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First published online 30 October 2007
doi: 10.1242/jcs.019729

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NCAM is ubiquitylated, endocytosed and recycled in neurons

Simone Diestel^1,*, Daniel Schaefer¹, Harold Cremer^2, and Brigitte Schmitz^1,,

¹ Institute of Animal Sciences, Department of Biochemistry, University of Bonn, Katzenburgweg 9a, 53115 Bonn, Germany
² Institut de Biologie du Développement de Marseille-Luminy, UMR 6216, CNRS/Université de la Méditeranée, Campus de Luminy-case 907, 13288 Marseille cedex 9, France

View larger version (37K): [in this window] [in a new window]   Fig. 1. Endocytosis of NCAM is developmentally regulated in cortical neurons. (A) Cortical neurons isolated from E15.5 mice (NCAM+/–) transiently expressing either human NCAM140 or NCAM180 were cultured for 1, 3 or 5 days (DIV1, DIV3 or DIV5, respectively). Endocytosis of NCAM was not induced or induced by an antibody specific for human NCAM for 30 minutes. The numbers of NCAM-positive vesicles per cell in cell somata were counted from at least three independent experiments with approximately 15 cells analysed in each experiment. ***P<0.0005; *P<0.05 (comparison between vesicle numbers of internalized NCAM at different cultivation times); +++P<0.0001 (comparison between vesicle numbers with or without endocytosis induction). (B,C) Representative images of endocytosed NCAM. Cell surface associated NCAM-antibody complexes were detected with Cy3-conjugated secondary antibodies, internalized NCAM with Alexa-Fluor-488-conjugated secondary antibodies (white arrows). Images were either taken from cell somata (B) or from growth cones or neurites of cortical neurons (C) using a confocal laser scanning microscope.

View larger version (60K): [in this window] [in a new window]   Fig. 2. NCAM is partially localized in early endosomes. Cortical neurons isolated from E15.5 mice or B35 cells were transiently co-transfected with GFP-Rab5 and human NCAM140 or NCAM180. NCAM endocytosis was induced with an NCAM-specific antibody for 30 (cortical neurons) or 60 minutes (B35 cells) and cells were directly processed for fluorescence analysis of NCAM with GFP-Rab5. Photos were taken either from entire cells of cortical neurons or B35 cells (A) or from neurites and growth cones of cortical neurons (B). The experiment was carried out three times with at least 15 cells analysed in each experiment.

View larger version (50K): [in this window] [in a new window]   Fig. 3. NCAM is localized only to a small extent in lysosomes. (A) Cortical neurons isolated from E15.5 mice (NCAM+/–) or B35 cells were transiently transfected with either NCAM140 or NCAM180. Endocytosis was induced for 1 hour by application of an NCAM-specific antibody in the presence of LysoTracker (200 nM). Cells were then processed for immunofluorescence analysis by visualization of NCAM with secondary Cy2-conjugated antibodies. (B) B35 cells were transiently transfected with either NCAM140-eGFP or NCAM180-eGFP. Endocytosis was induced for 1 hour by application of an NCAM-specific antibody in the presence of LysoTracker (200 nM). Cells were then directly embedded for confocal microscopy. Experiments were carried out three times with at least 15 cells analysed in each experiment. (C) B35 cells expressing either NCAM140 or NCAM180 were mock treated or treated with chloroquine at different concentrations or with 10 µM lactacystin for 4 hours. During the last hour of incubation an NCAM-specific antibody was added to induce NCAM endocytosis. After lysis, 15 µg of total protein of each sample were loaded onto an SDS gel and subjected to western blot analysis using an NCAM-specific antibody. As loading control for the NCAM140-specific blot, all lanes were blotted with an anti-actin antibody.

View larger version (68K): [in this window] [in a new window]   Fig. 4. NCAM is localized in Rab4- and transferrin receptor positive endosomes. (A) Cortical neurons isolated from E15.5 mice (NCAM+/–) or B35 cells were transiently co-transfected with NCAM140 and GFP-Rab4 or B35 cells were transiently co-transfected with either NCAM140 or NCAM180 and GFP-Rab4. Endocytosis was induced for 1 hour by application of an NCAM-specific antibody. Cells were then fixed and processed for immunofluorescence analysis by visualization of NCAM with secondary Cy3-conjugated antibodies. The experiment was carried out three times with at least 15 cells analysed in each experiment. (B) Cortical neurons from E15.5 mice (NCAM+/–) were transiently transfected with NCAM140 and endocytosis of NCAM was induced for 1 hour by application of an NCAM-specific antibody. Cells were then fixed and processed for immunofluorescence analysis using FITC-conjugated secondary antibodies for visualization of transferrin receptors (TfR) and Cy3-conjugated secondary antibodies to detect NCAM.

