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.
Highlights
Laminin Coclusters GM1 with the dystrophin-associated protein (DAP) Complex and aquaporin 4 (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]
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
Summary
Antibodies—The following antibodies were used in the present study: rabbit anti-AQP4 against rat glutathione S-transferase AQP4 corresponding to residues 249 –323 (Alomone Laboratories, Jerusalem, Israel), rabbit anti-laminin against purified mouse Engelbreth-Holm-Swarm sarcoma laminin, which recognizes laminin ␣1, 1, and ␥1 chains, rabbit polyclonal antibody to dystrophin 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. Astrocyte cultures were harvested and when their intensity was above background and above surlysed on ice for 30 min in TNE buffer supplemented with 1% rounding diffuse immunofluorescence and when their size was Triton X-100 and 1ϫ Complete protease inhibitor mixture. Nuclei and cellular debris were removed from the suspension random fields per treatment, and experiments were repeated at tures were subjected to a flotation assay The distribution of these proteins was evaluated in lipid raft fractions obtained using detergentfree astrocytes extracts. A pool of GraphPad Prism 3.00 software and unpaired Student’s t test. flotillin-1, caveolin-1, and GM1 translocated to non-DRM con-
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