Cholesterol controls lipid endocytosis through Rab11.
Cellular cholesterol increases when cells reach confluency in Chinese hamster ovary (CHO) cells. We examined the endocytosis of several lipid probes in subconfluent and confluent CHO cells. In subconfluent cells, fluorescent lipid probes including poly(ethylene glycol)derivatized cholesterol, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol, and fluorescent sphingomyelin analogs were internalized to pericentriolar recycling endosomes. This accumulation was not observed in confluent cells. Internalization of fluorescent lactosylceramide was not affected by cell confluency, suggesting that the endocytosis of specific membrane components is affected by cell confluency. The crucial role of cellular cholesterol in cell confluency-dependent endocytosis was suggested by the observation that the fluorescent sphingomyelin was transported to recycling endosomes when cellular cholesterol was depleted in confluent cells. To understand the molecular mechanism(s) of cell confluency- and cholesterol-dependent endocytosis, we examined intracellular distribution of rab small GTPases. Our results indicate that rab11 but not rab4, altered intracellular localization in a cell confluency-associated manner, and this alteration was dependent on cell cholesterol. In addition, the expression of a constitutive active mutant of rab11 changed the endocytic route of lipid probes from early to recycling endosomes. These results thus suggest that cholesterol controls endocytic routes of a subset of membrane lipids through rab11.
Takahashi
Miwa
M
Frontier Research System, RIKEN, Wako, Saitama, Japan.
Murate
Motohide
M
Fukuda
Mitsunori
M
Sato
Satoshi B
SB
Ohta
Akinori
A
Kobayashi
Toshihide
T
D000818:Animals; D016466:CHO Cells; D002784:Cholesterol / Q000172:deficiency / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis / Q000187:drug effects; D005456:Fluorescent Dyes / Q000378:metabolism; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D050356:Lipid Metabolism / Q000187:drug effects; D051379:Mice; D021381:Protein Transport / Q000187:drug effects; D012995:Solubility / Q000187:drug effects; D013109:Sphingomyelins / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000494:pharmacology; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
D002784:Cholesterol; D005456:Fluorescent Dyes; D020730:Guanine Nucleotide Dissociation Inhibitors; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D013109:Sphingomyelins; D047392:beta-Cyclodextrins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C031215:betadex