View larger version (38K): [in this window] [in a new window]   Fig. 5. NCAM is partially recycled to the plasma membrane by Rab11-positive endosomes. (A) Cortical neurons isolated from E15.5 mice (NCAM+/–) were transiently transfected with NCAM140. B35 cells were transiently co-transfected with either NCAM140 or NCAM180 and GFP-Rab11. Endocytosis was induced for 1 hour by application of an NCAM-specific antibody. Cells were then fixed and processed for immunofluorescence analysis by visualization of NCAM with secondary Cy3-conjugated antibodies and in the case of cortical neurons with anti-Rab11-antibodies and secondary Alexa-Fluor-488-conjugated antibodies. (B) Quantification of colocalization of NCAM-positive vesicles with Rab5, Rab4, Rab11 or LysoTracker. Data are the mean ±s.e.m. of three independent experiments with at least 15 cells analysed in each experiment.

View larger version (38K): [in this window] [in a new window]   Fig. 6. NCAM140 is recycled in growth cones and neurites of cortical neurons. Cortical neurons isolated from E15.5 mice (NCAM+/–) were transiently transfected with NCAM140. Endocytosis was induced for 1 hour by application of an NCAM-specific antibody. Cells were fixed and processed for immunofluorescence analysis. NCAM was visualized using secondary Cy3-conjugated antibodies, Rab11 was detected by Rab11-specific antibodies and Alexa-Fluor-488-conjugated secondary antibodies. Images were taken from growth cones or neurites of cortical neurons using a confocal laser scanning microscope.

View larger version (74K): [in this window] [in a new window]   Fig. 7. NCAM140 is re-expressed at the cell surface after endocytosis. (A) NCAM140 expressing B35 cells were biotinylated at 4°C and unreacted biotin was quenched. As positive control cells were directly fixed and stained for cell surface NCAM (a, anti-NCAM and anti-mouse Alexa-Fluor-488) and biotinylated cell surface proteins (a', streptavidin-Cy3). Biotin was removed from the cell surface using MESNA (+MESNA). (b-h and b'-h') Cell surface NCAM staining with anti-NCAM antibody and anti-mouse Alexa-Fluor-488-conjugated secondary antibodies (b-h); b'-h' shows the same images stained with Cy3-conjugated streptavidin for the detection of cell surface biotinylated proteins without or with induction of NCAM endocytosis (–E, E) and without or with additonal time for recycling (–R, R) for 30 or 60 minutes (30', 60'); b, b': without endocytosis and recycling (–E/–R); c, c', d, d': 30 or 60 minutes induction of NCAM endocytosis, no recycling time (30'E/–R or 60'E/–R, respectively); e, e', f, f': 30 minutes endocytosis and 30 or 60 minutes recycling time (30'E/30'R or 30'E/60'R, respectively); g, g', h, h': 60 minutes endocytosis and 30 or 60 minutes recycling time (60'E/30'R or 60'E/60'R, respectively). Overlays of the images are shown in insets. (B) Cortical neurons isolated from E15.5 mice (NCAM+/–) were transiently transfected with NCAM140 cDNA. Cells were processed as described in A. In all images the cell surface biotin was removed using MESNA (+MESNA). The endocytosis was either induced for 30 or 60 minutes followed by 0, 30 or 60 minutes recycling time; a, a': 60'E/–R; b, b': 30'E/60'R; c, c': 60'E/30'R; d, d': 60'E/60'R.

