Using quantitative light microscopy and a modified immunoelectron microscopic technique, we have characterized the entry pathway of the cholera toxin binding subunit (CTB) in primary embryonic fibroblasts. CTB trafficking to the Golgi complex was identical in caveolin-1null (Cav1−/−) mouse embryonic fibroblasts (MEFs) and wild-type (WT) MEFs. CTB entry in the Cav1−/− MEFs was predominantly clathrin and dynamin independent but relatively cholesterol dependent. Immunoelectron microscopy was used to quantify budded and surface-connected caveolae and to identify noncaveolar endocytic vehicles. In WT MEFs, a small fraction of the total Cav1-positive structures were shown to bud from the plasma membrane (2% per minute), and budding increased upon okadaic acid or lactosyl ceramide treatment. However, the major carriers involved in initial entry of CTB were identified as uncoated tubular or ring-shaped structures. These carriers contained GPI-anchored proteins and fluid phase markers and represented the major vehicles mediating CTB uptake in both WT and caveolae-null cells.
Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles
D.K. Sharma's present address is Photometrics, Roper Scientific, Tucson, AZ 85706.
Abbreviations used in this paper: AA, ascorbic acid; Cav1, caveolin-1; CT, cholera toxin; CTB, CT binding subunit; DN, dominant-negative; GEEC, GPI-AP–enriched early endosomal compartment; GPI-AP, GPI-anchored protein; LacCer, lactosyl ceramide; MEF, mouse embryonic fibroblast; OA, okadaic acid; PM, plasma membrane; SV40, simian virus 40; Tf, transferrin; TfR, transferrin receptor; WT, wild-type.
Matthew Kirkham, Akikazu Fujita, Rahul Chadda, Susan J. Nixon, Teymuras V. Kurzchalia, Deepak K. Sharma, Richard E. Pagano, John F. Hancock, Satyajit Mayor, Robert G. Parton; Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles . J Cell Biol 31 January 2005; 168 (3): 465–476. doi: https://doi.org/10.1083/jcb.200407078
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