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First published online December 31, 2008
doi: 10.1242/10.1242/jcs.030338

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Binding of laminin-1 to monosialoganglioside GM1 in lipid rafts is crucial for neurite outgrowth

¹ Research Institute for Diseases of Old Age, Juntendo University School of Medicine, Tokyo, Japan
² Department of Neurology, Juntendo University School of Medicine, Tokyo, Japan
³ Institute for Environmental and Gender Specific Medicine, Juntendo University School of Medicine, Tokyo, Japan
⁴ Department of Anatomy, Juntendo University School of Medicine, Tokyo, Japan
⁵ Sphingolipid Functions Laboratory, RIKEN Frontier Research System, Saitama, Japan
⁶ Shriners Hospitals for Children, Research Center, Portland, OR 97201, USA
⁷ Laboratory of Cell and Developmental Biology, NIDCR, NIH, Bethesda, MD 20892, USA

View larger version (46K): [in this window] [in a new window]   Fig. 1. Clustering of GM1 in laminin-1-treated DRG cells. (A) Immunostaining of endogenous GM1. DRG cells were treated with or without 5 µg/ml of laminin-1 in the presence of 100 ng/ml NGF. Cells were stained using anti-GM1 antibody (left) or CTxB (right) and analyzed by confocal laser microscopy. Bars, 5 µm. Arrowheads indicate clustering of GM1. (B) BODIPY-GM1 staining. Micrographs show DRG cells labeled with 500 nM BODIPY-GM1 (green) and then incubated with DiI-C18 (red). DRG cells were analyzed by confocal laser microscopy 5 minutes after the addition of 5 µg/ml of laminin-1. Arrowheads indicate clustering of BODIPY-GM1. Bar, 5 µm. Graphs show fluorescence intensities of BODIPY-GM1 (in arbitrary units; green curve) and DiI-C18 (in arbitrary units; red curve) versus the length of indicated lines within the area (indicated 1 and 2; in µm) of the two merged micrographs 5 minutes after addition of laminin-1. DiI-C18 fluorescence intensity was measured using Leica LCS software. Laminin-1 induced focal the aggregation of BODIPY-GM1 in the membrane, but not of DiI-C18 – which labeled the membrane in general. (C) Analysis of endogenous GM1 by immunogold electron microscopy. DRG cells were treated with 5 µg/ml of laminin-1 for 10 minutes. Cells were then fixed and stained with anti-GM1 antibody and secondary antibody conjugated to 5-nm gold particles to detect GM1. Labeled cells were analyzed by electron microscopy. Boxed areas 1 and 2 for NGF alone and NGF+laminin-1 treatment in the left panels are shown enlarged as the middle and right panels. Treatment with NGF alone showed no clustering of gold particles to detect GM1 (arrows). Treatment with NGF and laminin-1 together induced clustering of gold particles to detect GM1 (encircled areas).

View larger version (21K): [in this window] [in a new window]   Fig. 2. Binding of laminin-1 and recombinant LG4 protein to GM1. (A,B) Binding of (A) laminin-1 (1 ng/well) and (B) recombinant LG4 protein (1 ng/well) to glycosphingolipids (5 µg/well) was measured by ELISA. Data are given as the means + standard deviations (+ s.d.) of experiments performed in triplicate. (C) Dose-dependent inhibitory effects of various amounts of the recombinant LG4 protein on the binding of laminin-1 (1 ng/well) to GM1 (5 µg/well)-coated plates. Bound laminin-1 was immunostained using antibody aganst laminin-1–LG1-3, which recognizes laminin-1 but not LG4, and then quantified. (D) Dose-dependent inhibitory effects of laminin-1 on binding of biotin-bound AG73 to GM1-coated plates (5 µg/well). Each value is given as the mean (+ s.d.) of three experiments.

View larger version (37K): [in this window] [in a new window]   Fig. 3. Structures of colipids and sugar chain of -dystroglycan.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Laminin-1 induces clustering of β1 integrin in lipid rafts. (A) PC12 cells were treated with 100 ng/ml of NGF and/or 5 µg/ml of laminin-1. Cells were lysed in 1% Triton X-100 and fractionated on discontinuous sucrose gradients. Ten fractions were collected from top to bottom. Each fraction was subjected to immunoblotting using the indicated antibodies (lanes 1-10). Flotillin-1, a raft marker, was detected in fractions 2 and 3. (B) DRG cells, labeled with BODIPY-GM1 (green) and Alexa-Fluor-546-conjugated anti-β1 integrin antibody (red), were treated with 5 µg/ml of laminin-1. DRG cells were analyzed using confocal laser microscopy. Arrowheads indicate clustering and colocalization of BODIPY-GM1 and β1 integrin. Inset shows an enlarged image of the boxed area. Bar, 5 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 5. Clustering and colocalization of TrkA and/or β1 integrin induced by laminin-1. (A) TrkA clustering. DRG cells were treated with 5 µg/ml of laminin-1 for 10 minutes. Cells were then fixed and stained with anti-TrkA antibody and secondary antibody conjugated to 5-nm gold particles. Labeled cells were analyzed by electron microscopy. Boxed areas in left panels are shown enlarged in the middle panels. Gold-particle-positive areas are boxed and labeled 1 and 2 and are further enlarged in right panels. The number of gold particles in each cluster was counted within a 100-nm area of the luminal surface of the cells, as shown in the `calculation method' below, giving the percentage of clusters that contain the same number of gold particles. Total numbers of gold particles were not significantly different between cells treated with NGF only [59.07 +6.48 (s.d.)] and cells treated with NGF+laminin-1 [63.06 + 6.58 (s.d.)]; n=30. The graph gives the percentage of each cluster. Asterisks indicate significant differences of clusters containing 4 or >5 particles comparing NGF only and NGF + laminin-1 treatments (*P<0.00001; two-sided t-test). (B) TrkA and β1 integrin clustering. DRG cells were treated with 5 µg/ml of laminin-1 for 10 minutes. Cells were fixed and incubated with anti-TrkA antibody and anti-β1 integrin antibody. The cells were incubated with secondary antibody conjugated to 5-nm gold particles (TrkA, arrows) and 10-nm gold particles (β1 integrin, arrowheads). NGF+laminin-1 induced clustering of β1 integrin as well as TrkA, and both type of clusters localized closely to each other. Bar, 100 nm.

