Interdependence of Laminin-mediated Clustering of Lipid Rafts and the Dystrophin Complex in Astrocytes*

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.

The basement membrane is a specialized extracellular matrix (ECM) 2 composed of collagen, fibronectin, perlecan, agrin, and laminin. Several studies have focused on the involvement of these ECM molecules in the formation and maturation of neuromuscular junctions (1)(2)(3)(4) and interneuronal synapses (5). More recently, much effort has been made by our group and others to understand the role of these molecules at the interface of astroglia and blood vessels (6 -8). Laminin is highly expressed at the perivascular ECM, and the laminin receptor, dystroglycan (␣-DG), together with many other components of the dystrophin-associated protein (DAP) complex, is particularly enriched at astrocyte endfeet abutting the blood vessels (9 -11). The binding of laminin to ␣-DG at these specialized astrocyte domains in brain plays a key role in the polarized distribution of components of the DAP complex (6,12).
Multiple lines of evidence indicate that the DAP complex is crucial for the functional distribution both of the water-permeable channel, AQP4, and the inwardly rectifying potassium channel, Kir4.1, at astrocyte endfeet. Indeed, mutations in the dystrophin gene, deletion of ␣-syntrophin, or loss of laminin binding to ␣-DG caused by a mutation in the Large1 glycosyltransferase result in a dramatic reduction of the expression of AQP4 and Kir4.1 at perivascular astrocyte endfeet (6,7,(12)(13)(14)(15). The mislocalization of AQP4 in the dystrophin mutant and ␣-syntrophin null mice results in delayed onset of brain edema and K ϩ clearance (16 -18). Collectively, these studies highlight a cooperative role of the ECM and both the extracellular and cytoplasmic components of the DAP complex in the proper targeting of proteins to functional domains of astrocytes leading to the regulation of electrolyte balance and fluid movement.
Although the role of DG in targeting other members of the DAP complex (6) as well as AQP4 and Kir4.1 to astrocyte endfeet has been well established (12), the mechanisms underlying this highly organized distribution remain poorly understood. In C2C12 myotubes, agrin triggers AChR clustering, a DG-dependent process, through the coalescence of lipid rafts, which is necessary for proper AChR gating functions (19 -21). In oligodendrocytes, laminin induces the relocalization of ␣6␤1 integrin to lipid rafts containing PDGF␣R, thereby providing a potential mechanism for the incorporation of cell survival signals (22). Lipid rafts are defined as small (10 -200 nm), heterogeneous, highly dynamic, sterol-and sphingolipid-enriched domains that compartmentalize cellular processes. These small rafts can sometimes be stabilized to form larger platforms through protein-protein and protein-lipid interactions (23). Indeed, the immunological synapse is a good example where rafts are brought together to form large functional membrane domains (24). At the immunological synapse, agrin induces the clustering of lipid rafts and their colocalization with CD3 and CD28 complex surface antigens as well as with Lck tyrosine kinase leading to T cell activation (24). Together, these studies provide evidence for a functional role of ligand-induced clustering of lipid rafts.
We have previously shown that laminin induces the coclustering of the DAP complex with Kir4.1 and AQP4 in glial cell cultures (8,25). Moreover, in vivo studies have shown that the perivascular localization of these channels and several components of the DAP complex at astrocyte endfeet require the interaction of laminin with ␣-DG (6,12). In light of these data we asked whether lipid rafts contribute to the laminin-DG-dependent compartmentalization of the DAP complex and AQP4 to key active domains of astrocytes. We show here using fluorescently labeled cholera toxin subunit B (CtxB), a common marker for GM1-containing lipid rafts, that laminin induces a dramatic reorganization of GM1 into large clusters or macrodomains that colocalize extensively with components of the DAP complex in cortical astrocyte cultures. Laminin-mediated clustering of AQP4 is dependent both on cholesterol-sensitive lipid rafts and the DAP complex bringing novel insight into ECM-dependent membrane domain organization and the mechanisms underlying the polarized distribution of these proteins in astrocytes.
