Intracellular Ca2+ Release Triggers Translocation of Membrane Marker FM1-43 from the Extracellular Leaflet of Plasma Membrane into Endoplasmic Reticulum in T Lymphocytes

Department of Physiology and Membrane Biology, University of California, Davis, California 95616, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2005; 280(16):16377-82. DOI: 10.1074/jbc.M501202200
Source: PubMed


Stimulation of T cell receptor in lymphocytes enhances Ca(2+) signaling and accelerates membrane trafficking. The relationships between these processes are not well understood. We employed membrane-impermeable lipid marker FM1-43 to explore membrane trafficking upon mobilization of intracellular Ca(2+) in Jurkat T cells. We established that liberation of intracellular Ca(2+) with T cell receptor agonist phytohemagglutinin P or with Ca(2+)-mobilizing agents ionomycin or thapsigargin induced accumulation of FM1-43 within the lumen of the endoplasmic reticulum (ER), nuclear envelope (NE), and Golgi. FM1-43 loading into ER-NE and Golgi was not mediated via the cytosol because other organelles such as mitochondria and multivesicular bodies located in close proximity to the FM1-43-containing ER were free of dye. Intralumenal FM1-43 accumulation was observed even when Ca(2+) signaling in the cytosol was abolished by the removal of extracellular Ca(2+). Our findings strongly suggest that release of intracellular Ca(2+) may create continuity between the extracellular leaflet of the plasma membrane and the lumenal membrane leaflet of the ER by a mechanism that does not require global cytosolic Ca(2+) elevation.

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    • "Mechanistically, PMA/ionomycin provides a potent stimulation allowing us to bypass the T cell receptor activation essential for Treg cell development and it prevents the emergence of CD4-CD8+ cells in the culture [17]. PMA activates protein kinase C [38] while ionomycin is a Ca2+ mobilizing agent [39]. This combination has been shown to up-regulate CD25 on T lymphocytes [17] and a high CD25 expression is a marker of the Treg cell phenotype [40]. "
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    ABSTRACT: Background Induced regulatory T (iTreg) lymphocytes show promise for application in the treatment of allergic, autoimmune and inflammatory disorders. iTreg cells demonstrate advantages over natural Treg (nTreg) cells in terms of increased number of starting population and greater potential to proliferate. Different activation methods to generate iTreg cells result in iTreg cells that are heterogeneous in phenotype and mechanisms of suppression. Therefore it is of interest to explore new techniques to generate iTreg cells and to determine their physiological relevance. Methods Using phorbol myristate acetate (PMA)/ionomycin and anti-CD3 activation of CD4+CD25- cells we generated in vitro functional CD4+CD25+ iTreg (TregPMA) cells. Functionality of the generated TregPMA cells was tested in vivo in a mouse model of inflammatory bowel disease (IBD) - CD45RB transfer colitis model. Results TregPMA cells expressed regulatory markers and proved to ameliorate the disease phenotype in murine CD45RB transfer colitis model. The body weight loss and disease activity scores for TregPMA treated mice were reduced when compared to diseased control group. Histological assessment of colon sections confirmed amelioration of the disease phenotype. Additionally, cytokine analysis showed decreased levels of proinflammatory colonic and plasma IL-6, colonic IL-1 β and higher levels of colonic IL-17 when compared to diseased control group. Conclusions This study identifies a new method to generate in vitro iTreg cells (TregPMA cells) which physiological efficacy has been demonstrated in vivo.
    BMC Gastroenterology 12/2012; 12(1):172. DOI:10.1186/1471-230X-12-172 · 2.37 Impact Factor
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    • "STIM1 puncta in store-depleted cells were identifi ed at the EM level as regions of HRP-STIM1 accumulation in ER tubules lying in close proximity to the plasma membrane. Junctional ER has been observed before in many cell types, including Jurkat cells, Xenopus laevis oocytes, smooth muscle, neurons, and yeast, though a role in store-operated Ca 2+ entry has never been established (Henkart et al., 1976; Gardiner and Grey, 1983; Takeshima et al., 2000; Pichler et al., 2001; Dadsetan et al., 2005). We found that store depletion causes STIM1 to accumulate in preexisting junctions (comprising approximately two thirds of the total junctions in depleted cells) as well as in newly formed junctions (comprising approximately one third; Fig. 7 C). "
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    ABSTRACT: Stromal interacting molecule 1 (STIM1), reported to be an endoplasmic reticulum (ER) Ca(2+) sensor controlling store-operated Ca(2+) entry, redistributes from a diffuse ER localization into puncta at the cell periphery after store depletion. STIM1 redistribution is proposed to be necessary for Ca(2+) release-activated Ca(2+) (CRAC) channel activation, but it is unclear whether redistribution is rapid enough to play a causal role. Furthermore, the location of STIM1 puncta is uncertain, with recent reports supporting retention in the ER as well as insertion into the plasma membrane (PM). Using total internal reflection fluorescence (TIRF) microscopy and patch-clamp recording from single Jurkat cells, we show that STIM1 puncta form several seconds before CRAC channels open, supporting a causal role in channel activation. Fluorescence quenching and electron microscopy analysis reveal that puncta correspond to STIM1 accumulation in discrete subregions of junctional ER located 10-25 nm from the PM, without detectable insertion of STIM1 into the PM. Roughly one third of these ER-PM contacts form in response to store depletion. These studies identify an ER structure underlying store-operated Ca(2+) entry, whose extreme proximity to the PM may enable STIM1 to interact with CRAC channels or associated proteins.
    The Journal of Cell Biology 10/2006; 174(6):803-13. DOI:10.1083/jcb.200604014 · 9.83 Impact Factor
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    ABSTRACT: The research presented focusses on the self-weight consolidation and strength evolution of soft underwater mud beds. A series of experiments was carried out in 1.5 m tall consolidation columns for a maximum duration of 95 days. Segmented settling columns were designed and built in order to provide well-defined samples of the bed, the strength of which was measured using a high-precision rheometer equipped with a miniature vane.The process of consolidation was modelled as a one-dimensional process using the Gibson equation [9] written in a Eulerian reference frame and with the particle volume fraction as the dependent variable. The integration of the consolidation equation requires boundary conditions, initial conditions and constitutive equations for effective stress and permeability. The strength evolution is taken into account by a failure criterion which relates strength to effective stress.Constitutive equations for effective stress and permeability and a failure criterion were derived based on the concept of a scale invariant bed structure. The bed is assumed to be formed by aggregates. The properties of the aggregates are assumed to be determined by the clay fraction only. The averaged size of the aggregates determines the effective stress, permeability and shear strength. During consolidation the averaged size of the aggregates reduces, so that effective stress and shear strength increase and permeability decreases. The results of numerical modelling of the experiments agree well with measurements.
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