Microtubule-disrupting drugs blocked delivery of endocytosed transferrin to the cytocenter, but did not affect return of transferrin to plasma membrane.
The fluorescence of FL cells after endocytosis of rhodamine-labeled transferrin initially appeared as a dispersed punctate pattern over the whole cell and then accumulated in the cytocenter on further incubation. In nocodazole-treated cells, the punctate fluorescence appeared along the cell edges, and stayed there on further incubation but did not accumulate in the cytocenter. The localization of transferrin was examined at the electron microscopic level with horseradish peroxidase (HRP)-labeled transferrin. Nocodazole did not affect endosome formation but affected the distribution of the endosomes. Several types of endosomes (tubular, small spherical, and microvesicular endosomes) were observed in nocodazole-treated cells, as in control cells. The endosomes were in the Golgi area of the cytocenter and also in peripheral cytoplasm in control cells. In contrast, the endosomes were only in the periplasm, along the cell edges, in nocodazole-treated cells. The uptake and release of HRP-transferrin and the release of ferric ion into the cytoplasm in nocodazole-treated cells followed in the same time-course as those in control cells. The release of transferrin was the exponential with a half-time of 12 min. The activation energy of a rate-limiting step in the recycling was 5.5 kcal.mol-1 at around 37 degrees C and increased to 29 kcal.mol-1 below 25 degrees C. These results indicated that microtubule-dependent endosome transport was faster than the overall recycling process and was independent of the return event of transferrin to the plasma membrane.
Sakai
T
T
Department of Biophysics, Faculty of Science, Kyoto University.
Yamashina
S
S
Ohnishi
S
S
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D004705:Endocytosis / Q000187:drug effects*; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008854:Microscopy, Electron; D008870:Microtubules / Q000187:drug effects*; D015739:Nocodazole / Q000494:pharmacology*; D013816:Thermodynamics; D014168:Transferrin / Q000378:metabolism*
D014168:Transferrin; D007501:Iron; D015739:Nocodazole