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Cholesterol in T cells: homeostasis, plasma membrane organization and signaling
Stockholm University, Faculty of Science, The Wenner-Gren Institute . (Dr. Ingela Parmryd)
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.
Keyword [en]
Plasma membrane, T cells, Cholesterol, Lipid rafts, Membrane organization, Cholesterol homeostasis, cholesteryl esters
National Category
Cell Biology
Research subject
Cellbiology
Identifiers
URN: urn:nbn:se:su:diva-38357ISBN: 978-91-7447-055-0 (print)OAI: oai:DiVA.org:su-38357DiVA: diva2:309889
Public defence
2010-05-03, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
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
List of papers
1. Cholesterol homeostasis in T cells. Methyl-beta-cyclodextrin treatment results in equal loss of cholesterol from Triton X-100 soluble and insoluble fractions.
Open this publication in new window or tab >>Cholesterol homeostasis in T cells. Methyl-beta-cyclodextrin treatment results in equal loss of cholesterol from Triton X-100 soluble and insoluble fractions.
2008 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, 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.

Keyword
Cell Compartmentation, Cholesterol/*metabolism, Homeostasis, Humans, Jurkat Cells, Octoxynol/*chemistry, Solubility, Surface-Active Agents/*chemistry, T-Lymphocytes/drug effects/*metabolism, Temperature, beta-Cyclodextrins/*pharmacology
Identifiers
urn:nbn:se:su:diva-15809 (URN)10.1016/j.bbamem.2008.02.010 (DOI)000256211700006 ()18373974 (PubMedID)
Available from: 2008-12-10 Created: 2008-12-10 Last updated: 2017-12-13Bibliographically approved
2. Cholesterol Delivery From the Plasma Membrane To the ER Is the Rate Limiting Factor of Acyl-Coenzyme A Acyltransferase Activity In Vivo
Open this publication in new window or tab >>Cholesterol Delivery From the Plasma Membrane To the ER Is the Rate Limiting Factor of Acyl-Coenzyme A Acyltransferase Activity In Vivo
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Intracellular free cholesterol accumulation is toxic and has to be avoided. Acyl-CoA: cholesterol acyltransferase (ACAT) clears cells of cholesterol by forming cholesteryl esters (CE). Upon cold stress of Jurkat T cells at 0°C, plasma membrane cholesterol is reduced and both CEs and lipid droplets accumulate rapidly, resembling cells acutely loaded with exogenous cholesterol. ACAT is responsible for the increase, determined using the ACAT inhibitor Sandoz 58035. Cold stress accumulation of CEs requires the redistribution of plasma membrane cholesterol, shown by acute methyl-beta-cyclodextrin mediated cholesterol depletion. Filipin staining revealed that ACAT inhibition resulted in increased plasma membrane cholesterol levels. The plasma membrane of both cold stressed and cholesterol-loaded cells contained a lower proportion of ordered domains than control cells, assessed by laurdan staining. In vivo ACAT is limited by ER cholesterol availability that can be increased by redistribution of endogenous cholesterol.

Keyword
ACAT, Cholesterol, Plasma membrane, Cholesteryl esters
National Category
Cell and Molecular Biology
Research subject
Cellbiology
Identifiers
urn:nbn:se:su:diva-38350 (URN)
Available from: 2010-04-09 Created: 2010-04-09 Last updated: 2010-04-09Bibliographically approved
3. Limited cholesterol depletion causes aggregation of plasma membrane lipid raftsinducing T cell activation
Open this publication in new window or tab >>Limited cholesterol depletion causes aggregation of plasma membrane lipid raftsinducing T cell activation
2010 (English)In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, Vol. 1801, no 6, 625-634 p.Article in journal (Refereed) Published
Abstract [en]

Acute cholesterol depletion is generally associated with decreased or abolished T cell signalling but it can also cause T cell activation. This anomaly has been addressed in Jurkat T cells using progressive cholesterol depletion with methyl-beta-cyclodextrin (MBCD). At depletion levels higher than 50% there is substantial cell death, which explains reports of signalling inhibition. At 10–20% depletion levels, tyrosine phosphorylation is increased, ERK is activated and there is a small increase in cytoplasmic Ca2+. Peripheral actin polymerisation is also triggered by limited cholesterol depletion. Strikingly, the lipid raft marker GM1 aggregates upon cholesterol depletion and these aggregated domains concentrate the signalling proteins Lck and LAT, whereas the opposite is true for the non lipid raft marker the transferrin receptor. Using PP2, an inhibitor of Src family kinase activation, it is demonstrated that the lipid raft aggregation occurs independently of and thus upstream of the signalling response. Upon cholesterol depletion there is an increase in overall plasma membrane order, indicative of more ordered domains forming at the expense of disordered domains. That cholesterol depletion and not unspecific effects of MBCD was behind the reported results was confirmed by performing all experiments with MBCD–cholesterol, when no net cholesterol extraction took place. We conclude that non-lethal cholesterol depletion causes the aggregation of lipid rafts which then induces T cell signalling.

Keyword
Actin, Cholesterol, Colocalization, Lipid raft, Membrane order, Methyl-beta-cyclodextrin, T cell signalling
National Category
Cell Biology
Research subject
Cell Biology
Identifiers
urn:nbn:se:su:diva-38360 (URN)10.1016/j.bbalip.2010.02.003 (DOI)000277912600003 ()
Available from: 2010-04-09 Created: 2010-04-09 Last updated: 2017-12-12Bibliographically approved

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