Clathrin-coated pits with long, dynamin-wrapped necks upon expression of a clathrin antisense RNA.
To investigate the role of clathrin in coated vesicle formation, a cell line with inducible expression of clathrin heavy chain (CHC) antisense RNA was produced. After 18 h of CHC antisense RNA expression, the internalization of transferrin was inhibited by 90%. Although the amount of CHC was reduced by only 10%, the frequency of clathrin-coated pits at the cell surface increased by a factor of 3-5, and clathrin-coated structures also accumulated on a pleiomorphic, multivesicular, endosomal compartment. Remarkably, the coated pits were connected to the cell surface by long, tubular necks wrapped by dynamin rings, and the level of dynamin in the CHC antisense RNA-expressing cells was up-regulated 10-fold. In contrast, the amount of several other proteins associated with clathrin coat formation was unaffected. Thus, this study demonstrates that CHC antisense RNA causes accumulation of clathrin-coated pits with dynamin rings around the neck in intact cells not transfected with dynamin mutants, suggesting the existence of a previously uncharacterized functional interplay between clathrin and dynamin.
Iversen
T-G
TG
Institute for Cancer Research at the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.
Skretting
G
G
van Deurs
B
B
Sandvig
K
K
D000818:Animals; D015153:Blotting, Western; D002402:Cathepsin D / Q000378:metabolism; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D006224:Cricetinae; D011992:Endosomes / Q000378:metabolism; D008854:Microscopy, Electron; D016372:RNA, Antisense / Q000235:genetics / Q000378:metabolism*; D014168:Transferrin / Q000378:metabolism
D002966:Clathrin; D016372:RNA, Antisense; D014168:Transferrin; D002402:Cathepsin D