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  • 1.
    Adler, Jeremy
    et al.
    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 topology and membrane models2009Conference paper (Other academic)
  • 2.
    Adler, Jeremy
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Shevchuk, A
    Novak, P
    Korchev, Y
    Parmryd, I
    Stockholm University, Faculty of Science, The Wenner-Gren Institute, Cell Biology.
    Single Particle Tracking in 2D Produces Gibberish: A Solution2009Conference paper (Other academic)
  • 3.
    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.

  • 4.
    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.

  • 5.
    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)
  • 6.
    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.

  • 7.
    Tsarouhas, Vasilios
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Senti, Kirsten-André
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Jayaram, Satish Arcot
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Tiklová, Katarina
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Hemphälä, Johanna
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Adler, Jeremy
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Samakovlis, Christos
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Sequential Pulses of Apical Epithelial Secretion and Endocytosis Drive Airway Maturation in Drosophila2007In: Developmental Cell, ISSN 1534-5807, E-ISSN 1878-1551, Vol. 13, no 2, p. 214-225Article in journal (Refereed)
    Abstract [en]

    The development of air-filled respiratory organs is crucial for survival at birth. We used a combination of live imaging and genetic analysis to dissect respiratory organ maturation in the embryonic Drosophila trachea. We found that tracheal tube maturation entails three precise epithelial transitions. Initially, a secretion burst deposits proteins into the lumen. Solid luminal material is then rapidly cleared from the tubes, and shortly thereafter liquid is removed. To elucidate the cellular mechanisms behind these transitions, we identified gas-filling-deficient mutants showing narrow or protein-clogged tubes. These mutations either disrupt endoplasmatic reticulum-to-Golgi vesicle transport or endocytosis. First, Sar1 is required for protein secretion, luminal matrix assembly, and diametric tube expansion. Subsequently, a sharp pulse of Rab5-dependent endocytic activity rapidly internalizes and clears luminal contents. The coordination of luminal matrix secretion and endocytosis may be a general mechanism in tubular organ morphogenesis and maturation.

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