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  • 1.
    Dinic, Jelena
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Plasma membrane order; the role of cholesterol and links to actin filaments:  2011Doctoral 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.

  • 2.
    Dinic, Jelena
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Adler, Jeremy
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Parmryd, Ingela
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Plasma membrane order in T cell signalling2009Conference paper (Other academic)
    Abstract [en]

    Plasma membrane nanodomains, referred to as lipid rafts, more ordered than the bulk membrane play an important role in T cell signalling by forming signalling platforms in activated T cells. However, the existence of lipid rafts in resting T cells is contentious. Using laurdan, a membrane probe whose peak emission wavelength depends on the lipid environment, evidence is presented for the existence of ordered nanodomains in resting T cells.

    T cell signalling can be initiated by stimulating the T cell receptor (TCR), crosslinking the lipid raft markers GM1 (sphingolipid) or glycosylphosphatidylinositol (GPI) anchored proteins. The aggregation of lipid raft components induces the same response in Jurkat T cells as the ligation of an antigen to the TCR. Changes in membrane order linked with reorganization of the plasma membrane upon Jurkat T cell activation were followed at 37°C. Fluorescent images were analyzed for generalised polarisation values - a measure of the relative abundance of liquid ordered and liquid disordered domains. TCR patching does not increase the overall membrane order suggesting that membrane domains of high order are brought together in the patches. This supports the existence of small ordered membrane domains in resting T cells that aggregate upon activation. Patching of GM1, the GPI-anchored protein CD59 and the non lipid raft marker CD45 significantly increases the overall membrane order. So does general crosslinking of membrane components with Concanavalin A. Remodelling of the actin cytoskeleton is an integral part of TCR signaling and T cell activation. Disrupting actin polymerization using latrunculin B decreases membrane order and stabilizing actin filaments with jasplakinolide increases membrane order. An increase in membrane order appears to be a general effect of plasma membrane component patching and is likely due to a global induction of actin polymerization at the plasma membrane.

  • 3.
    Dinic, Jelena
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute.
    Biverståhl, Henrik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Parmryd, Ingela
    Stockholm University, Faculty of Science, The Wenner-Gren Institute.
    Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing2011In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1808, no 1, p. 298-306Article in journal (Refereed)
    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.

  • 4.
    Dinic, Jelena
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Parmryd, Ingela
    Uppsala universitet, Institutionen för medicinsk cellbiologi.
    Actin filaments at the plasma membrane in live cells cause the formation of ordered lipid domains via phosphatidylinositol 4,5-bisphosphateIn: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137Article in journal (Refereed)
    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.

  • 5.
    Dinic, Jelena
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Parmryd, Ingela
    Uppsala universitet, Institutionen för medicinsk cellbiologi .
    Riehl, Astrid
    Adler, Jeremy
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    The T cell receptor resides in small ordered plasma membrane domains that aggregate upon T cell activationManuscript (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.

  • 6.
    Mahammad, S
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Dinic, J
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Adler, J
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Parmryd, I
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Limited cholesterol depletion induces T cell activation and increases the plasma membrane fraction of higher order leading to clustering of signaling molecules2009In: The 49th Annual American Society of Cell Biology Meeting, San Diego, December 2009: Late abstracts, 2009, p. 2562/M-L20-Conference paper (Other academic)
    Abstract [en]

    The plasma membrane of eukaryotic cells contains nanodomains known as lipid rafts. Cholesterol depletion is a widely used technique for studying lipid rafts and their involvement in cellular processes. Cholesterol depletion has been reported to cause both increased and abolished T cell signaling. The abolished cell signaling upon cholesterol depletion is likely to be caused by substantial cell death as demonstrated by cell viability measurements. We have investigated how cholesterol depletion alters T cell activation by analyzing Jurkat T cells upon extraction of 10, 20, 30, 40 and 50% of total cholesterol using methyl β cyclodextrin (MBCD), a protocol in which cholesterol depletion does not have any adverse effect on cell viability.Upon cholesterol depletion peripheral actin polymerization and aggregation of the lipid raft marker GM1 in the plasma membrane is observed. The aggregation of GM1 upon cholesterol depletion is dependent on signaling protein Lck. The aggregated GM1 domains colocalize with signaling proteins such as Lck and LAT. To confirm that the effects seen by cholesterol depletion using cyclodextrin are actually due to cholesterol depletion and not cyclodextrin treatment itself, control experiments having Jurkat T cells treated with MBCD-cholesterol complexes to keep the cellular cholesterol content at equilibrium. A larger fraction of ordered (lo) plasma membrane is observed upon cholesterol depletion, a study performed by using laurdan. A relative membrane order is given by normalized ratio of the two emission regions termed as general polarization (GP). GP is defined analogously to fluorescence polarization by measuring the intensities (I) between 385 and 470 nm and 480 and 508 nm. Change in the membrane order and increased peripheral actin polymerization indicates that actin polymerization is in correlation to the formation of liquid ordered (lo) domains in the plasma membrane upon cholesterol depletion. Our results conclude that limited cholesterol depletion leads to T cell activation and an increase in the amount of liquid ordered domains in the plasma membrane. This activation is followed by aggregation of GM1 enriched domains.

  • 7.
    Mahammad, S.
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Dinic, J
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Adler, J
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Parmryd, I.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Limited cholesterol depletion induces T cell activation by increasing the plasma membrane fraction of higher order leading to clustering of signaling molecules2009Conference paper (Other academic)
  • 8.
    Mahammad, Saleemulla
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Dinic, Jelena
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Adler, Jeremy
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Parmryd, Ingela
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Limited cholesterol depletion causes aggregation of plasma membrane lipid raftsinducing T cell activation2010In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, Vol. 1801, no 6, p. 625-634Article in journal (Refereed)
    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.

1 - 8 of 8
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