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

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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°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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Authors and Affiliations

  1. Department of Pathophysiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Währinger Gürtel 18–20, 1090, Vienna, Austria

    Günther Baravalle, Daniela Schober, Marlis Huber, Nora Bayer & Renate Fuchs

  2. PerkinElmer Life and Analytical Sciences Vienna, Vienna, Austria

    Nora Bayer

  3. Departments of Biological Sciences and Biomedical Engineering, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pa., USA

    Robert F. Murphy

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  1. Günther Baravalle
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  2. Daniela Schober
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  3. Marlis Huber
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  4. Nora Bayer
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  5. Robert F. Murphy
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  6. Renate Fuchs
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Correspondence to Renate Fuchs.

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This work was supported by grants from the Austrian Science Fund P12967 and P17590 to R.F.

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Baravalle, G., Schober, D., Huber, M. et al. Transferrin recycling and dextran transport to lysosomes is differentially affected by bafilomycin, nocodazole, and low temperature. Cell Tissue Res 320, 99–113 (2005). https://doi.org/10.1007/s00441-004-1060-x

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  • DOI: https://doi.org/10.1007/s00441-004-1060-x

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