A02

A02 Project movie

A02

From Diercks, B.-P. et al. (2018). ORAI1, STIM1/2, and RYR1 shape subsecond Ca2+ microdomains upon T cell activation. Sci. Signal. 11, eaat0358.

Project A02 set out to disentangle the dynamic architecture of Ca2+ microdomains in T cells, focusing on Ca2+ entry and on development of tailor-made image processing for the analysis of Ca2+ microdomains. During the 1st funding period, in project A02 two Ca2+ entry systems were discovered to significantly contribute to TCR/CD3 evoked Ca2+ microdomains. By using T cells devoid of ORAI1, ORAI2, stromal interaction molecule 1 (STIM1) and STIM2, both STIM molecules and ORAI1 were identified as SOCE components required for Ca2+ microdomain formation. More unexpected, in P2x4-/- or P2x7-/- T cells, the number of Ca2+ microdomains was significantly decreased. Accordingly, also inhibition of pannexin1, a Ca2+ sensitive ATP-channel in the plasma membrane, as well as addition of extracellular apyrase diminished Ca2+ microdomain numbers, indicating TCR/CD3 evoked ATP release via pannexin1 and autocrine purinergic Ca2+ signaling via P2X4 and P2X7 to amplify Ca2+ microdomains numbers. Further, in project A02 Ca2+ microdomain formation in the absence of TCR/CD3 stimulation was observed. These ‘spontaneous’ Ca2+ microdomains do not require expression of RYR1 or P2X7, but depend on STIM1, STIM2, ORAI1, and on P2X4. They were also detectable by a genetically encoded Ca2+ indicator fused to ORAI1 (ORAI1-G-GECO1.2). These results provide evidence for a pre-activated T cell state that enables fast and efficient activation of Ca2+ signaling once the TCR/CD3 complex is triggered.

The 2nd major aim of project A02 has been to develop tailor-made image processing algorithms for the analysis of Ca2+ microdomains. To reduce uncertainties resulting from the enormous challenges of high-resolution live cell imaging, e.g. minimum emission times, photo bleaching, out-of-focus light, all leading to low signal-to-noise ratio, project A02 developed image processing approaches for run time-efficient advanced spatio-temporal variational deconvolution and image restoration specifically tailored to fluorescence live cell microscopy. Application to synthetic as well as Ca2+ microdomain data sets demonstrated superiority in terms of image quality compared to conventional image restoration and deconvolution, in particular for the challenging imaging conditions common in projects A02 and A01.

Dr. Björn-Philipp Diercks

Selected publications (*equal contribution)

 

  1. Gu F, Krüger A, Roggenkamp HG, Alpers R, Lodygin D, Jaquet V, Möckl F, Hernandez C LC, Winterberg K, Bauche A, Rosche A, Grasberger H, Kao JY, Schetelig D, Werner R, Schröder K, Carty M, Bowie AG, Huber S, Meier C, Mittrücker HW, Heeren J, Krause KH, Flügel A, Diercks B-P*, Guse AH*. Dual NADPH oxidases DUOX1 and DUOX2 synthesize NAADP and are necessary for Ca2+ signaling during T cell activation. Science Signal. 2021; 14 eabe3800.
  2. Roggenkamp HG, Khansahib I, Hernandez C LC, Zhang Y, Lodygin D, Krüger A, Gu F, Möckl F, Löhndorf A, Wolters V, Woike D, Rosche A, Bauche A, Schetelig D, Werner R, Schlüter H, Failla AV, Meier C, Fliegert R, Walseth TF, Flügel A, Diercks B-P*, Guse AH*. HN1L/JPT2: A signaling protein that connects NAADP generation to Ca2+ microdomain formation. Science Signal. 2021; 14:eabd5647.
  3. Woelk LM, Kannabiran SA, Brock VJ, Gee CE, Lohr C, Guse AH, Diercks B-P, Werner R. Time Dependent Image Restoration of Low-SNR Live-Cell Ca2+ Fluorescence Microscopy Data. Int. J. Mol. Sci. 2021; 22, 11792.
  4. Nawrocki M, Lory N, Bedke T, Stumme F, Diercks B-P, Guse AH, Meier C, Gagliani N, Mittrücker HW, Huber S. Trans-Ned 19-Mediated Antagonism of Nicotinic Acid Adenine Nucleotide - Mediated Calcium Signaling Regulates Th17 Cell Plasticity in Mice. Cells 2021; 10, 3039.
  5. Löhndorf A, Hosang L, Dohle W, Odoardi F, Waschkowski SA, Rosche A, Bauche A, Winzer R, Tolosa E, Windhorst S, Marry S, Flügel A, Potter BVL, Diercks B-P*, Guse AH*. 2-Methoxyestradiol and its derivatives inhibit store-operated Ca2+ entry in T cells: identification of a new and potent inhibitor. BBA Mol Cell Res. 2021; 118988.
  6. Hadaeghi F, Diercks B-P, Schetelig D, Damicelli F, Wolf IMA, Werner R (2021) Spatio-temporal feature learning with reservoir computing for T-cell segmentation in live-cell Ca2+ fluorescence microscopy. Sci Rep. 2021; 11:8233.
  7. Diercks BP, Werner R, Schetelig D, Wolf IMA, Guse AH (2019) High-Resolution Calcium Imaging Method for Local Calcium Signaling. Methods Mol Biol. 1929:27-39.
  8. Diercks BP, Werner R, Weidemüller P, Czarniak F, Hernandez L, Lehmann C, Rosche A, Krüger A, Kaufmann U, Vaeth M, Failla AV, Zobiak B, Kandil FI, Schetelig D, Ruthenbeck A, Meier C, Lodygin D, Flügel A, Ren D, Wolf IMA, Feske S, Guse AH (2018) ORAI1, STIM1/2, and RYR1 shape subsecond Ca2+ microdomains upon T cell activation. Sci Signal 18;11(561).
  9. Diercks BP, Fliegert R, Guse AH (2016) Mag-Fluo4 in T Cells: Imaging of Intra-Organelle Free Ca2+Concentrations. Biochim Biophys Acta Mol Cell Res. 2017 Jun;1864(6):977-986.
  10. Wolf IMA, Diercks BP, Gattkowski E, Czarniak F, Kempski J, Werner R, Schetelig D, Mittrücker HW, Schumacher V, von Osten M, Lodygin D, Flügel A, Fliegert R, Guse AH (2015) Frontrunners of T cell activation: Initial, localized Ca2+ signals mediated by NAADP and the type 1 ryanodine receptor. Sci Signal 8: ra102.

