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First published online January 10, 2007
doi: 10.1242/10.1242/jcs.03316

Journal of Cell Science 120, 353-365 (2007)
Published by The Company of Biologists 2007
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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,{ddagger},§ and Philippe Pierre1,2,3,{ddagger},§

1 Centre d'Immunologie de Marseille-Luminy, Université de la Méditerranée, Case 906, 13288 Marseille cedex 9, France
2 INSERM, U631, 13288 Marseille, France
3 CNRS, 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


Figure 1
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  		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.

 

Figure 2
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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 µm.

 

Figure 3
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  		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-BAD-LAMP (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 µm in A; 200 µm in B; 100 µm (left panel) and 10 µm (right panel) in C; 10 µm in D.

 

Figure 4
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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 BAD-LAMP (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) co-localization. Bars, 20 µm; 10 µm for THY1 high magnification.

 

Figure 5
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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 phospho-epitope 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 (beta-tubulin, red) and weak GM1 staining (green). Higher magnification (Z1) shows that BAD-LAMP vesicles align along microtubules. (Bottom) Nocodazole treatment induces microtubule destabilization and disorganization of BAD-LAMP vesicle clusters. Bars, 20 µm; 10 µm for Z1.

 

Figure 6
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  		Fig. 6. Confocal immunofluorescence microscopy analysis of BAD-LAMP 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 µm in A,B,C,F, 10 µm in D,E.

 

Figure 7
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  		Fig. 7. Surface biotinylation reveals the endocytic nature of BAD-LAMP-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 BAD-LAMP was observed. Co-localization 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 µm.

 

Figure 8
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  		Fig. 8. Localization of FLAG-tagged BAD-LAMP in transfected cortical neurons. Cortical neurons co-transfected with BAD-GFP and FLAG-BAD-LAMP were visualized by confocal microscopy. (A) Staining for FLAG antibody (red), BAD-GFP (green) and LAMP2 (white). FLAG-BAD-LAMP does not co-localize with LAMP 2. BAD-GFP is targeted to lysosomes upon addition of the GFP moiety at the C-terminal end of BAD-LAMP. Bar, 20 µm; 10 µm for high magnification of Z1 and 5 µm for Z2. (B) Internalization of FLAG antibody (red) in transfected neurons for indicated times and staining for LAMP2 (white). High magnifications reveal a late accessibility of BAD-LAMP into LAMP2 positive compartments in neurites. Bars, 20 µm; 5 µm for high magnification.

 

Figure 9
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  		Fig. 9. BAD-LAMP is targeted to early endosomes and recycles in HeLa cells. HeLa cells transfected with FLAG-BAD-LAMP 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 co-localization 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) Co-transfection of HeLa cells with FLAG-BAD-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 µm.

 

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© The Company of Biologists Ltd 2007