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    Adsorptive endocytosis and membrane recycling by cultured primary bovine brain microvessel endothelial cell monolayers

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    Audus_JCS_1990.pdf (5.040Mb)
    Issue Date
    1990
    Author
    Raub, Thomas J.
    Audus, Kenneth L.
    Publisher
    Company of Biologists
    Type
    Article
    Article Version
    Scholarly/refereed, publisher version
    Published Version
    http://jcs.biologists.org/cgi/reprint/97/1/127
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    Abstract
    The dynamics of membrane recycling were examined in primary cultures of brain microvessel endothelial cells (BMECs). Because the BMEC surface was dominated by galactosylated glycoconjugates, ricin agglutinin (RCAI) was used as a tracer to follow the endocytosis and recycling of RCAI binding sites. These binding sites accounted for 75 % of the iodinatable or most externally disposed plasma membrane proteins. Because greater than 90 % of the RCAI that had bound to BMECs was removed by a brief, nontoxic treatment with galactose, the amounts and kinetics for internalization and efflux of [125I]RCAI were measured. Both endocytosis and efflux were energy dependent. By using pseudo-first-order kinetics, the £j values for RCAI binding, internalization and efflux were 5, 18 and 13-14 min, respectively. By comparing efflux with and without galactose present, we found that 60 % of the RCAI binding sites that had been internalized were returned to the cell surface and reinternalized. Quantifying the distribution of gold-RCAI following internalization showed kinetics consistent with that obtained using radiolabeled RCAI. Both horseradish peroxidase (HRP) and gold-conjugated RCAI that had bound BMEC at 4°C became localized within more caveolae within 2.5 min of warming to 37 °C to permit endocytosis. With time, RCAI appeared within endosomes and tubules and vesicles of which some were located in the trans-Golgi network (TGN). The distribution of HRP-RCAI contrasted with that of free HRP, which was not routed to the TGN. The absence of RCAI conjugates in association with the basolateral membrane domain suggested the presence of functional tight junctions and maintenance of polarity throughout the duration of these experiments. These results showed that membrane recycling was more extensive and much slower than fluid-phase endocytosis in cultured BMECs. Moreover, we found that endocytosis of membrane by BMECs in culture was similar to that reported for brain endothelium in vivo in that a fraction of the cell surface membrane was routed to the TGN.
    URI
    http://hdl.handle.net/1808/7363
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    • Pharmacy Scholarly Works [275]
    Citation
    Raub, T.J. and Audus, K.L. (1990) Adsorptive endocytosis and membrane recycling by cultured primary bovine brain microvessel endothelial cell monolayers. J. Cell. Sci. 97, 127-138. PMID: 2258384

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    Lawrence, KS 66045
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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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