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. 2009 Jul 17;284(29):19694-704.
doi: 10.1074/jbc.M109.010090. Epub 2009 May 18.

Interdependence of laminin-mediated clustering of lipid rafts and the dystrophin complex in astrocytes

Geoffroy Noël  1 , Daniel Kai Long Tham, Hakima Moukhles
Affiliations

Interdependence of laminin-mediated clustering of lipid rafts and the dystrophin complex in astrocytes

Geoffroy Noël et al. J Biol Chem. .

Abstract

Astrocyte endfeet surrounding blood vessels are active domains involved in water and potassium ion transport crucial to the maintenance of water and potassium ion homeostasis in brain. A growing body of evidence points to a role for dystroglycan and its interaction with perivascular laminin in the targeting of the dystrophin complex and the water-permeable channel, aquaporin 4 (AQP4), at astrocyte endfeet. However, the mechanisms underlying such compartmentalization remain poorly understood. In the present study we found that AQP4 resided in Triton X-100-insoluble fraction, whereas dystroglycan was recovered in the soluble fraction in astrocytes. Cholesterol depletion resulted in the translocation of a pool of AQP4 to the soluble fraction indicating that its distribution is indeed associated with cholesterol-rich membrane domains. Upon laminin treatment AQP4 and the dystrophin complex, including dystroglycan, reorganized into laminin-associated clusters enriched for the lipid raft markers GM1 and flotillin-1 but not caveolin-1. Reduced diffusion rates of GM1 in the laminin-induced clusters were indicative of the reorganization of raft components in these domains. In addition, both cholesterol depletion and dystroglycan silencing reduced the number and area of laminin-induced clusters of GM1, AQP4, and dystroglycan. These findings demonstrate the interdependence between laminin binding to dystroglycan and GM1-containing lipid raft reorganization and provide novel insight into the dystrophin complex regulation of AQP4 polarization in astrocytes.

