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First published online 30 March 2004
doi: 10.1242/jcs.01063

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Amplification and propagation of pacemaker Ca²⁺ signals by cyclic ADP-ribose and the type 3 ryanodine receptor in T cells

Svenja Kunerth¹, Matthias F. Langhorst¹, Nadine Schwarzmann^1,*, Xianfeng Gu², Lijun Huang², Zhenjun Yang², Liangren Zhang², Steven J. Mills³, Li-he Zhang², Barry V.L. Potter³ and Andreas H. Guse^1,

¹ University Hospital Hamburg-Eppendorf, Center for Experimental Medicine, Institute of Biochemistry and Molecular Biology I: Cellular Signal Transduction, Martinistr. 52, 20246 Hamburg, Germany
² National Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xue Yuan Road, Beijing 100083, China
³ Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, BA2 7AY, UK

View larger version (25K): [in a new window]   Fig. 1. Effect of type 3 RyR knockdown, RyR inhibition and Ins(1,4,5)P3 R inhibition on early global Ca2+ signaling upon TCR/CD3 ligation. T cells [control clone E2 (a,c,d) or type 3 RyR-knockdown clone 25 (b)] were loaded with Fura-2/AM and analyzed by single-cell Ca2+ imaging as described in the Materials and Methods. OKT3 was added to a final concentration of 10 µg/ml as indicated. Inhibitors were microinjected directly before start of the measurement: (c) Ruthenium Red (RuRed; pipette concentration 10 µM), (d) D-Ins(1,4,6)PS3 (pipette concentration 80 µM). Data acquisition rate was 0.1 ratios/second before OKT3 addition, and 2 ratio/second thereafter. Characteristic tracings out of 18-39 cells are displayed.

View larger version (45K): [in a new window]   Fig. 2. Effect of type 3 RyR knockdown, RyR inhibition and Ins(1,4,5)P3R inhibition on early local Ca2+ signaling upon TCR/CD3 ligation. T cells [control clone E2 (A) or type 3 RyR-knockdown clone 25 (B)] were loaded with Fura-2/AM and analyzed by single-cell confocal Ca2+ imaging as described in the Materials and Methods. OKT3 (final concentration 10 µg/ml) was added as indicated (Ab,Bb). Inhibitors were microinjected directly before start of the measurement: Ruthenium Red (RuRed; pipette concentration 10 µM), D-Ins(1,4,6)PS3 (pipette concentration 80 µM). Data acquisition rate was 0.1 ratios/second before OKT3 addition, and 2 ratios/second thereafter. Staining for RyR expression was performed using BODIPY FL-X-ryanodine (Aa,Ba). Characteristic confocal ratio Ca2+ images acquired in the pacemaker phase are displayed (Ab,Bb; bar, 5 µm) and amplitudes of selected ROIs (see inset) are plotted against time for a 15-second time interval. Data in C and D are presented as mean±s.d. [n=9 ROIs for each subcellular region (edge, cytosol, nucleus) taken from three different individual cells]. Asterisks denote significant differences as indicated (P0.05, Student's t test).

View larger version (24K): [in a new window]   Fig. 3. Effect of type 3 RyR knockdown or RyR inhibition on global Ca2+ signaling upon activation of the cADPR/Ca2+-signaling system. T cells [control clone E2 (a,c) or type 3 RyR-knockdown clone 25 (b,d)] were loaded with Fura-2/AM and analyzed by single-cell Ca2+ imaging as described in the Materials and Methods. cADPR was microinjected (a,b; pipette concentration 20 µM) or cIDPRE was added extracellularly (c,d; final concentration 500 µM) as indicated. Inhibitors were microinjected directly before start of the measurement: (c) Ruthenium Red (RuRed; pipette concentration 10 µM). Data acquisition rate was 0.1 ratios/second before OKT3 addition, and 2 ratios/second thereafter. Characteristic tracings out of 15-24 cells are displayed.

View larger version (49K): [in a new window]   Fig. 4. Effect of type 3 RyR knockdown, RyR inhibition and Ins(1,4,5)P3R inhibition on early local Ca2+ signaling upon activation of the cADPR/Ca2+-signaling system. T cells [control clone E2 (A) or type 3 RyR-knockdown clone 25 (B)] were loaded with Fura-2/AM and analyzed by single-cell confocal Ca2+ imaging as described in the Materials and Methods. Either cADPR was microinjected (pipette concentration 20 µM) or cIDPRE was added extracellularly (final concentration 500 µM). During a 15-second time interval, characteristic confocal ratio Ca2+ images acquired in the pacemaker phase are displayed (A,B; bar, 5 µm) and amplitudes of selected ROIs (see inset) are plotted. Ruthenium Red (RuRed; pipette concentration 10 µM) was microinjected directly before start of the measurement. Data acquisition rate was 0.1 ratios/second before cADPR microinjection or cIDPRE addition, and 2 ratios/second thereafter. Data in C and D are presented as mean±s.d. [n=9 ROIs for each subcellular region (edge, cytosol, nucleus) taken from three different individual cells]. Asterisks denote significant differences as indicated (P0.05, Student's t test).

View larger version (26K): [in a new window]   Fig. 5. Effect of type 3 RyR knockdown or RyR inhibition on global Ca2+ signaling upon activation of the Ins(1,4,5)P3/Ca2+-signaling system. T cells [control clone E2 (a) or type 3 RyR-knockdown clone 25 (b), or Jurkat T cells (c,d)] were loaded with Fura-2/AM and analyzed by single-cell Ca2+ imaging as described in the Materials and Methods. Ins(1,4,5)P3 was microinjected (pipette concentration 4 µM) as indicated. Ruthenium Red (RuRed; pipette concentration 10 µM) was co-injected together with Ins(1,4,5)P3. Data acquisition rate was 0.75 ratios/second. Characteristic tracings out of 5-9 cells are displayed.

View larger version (43K): [in a new window]   Fig. 6. Localization of organelles and Ca2+-signaling proteins in T cells. T cells were fixed by p-formaldehyde, permeabilized by methanol (except for CD3 staining), and stained for RyR using BODIPY FL-X ryanodine (n=58; B), for Ins(1,4,5)P3R using anti-Ins(1,4,5)P3R antiserum and Rhodamine-conjugated secondary antibody (n=46; C), for CD3 using anti-CD3 antibody (OKT3) and a FITC-conjugated secondary antibody (n=17; D), for SERCA using BODIPY-thapsigargin (n=37; E), and for nuclei using Hoechst stain H33258 (n=37; F). Shown are characteristically stained single cells (bar, 5 µm). No or only weak fluorescence was seen in the control cells incubated without primary antibody or a surplus of ryanodine (10-fold) or thapsigargin (100-fold), respectively. All images were acquired from different individual cells. A representative confocal Ca2+ image (A; taken from Fig. 2Ab) facilitates comparison of protein and pacemaker signal localization.

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