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First published online April 5, 2005
doi: 10.1242/10.1242/jcs.02296

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DCC association with lipid rafts is required for netrin-1-mediated axon guidance

Zoltán Hérincs^1,*, Véronique Corset^2,*, Nathalie Cahuzac¹, Céline Furne², Valérie Castellani³, Anne-Odile Hueber^1,*, and Patrick Mehlen^2,*,

¹ Death Receptor Signalling Laboratory – Equipe ATIP CNRS, Institute of Signalling, Developmental Biology and Cancer Research, CNRS UMR 6543 Centre A. Lacassagne 33 Avenue Valombrose 06189 Nice, France
² Apoptosis, Cancer and Development Laboratory – Equipe labellisée `La Ligue', CNRS FRE 2870, Centre Léon Berard, 69008 Lyon, France
³ Axonal Guidance and Signalling, Molecular and Cellular Genetic Center, CNRS UMR 5534, University of Lyon, 69622 Villeurbanne, France

View larger version (52K): [in a new window]   Fig. 1. Ligand-independent partitioning of DCC in lipid membrane rafts. (A) HEK293 cells were transfected with a DCC-expressing construct and 24 hours after transfection, cell were treated or not with either MßCD or CO as described in the Materials and Methods. The cell lysates were solubilized in Brij 98 and subjected to sucrose gradient separation. Immunoblots performed on the different sucrose fractions were revealed with HRP conjugated anti-DCC, anti-Fyn or anti-TfR antibodies, or with cholera toxin B-HRP (GM1). (B) Same as A but Triton X-100 was used instead of Brij 98. (C) HEK293 cells were transfected with netrin-1 and/or DCC-expressing constructs and 24 hours after transfection cell lysates were solubilized in Brij 98 and subjected to sucrose gradient separation. Immunoblots performed on pooled heavy fractions (8 and 9) and light fractions (1-4) were revealed with HRP-conjugated anti-netrin-1 and anti-Rab5 antibodies. (D) DCC is in lipid rafts in commissural neurons. Same as in C but 3 x106 commissural neurons dissociated from rat E13 embryos were used instead of HEK293 cells. Raft, raft containing fraction; HF, heavy fraction. Raft inhibitors MßCD (10 mM for 12 minutes) or CO (2 U/ml for 1 hour) were included in the incubation at and just before the PNS preparation.

View larger version (38K): [in a new window]   Fig. 2. DCC is palmitoylated. (A) Schematic representation of DCC. (B) Wild-type DCC, mutant C1121V DCC, or Fyn-transfected HEK293 cells were labelled with [3H]palmitate. Cell lysates were immunoprecipitated as described in the Materials and Methods and analysed by both autoradiography and western blotting. (C,D) HEK293 cells were transiently transfected with wild-type DCC or the DCC C1121V mutant. (C) Rafts were then isolated as in Fig. 1A and analysed for DCC, Fyn, Rab5 and TfR expression by western blotting. (D) Raft-containing fractions (Raft) and heavy fractions (HF) were pooled as in Fig. 1C. A quantification of DCC in raft fractions was performed using the GeneTools software. Right: a flow cytometric analysis of DCC expression performed on non-permeabilized wild-type DCC- or DCC C1121V mutant-transfected HEK293 cells (Immunostaining performed using anti-DCC antibody). The experiments shown in C and D are representative of at least three experiments.

View larger version (89K): [in a new window]   Fig. 3. Netrin-1-mediated DCC-dependent commissural axon outgrowth requires lipid raft integrity. E13 dorsal spinal cord explants were cultured for 18 hours (A,B) or 40 hours (C) in collagen gel either without a netrin-1 source (–), or with purified netrin-1 (Net-p) or next to ventral spinal cord explants (FP). As described in the Materials and Methods, explants were either left untreated (–), treated for 1 hour with 2 U/ml of CO (CO1), 1.5 hours with 1 U/ml of CO (CO2) or 12 minutes with 10 mM MßCD. (B) Quantification of A. The total number of explants that were quantified from four distinct experiments varies from 10 to 15 per tested condition. Values shown are means ± s.e.m. Bars, 170 µm. (D) Quantification (as in B) of the effect of depletion-repletion in cholesterol on E13 dorsal spinal cord explants. Explants were incubated with MßCD as in A and further treated with 1 mM cholesterol and then cultured for 18 hours in collagen gel. Values shown are percentages of netrin-1 treated explants (100%) ± s.e.m.

View larger version (19K): [in a new window]   Fig. 4. Netrin-1-induced DCC-mediated ERK-1/2 activation is dependent on DCC association with lipid rafts. (A) Wild-type DCC- or mutant C1121V DCC-expressing HEK293 cells were pre-treated or not for 1 hour with 2 U/ml of CO and then treated for 15 minutes with purified netrin-1. Phosphorylated ERK-1/2 was compared with total ERK-1/2 according to the Face ERK-1/2 kit instructions. An index of ERK-1/2 phosphorylation is presented as the ratio between netrin-1-treated cells and untreated cells. (B) Dissociated E13 commissural neurons were pre-treated or not for 1 hour with 2 U/ml of CO and then treated for 15 minutes with purified netrin-1. To confirm the role of DCC in the ERK-1/2 phosphorylation observed in commissural neurons, a blocking DCC antibody was also added (-DCC) as previously described (Forcet et al., 2002). Phosphorylated ERK-1/2 was compared with total ERK-1/2 according to Face ERK-1/2 kit instructions. An index of ERK-1/2 phosphorylation is presented as the ratio between netrin-1 treated cells and untreated cells. (C) Same as A except than ERK-1/2 activity was analysed by an in vitro MBP kinase assay following ERK-1/2 immunoprecipitation.

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