Astrocyte Primary Cultures-Primary cortical astrocyte cultures were prepared from embryonic day 18 rats (E18) (Sprague-Dawley, Charles River). Cortices were dissected, and meninges and choroid plexus were removed. They were then cut into small pieces and incubated for 25 min with trypsin (3.0 mg/ml, Invitrogen). Dissociated cortices were then plated in culture flasks and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1 mM L-glutamine (Invitrogen) for 2-3 weeks. The culture medium was changed every 3 days. To remove microglia and oligodendrocyte progenitors, the flasks were shaken the day following the plating. After trypsinization, the cells were plated on glass coverslips coated with poly-Dlysine (0.1 mg/ml, Sigma) in 24-well plates at a density of 200 -250 ϫ 10 3 cells/ml. Two days after plating, the cells were treated for 8 h with either 30 nM Engelbreth-Holm-Swarm sarcoma laminin-1 (Sigma-Aldrich), C-agrin 4,8 (10 nM), C-agrin 0,0 (10 nM, generous gift from Dr. M. Ferns, University of California, Davis), or with 100 nM fibronectin (Sigma).
siRNA Transfections and Cholesterol Depletion and Repletion-Astrocytes were transfected in suspension before plating with 100 nM Dag1 and Aqp4 siRNAs (siGENOME and ON-TARGETplus SMARTpool siRNA reagents, Dharmacon) and control siRNA (ON-TARGETplus siCONTROL nontargeting siRNA, Dharmacon) using Lipofectamine 2000 (Invitrogen), following the manufacturer's protocol. One day and a half after plating, astrocytes were treated for 8 h with 30 nM laminin-1 (Sigma-Aldrich) and subsequently analyzed by immunofluorescence. The efficiency of transfection was assessed by immunoblot analysis of proteins extracted on ice using an extraction buffer (25 mM Tris, pH 7.4, 25 mM glycine, and 150 mM NaCl) containing 1% Triton X-100, 1ϫ Complete protease inhibitor mixture, and 5 mM EDTA.
For the cholesterol depletion experiments, the cells were incubated in the presence of 10 M mevastatin or 0.5 g/ml filipin for 8 h or with 20 mM methyl ␤-cyclodextrin (Sigma) for 1 h at 37°C. For the cholesterol repletion experiments, the cells were incubated in the presence of 10 M mevastatin and 50 g/ml cholesterol (Sigma) for 8 h at 37°C.
Fluorescence Recovery after Photobleaching Analysis-Cortical astrocytes plated in 8-well -slides (Ibidi, Munich, Germany) were incubated with 10 g/ml FITC-CtxB for 20 min. Images were acquired on a confocal microscope (FV1000, Olympus) with an Uplan Apochromat 1.35 numerical aperture 60ϫ objective and fully opened pinhole. Photobleaching of FITC-CtxB was performed for 50 ms using a 405 simultaneous scanner set at 60% power within a circular area of 15-pixel diameter. Fluorescence recovery data were collected every second for over 120 s from 8 cells per experiment and analyzed with Prism software (GraphPad) using non-linear regression.
Subcellular Fractionation of Proteins from Astrocyte Cultures-To separate detergent-soluble from -insoluble fractions, the cultured astrocytes were harvested and suspended in 0.5 ml of ice-cold TNE buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA) supplemented with 0.5% Triton X-100 and 1ϫ Complete protease inhibitor mixture. After 20-min incubation on ice, the lysates were centrifuged at 800 ϫ g for 10 min. The supernatant was further centrifuged at 100,000 ϫ g for 1 h at 4°C to obtain detergent-resistant membranes (DRMs) and non-detergent-resistant membranes (non-DRMs).
For the flotation assay, astrocyte cultures were harvested and lysed on ice for 30 min in TNE buffer supplemented with 1% Triton X-100 and 1ϫ Complete protease inhibitor mixture. Nuclei and cellular debris were removed from the suspension by centrifugation at 1000 ϫ g for 10 min. Other astrocyte cultures were harvested and lysed in a detergentfree buffer using 500 mM sodium carbonate, pH 11.0, as previously described (26). The protein extracts were collected and subjected to a flotation assay using a discontinuous sucrose gradient. To prepare the latter, 1 ml of supernatant was mixed with 3 ml of 85% sucrose in TNE and transferred to the bottom of a Beckman centrifuge tube. The diluted cell lysate was overlaid with 2 ml of 50% sucrose, 3 ml of 35% sucrose, 1 ml of 15% sucrose and finally with 200 l of TNE buffer. The samples were centrifuged in an SW41 rotor at 200,000 ϫ g for 20 h at 4°C. Fractions numbered 1-8 were collected from the top of the gradient and analyzed by Western and dot blots.
