Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles.
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.
Kirkham
Matthew
M
Institute for Molecular Bioscience, University of Queensland, Queensland 4072, Australia.
Fujita
Akikazu
A
Chadda
Rahul
R
Nixon
Susan J
SJ
Kurzchalia
Teymuras V
TV
Sharma
Deepak K
DK
Pagano
Richard E
RE
Hancock
John F
JF
Mayor
Satyajit
S
Parton
Robert G
RG
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000502:physiology; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001324:Autoantigens; D002135:Calcium-Binding Proteins / Q000235:genetics; D021941:Caveolae / Q000502:physiology / Q000648:ultrastructure; D051242:Caveolin 1; D022461:Caveolins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000378:metabolism; D002784:Cholesterol / Q000172:deficiency / Q000502:physiology; D002966:Clathrin / Q000502:physiology; D018699:Coated Vesicles / Q000502:physiology* / Q000648:ultrastructure; D003911:Dextrans / Q000378:metabolism; D034281:Dynamins / Q000235:genetics / Q000502:physiology; D004622:Embryo, Mammalian / Q000166:cytology; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006056:Golgi Apparatus / Q000737:chemistry / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007790:Lactosylceramides / Q000494:pharmacology; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D019319:Okadaic Acid / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics; D010873:Pinocytosis / Q000502:physiology; D011247:Pregnancy; D021381:Protein Transport / Q000502:physiology; D014162:Transfection; D014168:Transferrin / Q000378:metabolism; D022161:Transport Vesicles / Q000502:physiology* / Q000648:ultrastructure
D000089982:ADP-Ribosylation Factor 6; D048868:Adaptor Proteins, Signal Transducing; D001324:Autoantigens; D002135:Calcium-Binding Proteins; C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022461:Caveolins; D002966:Clathrin; D003911:Dextrans; C082729:Eps15 protein, mouse; D017261:Glycosylphosphatidylinositols; C097741:Golgin subfamily A member 2; D047908:Intracellular Signaling Peptides and Proteins; D007790:Lactosylceramides; D008565:Membrane Proteins; D010750:Phosphoproteins; D014168:Transferrin; D019319:Okadaic Acid; D002772:Cholera Toxin; D002784:Cholesterol; D006735:Horseradish Peroxidase; D020727:ADP-Ribosylation Factors; D034281:Dynamins