Article

Cholesterol controls lipid endocytosis through Rab11

Frontier Research System, RIKEN, Wako, Saitama, Japan.
Molecular Biology of the Cell (Impact Factor: 4.55). 08/2007; 18(7):2667-77. DOI: 10.1091/mbc.E06-10-0924
Source: PubMed

ABSTRACT 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.

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    • "This result demonstrated the specificity and efficacy of the purification procedure and was consistent with the data from immunocytochemistry Rab11a codistribution with the ACEC is critical for ciliogenesis So far, our data suggested that endocytosis of SM and then ASMase-catalyzed hydrolysis of SM to ceramide is the first step in ceramide-dependent regulation of ciliogenesis. Previous studies provided evidence that Rab11a is involved in the endocytosis of SM/ cholesterol-containing lipid vesicles, mediates vesicle transport in ciliogenesis, and is highly enriched at the base of the primary cilium (Takahashi et al., 2007; Nachury et al., 2010). Because the generation or enrichment of ciliogenic ceramide in the ACEC was likely to involve endolysosomal degradation of SM by ASMase and metabolic recycling via the salvage ceramide biosynthesis pathway, we postulated that there is a codistribution and association of SM and ceramide with Rab11a. "
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    • "The GTP and GDP in the supernatant were separated by polyethyleneimine– cellulose TLC (1057280001; Merck, Rahway, NJ, USA), with a solvent of 0.75 M KH 2 PO 4 , and were detected by autoradiography using an FLA-7000 image analyzer (Fuji Film, Tokyo, Japan). The proportion of GTP-bound Rab11A was calculated as reported previously (Takahashi et al. 2007). "
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