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.
Noël
Geoffroy
G
Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver V6T 1Z3, Canada.
Tham
Daniel Kai Long
DK
Moukhles
Hakima
H
D000818:Animals; D051401:Aquaporin 4 / Q000235:genetics / Q000378:metabolism; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D003902:Detergents / Q000737:chemistry / Q000494:pharmacology; D049030:Dystroglycans / Q000235:genetics / Q000378:metabolism; D016189:Dystrophin / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D036681:Fluorescence Recovery After Photobleaching; D005455:Fluorescent Antibody Technique; D005677:G(M1) Ganglioside / Q000378:metabolism; D007797:Laminin / Q000378:metabolism* / Q000494:pharmacology; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection
C496032:Aqp4 protein, rat; D051401:Aquaporin 4; D003902:Detergents; D016189:Dystrophin; D007797:Laminin; D034741:RNA, Small Interfering; C121346:laminin 1; D049030:Dystroglycans; D005677:G(M1) Ganglioside; D002784:Cholesterol; D016650:Fluorescein-5-isothiocyanate