Quantitative Analyses-The number of clusters, surface area of the clusters, and the colocalization between clusters containing GM1 and ␤-DG, ␤-DG and laminin, ␤-DG and AQP4, laminin and GM1, as well as AQP4 and GM1 were determined on images subjected to a threshold using ImagePro Plus software (Media Cybernetics, Inc.). Signals were considered as clusters when their intensity was above background and above surrounding diffuse immunofluorescence and when their size was at least 1.5 m 2 . Clusters were counted in an average of 15 random fields per treatment, and experiments were repeated at 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 M mevastatin for 8 h, and proteins were harvested in DRMs and non-DRMs. Immunoblots were probed for the TfR, ␣-tubulin, ␤-DG, AQP4, flotillin-1, and caveolin-1, and the dot blot was labeled for GM1. Representative blots from three independent experiments are shown.
least three times. Images from the same experiment were captured using identical acquisition parameters and subjected to the same threshold. Statistical analyses were performed using GraphPad Prism 3.00 software and unpaired Student's t test.

Cholesterol Regulates AQP4 Distribution to Detergent-resistant Membrane Domains in Astrocytes-
To determine the distribution pattern of ␤-DG and AQP4 in lipid raft-containing fractions, Triton X-100 extracts from primary astrocyte cul-tures were subjected to a flotation assay. The distribution of these proteins was also evaluated in lipid raft fractions obtained using detergentfree astrocytes extracts. The efficiency of the subcellular fractionation was confirmed by probing the obtained fractions with antibodies to the TfR, ␣-tubulin, flotillin-1, caveolin-1, as well as with horseradish peroxidase-CtxB to detect GM1 (Fig. 1A). Fractions 1-4 obtained from Triton X-100 and detergentfree extracts were both enriched for the lipid raft markers flotillin-1, caveolin-1, and GM1 (Fig. 1A). As previously described (27), AQP4 was enriched in low density lipid raft-containing fractions 1 to 4 and was not detected in non-lipid raft fractions 5-8 (Fig. 1A). However, ␤-DG co-distributed with the TfR and ␣-tubulin to fractions 5-8 (Fig.  1A). This is in agreement with previous findings showing that ␤-DG was not found in the GM1-containing lipid raft fractions isolated from embryonic stem cell cultures (28).

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Laminin Induces the Reorganization and Stabilization of GM1 in Astrocytes-To investigate the effect of laminin on the organization of lipid rafts containing the ganglioside GM1, we used FITC-CtxB. In laminin-treated astrocytes, GM1 presented a robust clustering (Fig. 2E) compared with control untreated astrocytes where the distribution of GM1 was diffuse ( Fig. 2A). These clusters presented a high degree of colocalization with the laminin clusters (Fig. 2, E-H). Furthermore, laminin did not induce a detectable change in the morphology of astrocytes, because laminin-treated astrocytes presented a flat and polygonal shape similar to that of control untreated astrocytes as assessed by wheat germ agglutinin labeling (supple-mental Fig. S2). Together these data show that the laminin-induced reorganization of the raft component GM1 is not associated with a change in astrocyte morphology.
To determine whether laminininduced clustering of GM1 affected its diffusion in the membrane (29,30), we performed fluorescence recovery after photobleaching of FITC-CtxB in untreated and laminin-treated astrocytes (Fig. 3). Laminin treatment resulted in a dramatic reduction in the rate of FITC-CtxB recovery but only for FITC-CtxB localized within the laminin-induced clusters (Fig. 3,  A-C). This shows that laminin stabilizes GM1-bound FITC-CtxB within the clusters and reduces the exchange rate of FITC-CtxB within the bleached region with FITC-CtxB outside the bleached region. Therefore, the colocalization of CtxB and laminin clusters not only reflects a laminin-induced redistribution of GM1 but also reveals a role for laminin in the stabilization of this raft component in astrocyte membrane domains.
