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Vol. 9, Issue 4, 957-975, April 1998
Centre for Molecular and Cellular Biology, University of
Queensland, Brisbane, Queensland 4072, Australia
To investigate the role of filamentous actin in the endocytic
pathway, we used the cell-permeant drug Jasplakinolide (JAS) to
polymerize actin in intact polarized Madin-Darby canine kidney (MDCK)
cells. The uptake and accumulation of the fluid-phase markers fluorescein isothiocyanate (FITC)-dextran and horseradish peroxidase (HRP) were followed in JAS-treated or untreated cells with confocal fluorescence microscopy, biochemical assays, and electron microscopy. Pretreatment with JAS increased the uptake and accumulation of fluid-phase markers in MDCK cells. JAS increased endocytosis in a
polarized manner, with a marked effect on fluid-phase uptake from the
basolateral surface but not from the apical surface of polarized MDCK
cells. The early uptake of FITC-dextran and HRP was increased more than
twofold in JAS-treated cells. At later times, FITC-dextran and HRP
accumulated in clustered endosomes in the basal and middle regions of
JAS-treated cells. The large accumulated endosomes were similar to late
endosomes but they were not colabeled for other late endosome markers,
such as rab7 or mannose-6-phosphate receptor. JAS altered transport in
the endocytic pathway at a later stage than the microtubule-dependent step affected by nocodazole. JAS also had a notable effect on cell
morphology, inducing membrane bunching at the apical pole of MDCK
cells. Although other studies have implicated actin in endocytosis at
the apical cell surface, our results provide novel evidence that
filamentous actin is also involved in the endocytosis of fluid-phase
markers from the basolateral membrane of polarized cells.
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