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Plasma membrane order; the role of cholesterol and links to actin filaments:  
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The connection between T cell activation, plasma membrane order and actin filament dynamics was the main focus of this study. Laurdan and di-4-ANEPPDHQ, membrane order sensing probes, were shown to report only on lipid packing rather than being influenced by the presence of membrane-inserted peptides justifying their use in membrane order studies. These dyes were used to follow plasma membrane order in live cells at 37°C. Disrupting actin filaments had a disordering effect while stabilizing actin filaments had an ordering effect on the plasma membrane, indicating there is a basal level of ordered domains in resting cells. Lowering PI(4,5)P2 levels decreased the proportion of ordered domains strongly suggesting that the connection of actin filaments to the plasma membrane is responsible for the maintaining the level of ordered membrane domains. Membrane blebs, which are detached from the underlying actin filaments, contained a low fraction of ordered domains. Aggregation of membrane components resulted in a higher proportion of ordered plasma membrane domains and an increase in cell peripheral actin polymerization. This strongly suggests that the attachment of actin filaments to the plasma membrane induces the formation of ordered domains. Limited cholesterol depletion with methyl-beta-cyclodextrin triggered peripheral actin polymerization. Cholesterol depleted cells showed an increase in plasma membrane order as a result of actin filament accumulation underneath the membrane. Moderate cholesterol depletion also induced membrane domain aggregation and activation of T cell signaling events. The T cell receptor (TCR) aggregation caused redistribution of domains resulting in TCR patches of higher order and the bulk membrane correspondingly depleted of ordered domains. This suggests the preexistence of small ordered membrane domains in resting T cells that aggregate upon cell activation. Increased actin polymerization at the TCR aggregation sites showed that actin polymerization is strongly correlated with the changes in the distribution of ordered domains. The distribution of the TCR in resting cells and its colocalization with actin filaments is cell cycle dependent. We conclude that actin filament attachment to the plasma membrane, which is regulated via PI(4,5)P2, plays a crucial role in the formation of ordered domains.

Place, publisher, year, edition, pages
Stockholm: The Wenner-Gren Institute, Stockholm University , 2011. , 56 p.
Keyword [en]
Membrane Organization, Lipid rafts, Actin, Laurdan, di-4-ANEPPDHQ, Cholesterol, T cell signaling, Colocalization, Generalized Polarization
National Category
Cell Biology
Research subject
Cellbiology
Identifiers
URN: urn:nbn:se:su:diva-62279ISBN: 978-91-7447-365-0 (print)OAI: oai:DiVA.org:su-62279DiVA: diva2:441173
Public defence
2011-10-14, E306, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript. Available from: 2011-09-22 Created: 2011-09-13 Last updated: 2011-09-16Bibliographically approved
List of papers
1. Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing
Open this publication in new window or tab >>Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing
2011 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, Vol. 1808, no 1, 298-306 p.Article in journal (Refereed) Published
Abstract [en]

Laurdan and di-4-ANEPPDHQ are used as probes for membrane order, with a blue shift in emission for membranes in liquid-ordered (lo) phase relative to membranes in liquid-disordered (ld) phase. Their use as membrane order probes requires that their spectral shifts are unaffected by membrane proteins, which we have examined by using membrane inserting peptides and large unilamellar vesicles (LUVs). The transmembrane polypeptides, mastoparan and bovine prion protein-derived peptide (bPrPp), were added to LUVs of either lo or ld phase, up to 1:10 peptide/total lipid ratio. The excitation and emission spectra of laurdan and di-4-ANEPPDHQ in both lipid phases were unaltered by peptide addition. The integrity and size distribution of the LUVs upon addition of the polypeptides were determined by dynamic light scattering. The insertion efficiency of the polypeptides into LUVs was determined by measuring their secondary structure by circular dichroism. Mastoparan had an α-helical and bPrPp a β-strand conformation compatible with insertion into the lipid bilayer. Our results suggest that the presence of proteins in biological membranes does not influence the spectra of laurdan and di-4-ANEPPDHQ, supporting that the dyes are appropriate probes for assessing lipid order in cells.