Quantitative analysis revealed over 2-fold increase in the number of GM1 clusters in the laminin-treated compared with the untreated astrocytes that parallels the increase in the number of ␤-DG, laminin, and AQP4 clusters, as previously reported (Fig. 5A) (8, 25). The surface area of GM1-containing clusters increased significantly by Ͼ6-fold in the laminintreated compared with untreated astrocytes, as did the surface area of ␤-DG and AQP4 (Fig. 5B). The higher laminin concen-tration used in the present study (30 nM instead of 15 nM) may account for the increase in the surface area of these AQP4 clusters relative to a previous study (8). A significant degree of colocalization was found between ␤-DG, GM1, and AQP4 upon laminin treatment (Fig. 5C). These results demonstrate an important role for the ECM and more specifically for laminin in the coclustering of the DAP complex and GM1-containing lipid rafts in astrocytes. In addition, the fluorescence data in Fig. 6 show that flotillin-1, another marker of lipid rafts, underwent clustering upon laminin treatment ( Fig. 6G compared with Fig. 6A) and that these clusters colocalized extensively with AQP4 clusters (Fig. 6, G-I). In contrast, the distribution of the lipid raft marker, caveolin-1, did not change upon laminin treatment (Fig. 6, compare J to D) and remained distinct from ␤-DG clusters (Fig. 6, J-L).
We also observed that laminin induces the coclustering of GM1-containing lipid rafts with ␤-DG and AChR in C2C12 myotubes (supplemental Fig. S3) as has been previously described for the ECM protein agrin (21,24,31,32). However, this is not the case for GM1 in astrocytes treated with either soluble recombinant C-agrin 4,8 (supplemental Fig. S4E) or C-agrin 0,0 (data not shown). Similarly, fibronectin did not induce GM1 clustering (supplemental Fig. S4I). These data argue that, in the astrocytes examined in the present study, laminin plays a primary role in GM1 reorganization.
Cholesterol Is Required for the Laminin-induced Coclustering of ␤-DG, AQP4, and GM1-containing Lipid Rafts-Based on the laminin-induced colocalization of GM1-containing lipid raft clusters with the DAP complex and AQP4 clusters (Figs. 4, M-X, and 5C), we examined qualitatively and quantitatively whether lipid raft integrity is crucial for the DAP/AQP4 clustering. To disrupt lipid rafts, we used the cholesterol-depleting and -sequestering agents mevastatin and filipin, respectively. Astrocytes treated both with laminin and mevastatin (Fig. 7, I-P) or laminin and filipin (supplemental Fig. S5, G-L) were fluorescently labeled for GM1, ␤-DG, laminin, and AQP4. We found that GM1 (Fig. 7, compare I and M to A and E), ␤-DG (Fig. 7, compare J and N to B and F), and AQP4 (Fig. 7, compare O to G) had a uniform distribution and virtually no clustering throughout the cells similar to untreated astrocytes (Fig. 4, I-L). Likewise, laminin did not cluster (Fig. 7, compare K to C) and presented a diffuse immunolabeling pattern. The diffuse immunolabeling is indicative of binding of exogenous laminin that is, however, ineffective in terms of assembling at the cell surface and clustering the DAP complex and AQP4. When astrocytes were incubated in the presence of laminin, mevastatin, and cholesterol, clustering of GM1, ␤-DG, laminin, and AQP4 was significantly rescued (Fig. 7, Q-Z). These data   JULY 17, 2009 • VOLUME 284 • NUMBER 29

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show that the laminin-induced cluster formation requires membrane cholesterol.
To assess the effect of cholesterol depletion on the expression levels of ␣-DG, ␤-DG, and AQP4, we compared by Western blot the amounts of these proteins in mevastatintreated astrocytes versus untreated astrocytes (supplemental Fig. S6). We found that the expression levels of these proteins are similar in treated and untreated astrocytes, indicating that the decrease in the clustering of DG and AQP4 in mevastatin-treated astrocytes is not a consequence of a reduction in their expression levels. These data demonstrate the cholesterol dependence of laminin-induced cell surface organization and DG and AQP4 clustering.
Dystroglycan Is Required for the Laminin-induced Clustering of GM1-We have previously shown both in astrocytes and Müller glia that the laminin binding receptor ␣-DG, but not ␤1-integrin, clusters extensively upon laminin treatment (8,25). We therefore asked whether DG had a role in the laminin-mediated clustering of GM1. To address this question, we used siRNA to silence DG expression (siDag1) and determined whether this altered the laminin-mediated clustering of GM1 by quantitative fluorescence analysis. As previous evidence demonstrated a dramatic alteration of the morphology of siAqp4-transfected rat astrocytes that occurs between 2 and 6 days after transfection, we carried out our analysis at 2 days following transfection (33,34). First, we assessed by Western blot the efficiency of the siDag1 transfection and found that ␤-DG could not be detected in the siDag1-transfected cells when compared with the control scrambled siRNA-transfected astrocytes (siCTL, Fig. 8A) and untransfected astrocytes (data not shown). As an additional control, we used siRNA to silence AQP4 expression (siAqp4) and found that, although this inhibited AQP4 expression, it had no impact on ␤-DG expression (Fig. 8A). Second,  F, N, and V), and AQP4 (G, O, and W). Scale bar, 50 m. Y and Z, the histograms represent the mean number of clusters as well as their mean surface area Ϯ 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 Ͻ 0.0001; **, p Ͻ 0.001; #, p Ͻ 0.03; ##, p Ͻ 0.005; ###, p Յ 0.0001).
