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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    ABSTRACT: We show that in Madin-Darby canine kidney (MDCK) cells, an apical ceramide-enriched compartment (ACEC) at the base of primary cilia is colocalized with Rab11a. Ceramide and Rab11a vesicles isolated by magnetic sorting contain a highly similar profile of proteins (atypical protein kinase C [aPKC], Cdc42, Sec8, Rab11a, and Rab8) and ceramide species, suggesting the presence of a ciliogenic protein complex associated with ceramide at the ACEC. It is intriguing that C16 and C18 ceramide, although less abundant ceramide species in MDCK cells, are highly enriched in ceramide and Rab11a vesicles. Expression of a ceramide-binding but dominant-negative mutant of aPKC suppresses ciliogenesis, indicating that the association of ceramide with aPKC is critical for the formation of this complex. Our results indicate that ciliogenic ceramide is derived from apical sphingomyelin (SM) that is endocytosed and then converted to the ACEC. Consistently, inhibition of acid sphingomyelinase with imipramine disrupts ACEC formation, association of ciliogenic proteins with Rab11a vesicles, and cilium formation. Ciliogenesis is rescued by the histone deacetylase (HDAC) inhibitor trichostatin A, indicating that ceramide promotes tubulin acetylation in cilia. Taken together, our results suggest that the ACEC is a novel compartment in which SM-derived ceramide induces formation of a ciliogenic lipid-protein complex that sustains primary cilia by preventing deacetylation of microtubules.
    Molecular biology of the cell 06/2012; 23(16):3156-66. DOI:10.1091/mbc.E12-02-0079 · 5.98 Impact Factor
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    • "All these findings call for a reconsideration of the high cholesterol model, and suggest that low rather than high levels of cholesterol in neuronal membranes could be detrimental for cells and might also promote neuronal degeneration. However, it has been demonstrated that for model cell lines the overall cholesterol content can change from 2 to 4-fold at different phases of cell growth (Cansell et al., 1997; Takahashi et al., 2007). Thus, a full understanding of the in vivo dynamic variability of cholesterol and lipid raft content in plasma or organelle membranes as a function of cellular physiological state is still lacking. "
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    ABSTRACT: Neurodegeneration, a common feature for many brain disorders, has severe consequences on the mental and physical health of an individual. Typically human neurodegenerative diseases are devastating illnesses that predominantly affect elderly people, progress slowly, and lead to disability and premature death; however they may occur at all ages. Despite extensive research and investments, current therapeutic interventions against these disorders treat solely the symptoms. Therefore, since the underlying mechanisms of damage to neurons are similar, in spite of etiology and background heterogeneous, it will be of interest to identify possible trigger point of neurodegeneration enabling development of drugs and/or prevention strategies that target many disorders simultaneously. Among the factors that have been identified so far to cause neurodegeneration, failures in cholesterol homeostasis are indubitably the best investigated. The aim of this review is to critically discuss some of the main results reported in the recent years in this field mainly focusing on the mechanisms that, by recovering perturbations of cholesterol homeostasis in neuronal cells, may correct clinically relevant features occurring in different neurodegenerative disorders and, in this regard, also debate the current potential therapeutic interventions.
    Frontiers in Physiology 01/2012; 3:486. DOI:10.3389/fphys.2012.00486 · 3.50 Impact Factor
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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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    ABSTRACT: Trafficking of recycling endosomes (REs) is regulated by the small GTPase, Rab11A; however, the regulatory mechanism remains elusive. Apoptosis-associated tyrosine kinase 1A (AATYK1A) is a Ser/Thr kinase expressed highly in brain. We have recently shown that AATYK1A localizes to Rab11A-positive RE and is phosphorylated at Ser34 by cyclin-dependent kinase 5 (Cdk5). Here, we have investigated a role of AATYK1A and its phosphorylation in recycling endosomal trafficking using Chinese hamster ovary-K1 (CHO-K1) cells. AATYK1A localizes predominantly to Rab11A-positive pericentrosomal endocytic recycling compartment (ERC). Phosphorylation at Ser34 of AATYK1A disrupts its accumulation in the pericentrosomal ERC. Consistently, phosphorylation-mimic mutant (AATYK1A-S34D) did not accumulate in the ERC and additionally attenuated ERC formation. ERC formation suppression can be reversed by constitutively active Rab11A-Q70L, suggesting a functional link between AATYK1A phosphorylation and Rab11A activity. Although no direct interaction between AATYK1A and Rab11A could be detected, the exchange of guanine nucleotides bound to Rab11A was significantly reduced in the presence of the phosphorylation-mimic AATYK1A-S34D. Together, our results reveal a regulatory role for AATYK1A in the formation of pericentrosomal ERC. They furthermore indicate that Cdk5 can disrupt ERC formation via Ser34 phosphorylation of AATYK1A. Finally, our data suggest a mechanism by which AATYK1A signaling couples Cdk5 to Rab11A activity.
    Genes to Cells 06/2010; 15(7):783-97. DOI:10.1111/j.1365-2443.2010.01419.x · 2.86 Impact Factor
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