View larger version (45K): [in this window] [in a new window]   Fig. 6. Inhibition of laminin-1-induced GM1 clustering with lyso-GM1. (A) DRG cells were treated with 2.5 µM lyso-GM1 or 2.5 µM lyso-LacCer, labeled with BODIPY-GM1 and immunostained with Alexa-Fluor-546-conjugated anti-TrkA antibody or with Alexa-Fluor-546-conjugated anti-β1 integrin antibody. Cells were then analyzed by confocal laser microscopy 5 minutes after the addition of 5 µg/ml of laminin-1. Arrowheads indicate clustering and colocalization of BODIPY-GM1 with TrkA or β1 integrin. Bar, 5 µm. (B) DRG cells were treated with 2.5 µM lyso-GM1 or 2.5 µM lyso-LacCer and incubated with 5 µg/ml of laminin-1 for 24 hours in the presence of 50 ng/ml NGF. Cells were stained with antibodies against the neurofilament proteins and with Hoechst dye 33258. Bar, 100 µm. Bar graph shows the mean length + s.d. of neurite outgrowth from 30 DRG cells. (*P<0.001; two-sided t-test).

View larger version (30K): [in this window] [in a new window]   Fig. 7. Depletion of endogenous GM1 or β1 integrin inhibits neurite outgrowth induced by laminin-1. (A,B) PC12 cells were transfected with the vector expressing EGFP and with (A) siRNA targeting GM1 synthase (β1,3-galactosyltransferase) or (B) β1 integrin. Expression levels of GM1 and β1 integrin were analyzed by western blotting 24 hours and 48 hours after the transfection. At 24 hours after each siRNA transfection, neurite outgrowth was analyzed and the percentage of EGFP-expressing cells with neurite outgrowth was determined as described in Materials and Methods. (C) Neurite outgrowth of PC12 cells was analyzed 24 hours after treatment with anti-β1-integrin antibody or normal mouse IgG. Data are given as the means + s.d. of triplicate results; *P<0.05 (A), P<0.01 (B,C); two-sided t-test. Bars, 50 µm.

View larger version (41K): [in this window] [in a new window]   Fig. 8. Activation of Lyn induced by clustering of GM1. (A, left panel) PC12 cells treated with 5 µg/ml of laminin-1 for the indicated times in the presence of 100 ng/ml of NGF. Lyn was immunoprecipitated and subjected to immunoblotting using antibody against phosphorylated (Y396) Lyn (pLyn). The same blot was reprobed with anti-Lyn antibody. (Right panel) PC12 cells treated with 100 ng/ml of NGF and/or 5 µg/ml of laminin-1. Cells were lysed in 1% Triton and were fractionated on discontinuous sucrose gradients. Raft (R) and non-raft (NR) fractions were collected and subjected to immunoblotting using the indicated antibodies. Flotillin-1 was detected in the raft fraction. (B) PC12 cells were treated with 2.5 µM lyso-GM1 and stimulated with 2 µg/ml of CTxB or 5 µg/ml of laminin-1 for 10 minutes in the presence of 100 ng/ml of NGF. Phosphorylated Lyn (pLyn) and total Lyn were detected as described above. Bar graph shows the means +s.d. of three experiments. Relative activity, expressed as the ratio phosphorylated Lyn to total Lyn, is given as the fold-increase relative to NGF treatment (defined as 1) (*P<0.05; **P<0.01; two-sided t-test). (C) PC12 cells were transfected with siRNA targeting GM1 synthase or β1 integrin and stimulated with laminin-1 for 10 minutes in the presence of 100 ng/ml of NGF. Phosphorylated Lyn and total Lyn were detected as described above.

View larger version (20K): [in this window] [in a new window]   Fig. 9. Depletion of endogenous Lyn inhibits laminin-induced neurite outgrowth. (A) PC12 cells were transfected with vector expressing EGFP and siRNA targeting Lyn. Lyn expression was analyzed by western blotting 24 hours and 48 hours after transfection. (B,C) At 24 hours after siRNA transfection, neurite outgrowth was analyzed and the percentage of EGFP-expressing cells with neurite outgrowth was determined as described in Materials and Methods. Data indicate the mean + s.d. of triplicate results (*P<0.05; two-sided t-test). Bars, 50 µm.

View larger version (19K): [in this window] [in a new window]   Fig. 10. Model for laminin-1-induced clustering of GM-1, TrkA, β1 integrin and signaling molecules in lipid rafts required for neurite outgrowth. Laminin-1 directly binds to GM1 and induces its focal aggregation, thereby enhancing the relocation of TrkA in the lipid rafts and the subsequent activation of NGF-signaling molecules such as Lyn. Clustering of GM1 with laminin-1 also promotes relocation and enrichment of β1 integrin in lipid rafts, and enhances the combined laminin-1–integrin signaling of laminin-1 and integrins. Laminin-1 self-assembly might stabilize and enhance the focal formation of the GM1-enriched microdomain in the membrane. The focal lipid-raft structure enriched with TrkA, integrin and signaling molecules provides a functional platform within the membrane that helps to link and activate NGF-TrkA and laminin-1–integrin signaling pathways that trigger neurite outgrowth in a cooperative manner.

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