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Figures

FIGURE 1.
FIGURE 1.
The association of AQP4 with the DRMs is dependent on cholesterol in astrocytes. A, rat cortical astrocytes were solubilized either in 1% Triton X-100 (left panel) or detergent-free buffer (right panel) and fractionated through a discontinuous sucrose gradient. B, astrocytes were incubated with 10 μm mevastatin for 8 h, and proteins were harvested in DRMs and non-DRMs. Immunoblots were probed for the TfR, α-tubulin, β-DG, AQP4, flotillin-1, and caveolin-1, and the dot blot was labeled for GM1. Representative blots from three independent experiments are shown.
FIGURE 2.
FIGURE 2.
Laminin organizes GM1-containing lipid rafts into clusters in astrocytes. A–D, rat cortical astrocytes incubated in the absence or the presence (E–H) of 30 nm laminin were labeled for GM1 using FITC-CtxB (A and E) and for laminin (B and F). High magnifications of the areas boxed in C and G are shown in D and H, respectively. Scale bar, 45 μm.
FIGURE 3.
FIGURE 3.
Laminin regulates the membrane diffusion of GM1-containing lipid rafts labeled with FITC-CtxB. A, rat cortical astrocytes treated with 30 nm laminin were incubated with 10 μg/ml FITC-CtxB at room temperature, and areas within CtxB clusters were bleached and imaged for fluorescence recovery. Areas with diffuse FITC-CtxB labeling in both astrocytes treated with laminin and untreated astrocytes were also bleached and imaged for over 120 s. Percent fluorescence intensity ± S.E. in the bleached area during recovery is shown for one representative experiment out of three (n = 8 cells/experiment). B, percent mobile fraction of FITC-CtxB in areas of untreated astrocytes (−LAM), diffuse areas (+LAM diff.), and clustered areas of laminin-treated astrocytes (+LAM clus.). C, representative images of FITC-CtxB-labeled astrocytes are shown before bleaching (−1 s), shortly after bleaching (2 s), and at various time points during fluorescence recovery.
FIGURE 4.
FIGURE 4.
Laminin-induced clustering of the dystrophin complex and AQP4 is associated with the organization of GM1-containing lipid rafts. R rat cortical astrocytes incubated in the absence (A–L) or the presence (M–X) of 30 nm laminin were first labeled for GM1 using FITC-CtxB and then double immunolabeled for utrophin (B and N) and dystrophin (C and O), syntrophin (F and R) and laminin (G and S), as well as for β-DG (J and V) and AQP4 (K and W). Scale bar, 50 μm.
FIGURE 5.
FIGURE 5.
Quantitative analysis of the laminin-induced clustering of GM1-containing lipid rafts, β-DG, laminin, and AQP4. A and B, the histograms represent the mean number of clusters ± S.E. and surface area of clusters ± S.E. in astrocyte cultures treated with laminin (+LAM) and control untreated cultures (−LAM) from three experiments. The asterisks represent statistically significant differences from control (−LAM) as assessed by Student's t test (***, p < 0.0001; **, p < 0.001). C, the table represents the mean Pearson's colocalization coefficient ± S.E. from three experiments. The asterisks represent statistically significant differences from control untreated cells as assessed by Student's t test (***, p < 0.0001). All quantifications were performed on 15 fields acquired randomly from each experiment.
FIGURE 6.
FIGURE 6.
Laminin induces the coclustering of the lipid raft marker flotillin-1 with AQP4 but not of caveolin-1 with β-DG. Astrocytes incubated in the absence (A–F) or the presence (G–L) of 30 nm laminin were double immunolabeled for flotillin-1 (A and G) and AQP4 (B and H) or caveolin-1 (D and J) and β-DG (E and K). Scale bar, 50 μm.
FIGURE 7.
FIGURE 7.
The laminin-induced clustering of GM1, β-DG, and AQP4 is dependent on cholesterol. A–H, Astrocytes incubated in the presence of 30 nm laminin and DMSO (A–H), 30 nm laminin and 10 μm mevastatin diluted in DMSO (I–P), or 30 nm laminin, 10 μm mevastatin, and 50 μg/ml cholesterol (Q–X) were labeled for GM1 (A, I, and Q), β-DG (B, J, and R), and laminin (C, K, and S) or GM1 (E, M, and U), β-DG (F, N, and V), and AQP4 (G, O, and W). Scale bar, 50 μm. Y and Z, the histograms represent the mean number of clusters as well as their mean surface area ± S.E. from three different experiments. The quantifications were performed on 15 fields acquired randomly from each experiment. The asterisks and number signs represent statistically significant differences from laminin and laminin plus mevastatin-treated astrocytes, respectively, as assessed by Student's t test (***, p < 0.0001; **, p < 0.001; #, p < 0.03; ##, p < 0.005; ###, p ≤ 0.0001).
FIGURE 8.
FIGURE 8.
Dystroglycan and AQP4 siRNAs mediate gene silencing in astrocytes. A, Western blot analysis of β-DG and AQP4 was performed 2 days following the transfection of astrocytes with siDag1, siAqp4, and siCTL (scrambled siRNA). B, the histograms represent the mean expression levels of β-DG and AQP4 ± S.E. from three experiments normalized to tubulin and compared with siCTL. The asterisks represent statistically significant differences from control as assessed by Student's t test (***, p < 0.0001).
FIGURE 9.
FIGURE 9.
Dystroglycan is essential for the laminin-induced GM1-containing lipid raft, β-DG, and AQP4 coclustering. Astrocytes were transfected with siCTL (A–H), siDag1 (I–P), or siAqp4 (Q–X) and incubated with 30 nm laminin. They were then incubated with FITC-CtxB to label GM1 (A, E, I, M, Q, and U) and double immunolabeled for β-DG (B, J, and R) and AQP4 (C, K, and S) or β-DG (F, N, and V) and laminin (G, O, and W). Scale bar, 50 μm.
FIGURE 10.
FIGURE 10.
Quantitative analysis of the effect of the dystroglycan silencing on the laminin-induced clustering of GM1-containing lipid rafts, β-DG, and AQP4. A and B, the histograms represent the mean number of clusters ± S.E. and surface area of clusters ± S.E. in astrocyte cultures from three experiments. The asterisks represent statistically significant differences from control siCTL-transfected cells as assessed by Student's t test (***, p < 0.0001). All quantifications were performed on 15 fields acquired randomly from each experiment.
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References

    1. Arikawa-Hirasawa E., Rossi S. G., Rotundo R. L., Yamada Y. (2002) Nat. Neurosci. 5, 119–123 - PubMed
    1. Marangi P. A., Wieland S. T., Fuhrer C. (2002) J. Cell Biol. 157, 883–895 - PMC - PubMed
    1. Nishimune H., Valdez G., Jarad G., Moulson C. L., Müller U., Miner J. H., Sanes J. R. (2008) J. Cell Biol. 182, 1201–1215 - PMC - PubMed
    1. Ruegg M. A., Bixby J. L. (1998) Trends Neurosci. 21, 22–27 - PubMed
    1. Huh K. H., Fuhrer C. (2002) Mol. Neurobiol. 25, 79–112 - PubMed

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