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Vol. 18, Issue 7, 2667-2677, July 2007

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Cholesterol Controls Lipid Endocytosis through Rab11

Miwa Takahashi^*,, Motohide Murate^*, Mitsunori Fukuda^,, Satoshi B. Sato^*,||, Akinori Ohta, and Toshihide Kobayashi^*,¶,#

*Frontier Research System, Fukuda Initiative Research Unit, and ^¶Lipid Biology Laboratory, RIKEN, Wako, Saitama 351-0198, Japan; Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan; Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Miyagi 980-8578, Japan; ^||Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan; and ^#Institut National de la Santé et de la Recherche Médicale U870, Institut National de la Recherche Agronomique U1235, Institut National des Sciences Appliquées de Lyon, University Lyon 1 and Hospices Civils de Lyon, 69621 Villeurbanne, France

Submitted October 17, 2006; Revised April 20, 2007; Accepted April 24, 2007
Monitoring Editor: Robert Parton

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-3-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.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Toshihide Kobayashi (kobayasi{at}riken.jp)

Abbreviations used: PEG-cholesterol, poly(ethylene glycol)-derivatized cholesterol; NBD-SM, N-((6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)hexanoyl)sphingosylphosphorylcholine; BODIPY-SM, N-(4,4- difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl) sphingosylphosphorylcholine; NBD-cholesterol, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3-ol.