we verified that the morphology and the GFAP immunolabeling of the siDag1-and siAqp4-transfected astrocytes were comparable to those of siCTL-transfected cells (supplemental Fig.  S7). The data obtained here show a high efficiency of siDag1 gene silencing and no detectable effect of either siDag1 or siAqp4 on astrocyte morphology when compared with siCTLtransfected (supplemental Fig. S7) or to untransfected astrocytes at 2 days post-transfection (data not shown).
The immunofluorescence analysis corroborates the Western blot data (Fig. 8) and shows virtually no immunolabeling for ␤-DG in the siDag1-transfected astrocytes (Fig. 9, J and N). However, some small clusters of laminin (Fig. 9O) were detected even in the absence of any signal for ␤-DG (Fig. 9N). Of particular interest, the laminin-induced GM1 clustering in the siCTL-transfected astrocytes (Fig. 9, A and E) was significantly reduced in the siDag1-transfected astrocytes (Fig. 9, I and M). In addition, the astrocytes deficient for DG exhibited reduced clustering of AQP4 compared with the siCTL astrocytes (Fig. 9, compare K to C). The siAqp4-transfected astrocytes presented a decrease in AQP4 immunolabeling (Fig. 9, compare S to C) and no change in ␤-DG (Fig. 9, R and V) and laminin immunolabeling and clustering (Fig. 9W) compared with control cells (Fig. 9, B, F, and G). In addition, the laminininduced clustering of GM1 was not affected by AQP4 silencing (Fig. 9, Q and U). It is noteworthy that the effect of siDag1 was more pronounced on the surface area of the GM1, ␤-DG, laminin, and AQP4 clusters than on their number (Fig. 10, compare  B to A). This suggests that, despite the fact that a small proportion of these clusters can form, their size is dramatically reduced in astrocytes deficient for DG.

DISCUSSION
Laminin Coclusters GM1 with the DAP Complex and AQP4 in Astrocytes-In brain, astrocytes are polarized and present an asymmetrical distribution of proteins such as DG and AQP4 that are diffusely distributed along the astrocyte processes in the parenchyma but highly concentrated at astrocyte endfeet abutting blood vessels. Astrocyte endfeet interact with the perivascular ECM, and previous studies have shown both in vivo and in vitro that astrocyte interaction with laminin via DG is essential for the concentration of several members of the DAP complex as well as Kir4.1 and AQP4 at specialized astrocyte membrane domains (6,8,12). Although such a distribution is functionally important in potassium ion and water homeostasis, the mechanisms regulating the formation of these Kir4.1-and AQP4-enriched domains remain to be elucidated. AQP4 is associated with lipid raft-containing fractions in both brain and retina (27,35) as is another aquaporin, AQP5, that participates in fluid secretion in parotid gland cells (36). We therefore undertook a study to evaluate the role of lipid rafts in laminin-DG-dependent AQP4 clustering, and our data show that laminin induced a dramatic redistribution of GM1 into large cell surface clusters or macrodomains that colocalized extensively with ␤-DG and AQP4. The recruitment of AQP4 to laminin-rich clusters is therefore interdependent on both DG and cholesterol-rich lipid raft domains.
The DAP complex and AQP4 associate in a subset of rafts containing GM1 and flotillin-1 distinct from caveolin-1-containing caveolae. Indeed, flotillin has been reported to be localized to lipid raft domains distinct from caveolin-1 (37). Laminin therefore promotes coclustering of the DAP complex and AQP4 with a select population of lipid rafts in astrocyte membrane domains. How these coclusters of the DAP complex and AQP4/GM1/flotillin-containing lipid rafts are organized remains uncertain. In fact, laminin-induced clustering does not result in the recruitment of ␤-DG to DRMs (supplemental Fig.  S8). Raft-dependent laminin remodeling may induce spatial reorganization in the membrane such that the DAP complex and lipid rafts coexist and interact within the same polarized macrodomain. Indeed, the fact that GM1-bound CtxB exhibits reduced exchange in laminin-induced clusters shows that lipid raft dynamics and behavior are altered within these large membrane domains.
