Cells were incubated for 5 and 45 minutes at 37°C to
allow endocytosis of biotinylated membrane proteins, prior to fixation and
immunostaining.
Immunoblots and immunoprecipitation
1% Triton X-100 cell extracts complemented with protease inhibitors cocktail
(Roche) were immunoblotted after separation by 12% or a 7-17% gradient SDSPAGE. Immunoprecipitation with anti-FLAG antibody and N-glycosidase F or
endoglycosidase H (Calbiochem) treatment were performed as described previously
(Cappello et al., 2004).
This work was supported by grants to P.P. from CNRS-INSERM,
the Ministère de la Recherche et de la Technologie (ACI), La Ligue
Nationale Contre le Cancer and the Human Frontier of Science
Program. A.D. is supported by the MRT and ARC. P.P. is part of the
EMBO Young Investigator Program. H.C. was supported by the
French Fondation pour la Recherche sur le Cerveau (FRC), the
Association Francaise contre le Myopathies (AFM) and the European
Community through the NOE NeuroNE. We thank the PICsL imaging
core facility for expert technical assistance. We are grateful to Vilma
Arce for expert technical advice.
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