Apical macropinocytosis in polarized MDCK cells: regulation by N-ethylmaleimide-sensitive proteins.
In cells tested so far endocytosis seems to be dependent on N-ethylmaleimide (NEM)-sensitive proteins, and treatment with NEM results in a complete block of endocytosis. We here demonstrate that treatment of polarized MDCK I cells with NEM strongly increased endocytosis of ricin and horseradish peroxidase at the apical side, and electron microscopy revealed NEM-induced formation of large macropinosomes at the apical pole. The NEM-stimulated apical endocytosis seemed to involve phosphatidylinositol-3 kinase, protein kinase C and phospholipase D and it was dependent on ATP. Moreover, in contrast to endocytosis in nonpolarized cells ricin endocytosis at the basolateral side continued in the presence of NEM whereas endocytosis of transferrin was blocked. Furthermore, recycling of ricin endocytosed in the absence of NEM was not inhibited on either side upon addition of NEM demonstrating the existence of a NEM-resistant fusion machinery. The results suggest that the fusogenic property of both the apical and the basolateral plasma membrane of MDCK cells differs from that typically observed in cells unable to polarize.
Sandvig
K
K
Institute for Cancer Research, The Norwegian Radium Hospital, Department of Biochemistry, Montebello, Oslo. ksandvig@radium.uio.no
Llorente
A
A
Rodal
S K
SK
Eker
P
P
Garred
O
O
Stahlhut
M
M
van Deurs
B
B
D000255:Adenosine Triphosphate / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D016764:Cell Polarity / Q000187:drug effects*; D002867:Chromones / Q000494:pharmacology; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology*; D005690:Galactose / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000493:pharmacokinetics; D006801:Humans; D008854:Microscopy, Electron; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism; D010873:Pinocytosis / Q000187:drug effects / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism; D012276:Ricin / Q000493:pharmacokinetics; D013997:Time Factors; D014407:Tumor Cells, Cultured; D000077191:Wortmannin; D020002:tert-Butyl Alcohol / Q000494:pharmacology
D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D000255:Adenosine Triphosphate; D012276:Ricin; D006735:Horseradish Peroxidase; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D010739:Phospholipase D; D020002:tert-Butyl Alcohol; D005033:Ethylmaleimide; D005690:Galactose; D000077191:Wortmannin