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Cholesterol homeostasis in T cells. Methyl-beta-cyclodextrin treatment results in equal loss of cholesterol from Triton X-100 soluble and insoluble fractions.
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
2008 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, Vol. 1778, no 5, 1251-8 p.Article in journal (Refereed) Published
Abstract [en]

Methyl-beta-cycloclextrin (MBCD) is frequently used to acutely deplete cells of cholesterol. A widespread assumption is that MBCD preferentially targets cholesterol in lipid rafts and that sensitivity to MBCD is proof of lipid raft involvement in a cellular process. To analyse any MBCD preference systematically, progressive cholesterol depletion of Jurkat T cells was performed using MBCD and [H-3]-cholesterol. It was found that at 37 degrees C, MBCD extracts similar proportions of cholesterol from the Triton X-100 resistant (lipid raft enriched) as it does from other cellular fractions and that the cells rapidly reestablish the relative differences in cholesterol concentration between different compartments. Moreover, cells restore the cholesterol level in the plasma membrane by mobilising cholesterol from intracellular cholesterol stores. Interestingly, mere incubation at 0 degrees C caused a loss of plasma membrane cholesterol with a concomitant increase in cholesteryl esters and adiposomes. Moreover, only 35% of total cholesterol could be extracted by MBCD at 0 degrees C and was accompanied by a complete loss of plasma membrane and endocytotic recycling centre filipin staining. This study clearly shows that MBCD does not specifically extract cholesterol from any cellular fraction, that cholesterol redistributes upon temperature changes and that intracellular cholesterol stores can be used to replenish plasma membrane cholesterol.

Place, publisher, year, edition, pages
2008. Vol. 1778, no 5, 1251-8 p.
Keyword [en]
Cell Compartmentation, Cholesterol/*metabolism, Homeostasis, Humans, Jurkat Cells, Octoxynol/*chemistry, Solubility, Surface-Active Agents/*chemistry, T-Lymphocytes/drug effects/*metabolism, Temperature, beta-Cyclodextrins/*pharmacology
URN: urn:nbn:se:su:diva-15809DOI: 10.1016/j.bbamem.2008.02.010ISI: 000256211700006PubMedID: 18373974OAI: diva2:182329
Available from: 2008-12-10 Created: 2008-12-10 Last updated: 2010-04-09Bibliographically approved
In thesis
1. Cholesterol in T cells: homeostasis, plasma membrane organization and signaling
Open this publication in new window or tab >>Cholesterol in T cells: homeostasis, plasma membrane organization and signaling
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The plasma membrane of eukaryotic cells contains cholesterol and glycosphingolipids enriched nanodomains known as lipid rafts; which are believed to exist in a liquid ordered (lo) state. Methyl-beta-cyclodextrin (MBCD) is used to deplete cellular cholesterol and a widespread assumption is that MBCD preferentially targets cholesterol in lipid rafts. To analyze this in T cells a progressive cholesterol extraction protocols was established. At 37ºC, MBCD treatment does not lead to the preferential loss of cholesterol from TX-DRMs. At 0ºC only 35% of total cholesterol could be extracted demonstrating that less than 35% of the cell’s cholesterol is found in the plasma membrane. Moreover, incubation of cells at 0ºC causes loss of plasma membrane cholesterol and an increase in cholesteryl esters. The increase in cholesterol esters upon cold exposure is linked to the cholesterol concentration induced activation of ACAT enzyme which converts cholesterol to cholesteryl esters. Cholesterol concentration specific activation of ACAT and conversion of cholesterol to cholesteryl esters during the loading of cholesterol onto T cells by MBCD was also observed. By using MBCD for progressive cholesterol depletion from T cells at 37ºC, the effect of cholesterol depletion on T cell signaling was addressed. At 10-20% cholesterol depletion levels, tyrosine phosphorylation is increased and ERK is activated. Peripheral actin polymerization, cell spreading and membrane protrusions are also triggered by limited cholesterol depletion. Upon limited cholesterol depletion aggregation of lipid rafts in the plasma membrane was observed. The aggregation of lipid rafts upon cholesterol depletion does not dependent on the signaling proteins such as Src-kinases. Upon cholesterol depletion there is an increase in overall plasma membrane order, indicative of more ordered domains forming at the expense of disordered domains.

Place, publisher, year, edition, pages
Stockholm: The Wenner-Gren Institute, Stockholm University, 2010. 76 p.
Plasma membrane, T cells, Cholesterol, Lipid rafts, Membrane organization, Cholesterol homeostasis, cholesteryl esters
National Category
Cell Biology
Research subject
urn:nbn:se:su:diva-38357 (URN)978-91-7447-055-0 (ISBN)
Public defence
2010-05-03, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: In press. Available from: 2010-04-11 Created: 2010-04-09 Last updated: 2010-04-12Bibliographically approved

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