Keyword
di-4-ANEPPDHQ, Laurdan, ld phase, Lipid rafts, lo phase, Membrane order
National Category
Cell Biology
Research subject
Cell Biology
Identifiers
urn:nbn:se:su:diva-51788 (URN)10.1016/j.bbamem.2010.10.002 (DOI)000285853800032 ()20937246 (PubMedID)
Funder
Swedish Research Council, 621-2011-5964
Available from: 2011-01-12 Created: 2011-01-12 Last updated: 2013-04-29Bibliographically approved
2. Actin filaments at the plasma membrane in live cells cause the formation of ordered lipid domains via phosphatidylinositol 4,5-bisphosphate
Open this publication in new window or tab >>Actin filaments at the plasma membrane in live cells cause the formation of ordered lipid domains via phosphatidylinositol 4,5-bisphosphate
(English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137Article in journal (Refereed) Submitted
Abstract [en]

The relationship between ordered plasma membrane nanodomains, known as lipid rafts, and actin filaments is the focus of this study. Plasma membrane order was followed in live cells at 37°C using laurdan and di-4-ANEPPDHQ to report on lipid packing. Disrupting actin polymerization decreased the fraction of ordered domains, which strongly argue that unstimulated cells have a basal level of ordered domains. Stabilising actin filaments had the opposite effect and increased the proportion of ordered domains. Decreasing the plasma membrane level of phosphatidylinositol 4,5-bisphosphate lowers the number of attachment points for actin filaments and reduced the proportion of ordered domains. Aggregation of plasma membrane molecules, both lipid raft and non-lipid raft markers, leads to the formation of ordered domains that is correlated with an increase in cell peripheral actin filaments. In membrane blebs, which are detached from the underlying actin filaments the fraction of ordered domains was low and GM1 could not be patched to form ordered domains. We conclude that ordered domains form where actin filaments attach to the plasma membrane via phosphatidylinositol 4,5-bisphosphate. This downplays lipid-lipid interactions as the main driving force behind the formation of ordered membrane domains in vivo, giving greater prominence to membrane-intracellular filament interactions.

Keyword
actin, generalised polarisation, laurdan, lipid rafts, liquid ordered phase, membrane order, phosphatidylinositol 4, 5-bisphosphate
National Category
Cell Biology
Research subject
Cellbiology
Identifiers
urn:nbn:se:su:diva-62286 (URN)
Available from: 2011-09-14 Created: 2011-09-13 Last updated: 2017-12-08Bibliographically 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 1388-1918, 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: 2011-12-05Bibliographically approved
4. The T cell receptor resides in small ordered plasma membrane domains that aggregate upon T cell activation
Open this publication in new window or tab >>The T cell receptor resides in small ordered plasma membrane domains that aggregate upon T cell activation
(English)Manuscript (preprint) (Other academic)
Abstract [en]

T cell signaling emanates from large lipid raft platforms. Whether lipid rafts form upon T cell receptor (TCR) engagement or exist in resting T cells was the focus of this study. Plasma membrane order was followed in live T cells at 37°C using laurdan to report on lipid packing. Patching of the TCR in both Jurkat and human primary CD4+ T cells resulted in higher fractions of ordered plasma domains in the patches but did not increase the overall membrane order. The TCR colocalized with actin filaments in unstimulated Jurkat T cells and this colocalization was most prominent for cells in G1 phase. Moreover, the TCR located to the nuclear envelope, in addition to the plasma membrane, in cells in S and G2/M phase. Our study suggests that the TCR resides in ordered plasma membrane domains/lipid rafts that are linked to actin filament and aggregate upon T cell activation.

Keyword
actin, generalised polarisation, lipid rafts, membrane order, T cell receptor
National Category
Cell Biology
Research subject
Cellbiology
Identifiers
urn:nbn:se:su:diva-62288 (URN)
Available from: 2011-09-14 Created: 2011-09-13 Last updated: 2011-09-16Bibliographically approved

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