Laminin-induced Coclustering of GM1, the DAP Complex, and AQP4 Depends on the Integrity of Lipid Rafts and Dystroglycan-Cholesterol depletion experiments show that the integrity of lipid rafts is essential not only for the recruitment of AQP4 to DRMs (27) but also for the laminin-dependent assembly of the AQP4, ␤-DG, and GM1-enriched domains. Conversely, DG silencing resulted in the disruption of laminin assembly and a significant decrease of clustering of not only AQP4 and ␤-DG at the cell surface but also the raft marker GM1. Coordinate plasma membrane domain organization by both lipid rafts and laminin binding to the DG complex may therefore restrict lateral diffusion of AQP4 within the plasma membrane shedding new light on the mechanisms involved in the polarized distribution of AQP4, and the DAP complex, at perivascular astrocyte endfeet. Interestingly, the coclustering of GM1 and ␤-DG with AQP4 and AChRs in astrocytes and C2C12 myotubes (supplemental Fig. S3), respectively, is consistent with a role for laminin in orchestrating the distribution of both lipids and a specific set of proteins to establish functional membrane domains. In C2C12 myotubes, laminin-induced coclustering of components of the DAP complex with AChRs requires Src-and Fyn-dependent tyrosine phosphorylation that enhances the association of postsynaptic proteins with cholesterol-rich membrane domains leading to the stabilization of AChR-containing synapses in muscle (32). Laminin-mediated clustering of ␣6␤1-integrin within the platelet-derived growth factor receptor-containing lipid rafts in oligodendrocytes creates a signaling environment that promotes survival of these cells (22). Furthermore, integrin-mediated adhesion to the ECM regulates Rac1 signaling by preventing internalization of lipid raft-containing membrane domains (38). More recently, the lamininmediated clustering of GM1 and the agrin association with lipid rafts have both been involved in promoting neurite outgrowth by activating Lyn/Akt/MAPK and Fyn/MAPK signaling pathways, respectively (39,40). These data define a role for the ECM in regulating raft-associated signaling. It is therefore likely that the laminin-mediated reorganization of GM1-containing lipid rafts and coclustering with the DAP complex provides a signaling environment that promotes the formation of AQP4-rich membrane domains in astrocytes.
Potential Role of Laminin and Lipid Raft-dependent Clustering of AQP4 on Its Activity-AQP4 is the main structural component of intramembrane particles called orthogonal arrays of particles (41)(42)(43). Recently, an increase of the density of orthogonal arrays of particles as well as an increase in AQP4 expression accompanied by a more efficient water transport have been reported in astrocytes treated with the ECM molecule, agrin (44). This substantiates further the role of the ECM in the formation of assemblies of orthogonal arrays of particles rich in AQP4 at astrocyte endfeet. Mislocalization of AQP4 at perivascular astrocyte endfeet in the Large myd mouse (6,12) and loss of its clustering in astrocytes deficient in DG may cause the dispersion or a decrease in the density of orthogonal arrays of particles at these specialized domains that will ultimately alter water transport.
Recent studies have reported that lack of the lipid raft gangliosides GM1 and GD1 (45) or depletion of cholesterol resulted in abnormal paranodal sodium channel clusters in the peripheral nervous system and prevented the agrin-induced clustering of AChRs in C2C12 myotubes, respectively (31,45,46). Moreover, cholesterol depletion in neuronal cultures decreases the number of synapses and ␣-amino-3-hydroxy-5methyl-4-isoxazolpropionic acid receptor stability (47). In some instances, it has been demonstrated that cholesterol can influence the activity of ion channels (19,48). These data raise the interesting hypothesis that lipid rafts may participate in the regulation of the AQP4 channel properties that define its role in water transport.
The present study demonstrates that loss of interaction between laminin and DG mimics the effect of membrane cholesterol depletion on AQP4 clustering and points to a concerted action between lipid rafts and ligand binding to DG that leads to the formation of membrane specializations at specific astrocytic sites. These findings therefore implicate lipid rafts in the polarization of the DAP complex and AQP4 in astrocyte endfeet and potentially in AQP4-mediated water transport.