 

 

 

 

PD Dr. René Werner

Selected publications (* equal contribution)

 

  1. Woelk LM, Kannabiran SA, Brock VJ, Gee CE, Lohr C, Guse AH, Diercks B-P, Werner R. Time Dependent Image Restoration of Low-SNR Live-Cell Ca2+ Fluorescence Microscopy Data. Int. J. Mol. Sci. 2021; 22, 11792.
  2. Hadaeghi F, Diercks B-P, Schetelig D, Damicelli F, Wolf IMA, Werner R (2021) Spatio-temporal feature learning with reservoir computing for T-cell segmentation in live-cell Ca2+ fluorescence microscopy. Sci Rep. 2021; 11:8233.
  3. Schmitz R, Madesta F, Nielsen M, Krause J, Steurer S, Krause J, Werner R*, Rösch T*. Multi-scale fully convolutional neural networks for histopathology image segmentation: From nuclear aberrations to the global tissue architecture. Med Image Anal. 2021; 70:101996.
  4. Schmitz R, Werner R, Repici A, Bisschops R, Meining A, Zornow M, Messmann H, Hassan C, Sharma P, Rösch T. Artificial intelligence in GI endoscopy: stumbling blocks, gold standards and the role of endoscopy societies. Gut. 2021; 0:1–4.
  5. Nielsen M, Waldmann M, Frölich AM, Flottmann F, Hristova E, Bendszus M, Seker F, Fiehler J, Sentker T, Werner R. Deep learning-based automated TICI scoring: a timely proof-of-principle study. Stroke 2021; 52:3497-3504.
  6. Madesta F, Schmitz R, Rösch T, Werner R. Widening the focus: biomedical image segmentation challenges and the underestimated role of patch sampling and inference strategies. In: MICCAI 2020, Lecture Notes in Computer Science (LNCS), 2020, Springer, vol. 12264, pp. 289-298.
  7. Diercks BP, Werner R, Schetelig D, Wolf IMA, Guse AH (2019) High-Resolution Calcium Imaging Method for Local Calcium Signaling. Methods Mol Biol. 1929:27-39.
  8. Diercks BP, Werner R, Weidemüller P, Czarniak F, Hernandez L, Lehmann C, Rosche A, Krüger A, Kaufmann U, Vaeth M, Failla AV, Zobiak B, Kandil FI, Schetelig D, Ruthenbeck A, Meier C, Lodygin D, Flügel A, Ren D, Wolf IMA, Feske S, Guse AH (2018) ORAI1, STIM1/2, and RYR1 shape subsecond Ca2+ microdomains upon T cell activation. Sci Signal 18;11(561).
  9. Wolf IMA, Diercks B-P, Gattkowski E, Czarniak F, Kempski J, Werner R, Schetelig D, Mittrücker HW, Schumacher V, von Osten M, Lodygin D, Flügel A, Fliegert R, Guse AH (2015) Frontrunners of T cell activation: Initial, localized Ca2+ signals mediated by NAADP and the type 1 ryanodine receptor. Sci Signal 8:ra120.
  10. Werner R, Schmidt-Richberg A, Handels H, Ehrhardt J (2014) Estimation of lung motion fields in 4D CT data by variational non-linear intensity-based registration. Phys Med Biol 59:4247-4260.

Our Team

Dr. Björn-Philipp Diercks

Department of Biochemistry and Molecular Cell Biology

University Medical Center Hamburg-Eppendorf (UKE)

PD Dr. René Werner

Department of Computational Neuroscience

University Medical Center Hamburg-Eppendorf (UKE)

M.Sc. Valerie Brock

 
Department of Biochemistry and Molecular Cell Biology

University Medical Center Hamburg-Eppendorf (UKE)

Dr. Lena Woelk

 
Department of Computational Neuroscience

University Medical Center Hamburg-Eppendorf (UKE)

Dr. Fatemeh Hadaeghi

Department of Computational Neuroscience

University Medical Center Hamburg-Eppendorf (UKE)

Alumni

PD Dr. Insa Wolf

Department of Biochemistry and Molecular Cell Biology

University Medical Center Hamburg-Eppendorf (UKE)

Dr. Daniel Schetelig

Department of Computational Neuroscience

University Medical Center Hamburg-Eppendorf (UKE)

Contact

University Medical Center Hamburg-Eppendorf 
Department of Biochemistry and Molecular Cell Biology 
Martinistrasse 52
20246 Hamburg

Scientific Coordinator

Dr. Björn-Philipp Diercks
Fon: +49  (0) 40 7410 54338
E-Mail: b.diercks©uke.de

Administration

Laura Mitsching
Fon: +49  (0) 40 7410 50301
E-Mail: l.mitsching©uke.de