FLUID-PHASE MARKERS IN THE BASOLATERAL ENDOCYTIC PATHWAY ACCUMULATE IN RESPONSE TO THE ACTIN ASSEMBLY-PROMOTING DRUG JASPLAKINOLIDE

To investigate the role of filamentous actin in the endocytic pathway, we used the cell-permeant drug Jasplakinolide (JAS) to polymerize act in in intact polarized Madin-Darby canine kidney (MDCK) cells. The upt ake and accumulation of the fluid-phase markers fluorescein isothiocya nate (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 inc reased the uptake and accumulation of fluid-phase markers in MDCK cell s. JAS increased endocytosis in a polarized manner, with a marked effe ct on fluid-phase uptake from the basolateral surface but not from the epical surface of polarized MDCK cells. The early uptake of FITC-dext ran and HRP was increased more than twofold in JAS-treated cells. At l ater times, FITC-dextran and HRP accumulated in clustered endosomes in the basal and middle regions of JAS-treated cells. The large accumula ted endosomes were similar to late endosomes but they were not colabel ed for other late endosome markers, such as rab7 or mannose-6-phosphat e 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 bunchi ng at the apical pole of MDCK cells. Although other studies have impli cated actin in endocytosis at the apical cell surface, our results pro vide novel evidence that filamentous actin is also involved in the end ocytosis of fluid-phase markers from the basolateral membrane of polar ized cells.