Identification of caveolae and caveolin in C6 glioma cells

Caveolae (CAV) constitute a novel subcellular transport vesicle that has re ceived special attention based on its proven and postulated participation i n transcytosis. potocytosis, and in cell signaling events. One of the princ ipal components of CAV are caveolin protein isoforms. Here, we have underta ken the immunochemical identification of CAV and the known caveolin isoform s (1 alpha, 1 beta, 2 and 3) in cultured rat C6 glioma cells. Immunoblot an alysis revealed that particulate fractions from rat C6 glioma cells express caveolin-1 and caveolin-2. The relative detergent-insolubility of these ca veolin isoforms was also determined by Western blot analysis. Indirect immu nofluorescence analysis with caveolin-1 and -2 antibodies revealed staining patterns typical of CAV's known subcellular distribution and localization. For both caveolin isoforms immunocytochemical staining was characterized b y intensely fluorescent puncta throughout the cytoplasm and diffuse micropa tches at ihc level of the plasmalemma. Perinuclear staining was also detect ed, consistent and suggestive of caveolin's localization in the trans Golgi region. The caveolin-1 and -2 immunoreactivity seen in Western blots and i mmunocytochemically is related to structurally relevant CAV as supported by the isolation of caveolin-enriched membrane complexes using two different methods. Light-density, Triton X-100-insoluble caveolin-1- and caveolin-2-e nriched fractions were obtained after fractionation of rat C6 glioma cells and their separation over 5-40% discontinuous sucrose-density gradients. Si milar fractions were obtained using a detergent-free, sodium carbonate-base d fractionation method. These results further support the localization of C AV and caveolins in glial cells. In addition, they demonstrate that culture d C6 glioma cells can be useful as a model system to study the role of CAV and caveolins in subcellular transport and signal transduction events in gl ial cells and the brain. (C) 1999 ISDN. Published bq Elsevier Science Ltd. All rights reserved.