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Adsorptive endocytosis and membrane recycling by cultured primary bovine brain microvessel endothelial cell monolayers
Raub, Thomas J. Audus, Kenneth L.
Raub, Thomas J.
Audus, Kenneth L.
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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.
Description
Date
1990
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Company of Biologists
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Research Projects
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Keywords
Blood-brain barrier, Brain, Endocytosis, Endothelial cell, Microvessel, Recycling, Ricin agglutinin, trans-Golgi network
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