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. 2005 Apr;320(1):99-113.
doi: 10.1007/s00441-004-1060-x. Epub 2005 Feb 16.

Transferrin recycling and dextran transport to lysosomes is differentially affected by bafilomycin, nocodazole, and low temperature

Günther Baravalle  1 , Daniela Schober, Marlis Huber, Nora Bayer, Robert F Murphy, Renate Fuchs
Affiliations

Transferrin recycling and dextran transport to lysosomes is differentially affected by bafilomycin, nocodazole, and low temperature

Günther Baravalle et al. Cell Tissue Res. 2005 Apr.

Abstract

The effects of bafilomycin, nocodazole, and reduced temperature on recycling and the lysosomal pathway have been investigated in various cultured cell lines and have been shown to vary dependent on the cell type examined. However, the way in which these treatments affect recycling and transport to lysosomes within the same cell line has not been analyzed. In the current study, we used fluorophore-labeled transferrin and dextran as typical markers for the recycling and the lysosomal pathways, respectively, to explore the morphology and the intravesicular pH of endocytic compartments in HeLa cells. The V-ATPase inhibitor bafilomycin selectively inhibited the transport of marker destined for lysosomal degradation in early endosomes, whereas the transport of transferrin to the perinuclear recycling compartment (PNRC) still occurred. The kinetics of transferrin acidification was found to be biphasic, indicative of fast and slow recycling pathways via early endosomes (pH 6.0) and PNRC (pH 5.6), respectively. Furthermore, the disruption of microtubules by nocodazole blocked the transport of transferrin to the PNRC in early endosomes and of lysosome-directed marker into endosomal carrier vesicles. In contrast, incubation at 20 degrees C affected the lysosomal pathway by causing retention of internalized dextran in late endosomes and a delay in transferrin recycling. Taken together, these data clearly demonstrate, for the first time, that the transferrin recycling pathway and transport of endocytosed material to lysosomes are differentially affected by bafilomycin, nocodazole, and low temperature in HeLa cells. Consequently, these treatments can be applied to investigate whether internalized macromolecules such as viruses follow a recycling or degradative pathway.

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