View larger version (35K): [in this window] [in a new window]   Fig. 8. NCAM is mono-ubiquitylated at the plasma membrane. (A,B) After either no induction (–) or induction of NCAM endocytosis for 60 minutes (+) in B35, NCAM140-expressing (A) or NCAM180-expressing (B) B35 cells, lysates were subjected to immunoprecipitation with an NCAM-specific antibody. Immunoblot analysis was carried out using an antibody specifically recognizing ubiquitin (P4D1, top). After antibody removal the blot was reprobed with an NCAM-specific antibody (123C3) as a control (bottom). (C) Endocytosis of NCAM140 was induced (+) for 60 minutes or not induced (–) in NCAM140-expressing B35 cells. Cell lysates were immunoprecipitated with an NCAM-specific antibody. Immunoblot analysis was carried out using an antibody recognizing specifically poly-ubiquitylated proteins (FK1). After antibody removal the blot was reprobed with an antibody recognizing mono- and polyubiquitylated proteins (FK2). After another antibody removal an incubation with an NCAM-specific antibody (123C3) was carried out as control. To control the functionality of the antibodies whole-cell lysates (L) were applied to the gel. (D) After no induction (–) or induction of NCAM endocytosis for 60 minutes (+) in NCAM140-expressing B35 cells, cell surface proteins were biotinylated at 4°C (lanes 1 and 2). After stopping the reaction, lysates were immunoprecipitated with an NCAM-specific antibody. Immunoprecipitated NCAM was eluted from the sepharose beads and a second precipitation was carried out using streptavidin-agarose beads to isolate cell surface NCAM proteins. In lanes 3 and 4, control immunoprecipitations were are shown with total NCAM [lanes 1 and 3 without (–) and lane 2 and 4 with (+) induction of endocytosis]. Immunoblot analysis was performed using an antibody specifically recognizing ubiquitin (P4D1, top). After antibody removal the blot was reprobed with NCAM-specific antibodies (123C3) as a control (bottom).

View larger version (30K): [in this window] [in a new window]   Fig. 9. NCAM is internalized by clathrin-dependent and caveolae-dependent pathways. (A) NCAM140 or NCAM180-expressing B35 cells were preincubated with MDC (300 µM, 10 minutes), nystatin (NYS, 50 µg/ml, 1 hour) or both inhibitors together (MDC 10 min, NYS 1 hour). Endocytosis was induced for either 30 (NCAM140) or 60 minutes (NCAM180). Cell surface NCAM was detected using Cy3-conjugated secondary antibodies and internalized NCAM using Cy2-conjugated secondary antibodies. Data are the mean ±s.e.m. of three independent experiments with at least 15 cells analysed in each experiment. *P<0.05, **P<0.005, ***P<0.001. (B) Representative images of endocytosed NCAM140 or NCAM180 in B35 cells in the presence or absence of MDC and nystatin. (C) Cortical neurons isolated from E15.5 mice (NCAM+/–) were transiently transfected with NCAM140 or NCAM180 cDNA. Cells were treated with MDC and nystatin as described in A and endocytosis was induced for 30 minutes. Cell surface and internalized NCAM was detected as described in A. Data are the mean ±s.e.m. of three independent experiments with at least 15 cells analysed in each experiment. *P<0.05, **P<0.005, ***P<0.001. (D) Representative images of endocytosed NCAM140 or NCAM180 in cortical neurons in the presence or absence of MDC and nystatin.

View larger version (61K): [in this window] [in a new window]   Fig. 10. Ubiquitin represents an internalization signal for NCAM. (A) Representative images of endocytosed NCAM140 or NCAM180 in the presence or absence of HA-ubiquitin (HA-ubi). B35 cells were co-transfected with HA-ubiquitin and NCAM140 or NCAM180 (second and fourth rows) or with NCAM140 or NCAM180 alone (first and third rows). Endocytosis of NCAM was induced by NCAM-specific antibodies for 60 minutes. HA-ubiquitin was visualized with FITC-conjugated secondary antibodies for identification of HA-ubiquitin-expressing cells, endocytosed NCAM with Cy3-conjugated antibodies and cell surface NCAM using Alexa-Fluor-633-conjugated secondary antibodies. (B) Quantification of endocytosed NCAM or L1 in the presence or absence of HA-ubiquitin. NCAM- or L1-positive vesicles were counted from at least three independent experiments with approximately 15 cells analysed in each experiment. Data are the mean ± s.e.m., ***P<0.0001, **P<0.01, n.s.: not significant.

View larger version (23K): [in this window] [in a new window]   Fig. 11. Proposed model of NCAM endocytosis and trafficking. Cell surface NCAM becomes phosphorylated in response to antibody-induced clustering. This allows a subsequent ubiquitylation of NCAM leading to its internalization. Ubiquitin is cleaved after NCAM is removed from the cell surface. A small part of NCAM is degraded in lysosomes whereas the major part is recycled to the cell surface. PM, plasma membrane; EC, extracellular; IC, intracellular; P, phosphorylation; U, ubiquitylation.

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