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Use of micro-rotation imaging to study JNK-mediated cell survival in Theileria parva-infected B-lymphocytes

Published online by Cambridge University Press:  16 February 2005

R. LIZUNDIA ,
L. SENGMANIVONG ,
J. GUERGNON ,
T. MÜLLER ,
T. SCHNELLE ,
G. LANGSLEY  and
S. L. SHORTE
R. LIZUNDIA
Affiliation:
Laboratoire du Signalisation Immunoparasitaire, URA CNRS 2851, Department Parasitologie, Institut Pasteur, Paris, France
L. SENGMANIVONG
Affiliation:
Platefome d'Imagerie Dynamique, Department Biologie Cellulaire et Infection, Institut Pasteur, 25–28 rue du Dr. Roux, 75015, Paris, France
J. GUERGNON
Affiliation:
Laboratoire du Signalisation Immunoparasitaire, URA CNRS 2851, Department Parasitologie, Institut Pasteur, Paris, France
T. MÜLLER
Affiliation:
Cell Handling and Analysis, Evotec Technologies GmbH, c/o Institut für Biologie der Humboldt-Universität zu Berlin, Invalidenstrasse 42, D-10115 Berlin, Germany
T. SCHNELLE
Affiliation:
Cell Handling and Analysis, Evotec Technologies GmbH, c/o Institut für Biologie der Humboldt-Universität zu Berlin, Invalidenstrasse 42, D-10115 Berlin, Germany
G. LANGSLEY
Affiliation:
Laboratoire du Signalisation Immunoparasitaire, URA CNRS 2851, Department Parasitologie, Institut Pasteur, Paris, France
S. L. SHORTE
Affiliation:
Platefome d'Imagerie Dynamique, Department Biologie Cellulaire et Infection, Institut Pasteur, 25–28 rue du Dr. Roux, 75015, Paris, France
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Abstract

Lymphocytes infected with the protozoan parasite Theileria parva are transformed to permanently proliferating cells, an event underlying the pathology of the disease. However, the molecular signalling mediating this process is complex and poorly understood. Here, we show that down-regulation of JNK signalling by transient over expression of a dominant-negative mutant of JNK (JNK-APF) significantly increases Annexin-V-phycoerythrin (V-PE) labelling on infected B cell populations observed using flow cytometry. To establish whether this increase was specifically due to apoptosis, we used a novel single-cell imaging method: micro-rotation (MR)-imaging, designed to allow high-resolution 3-dimensional imaging of single cells in suspension. With this method we visualized subcellular patterns of V-PE uptake and chromatin organization in lymphocytes co-transfected with JNK-APF and GFP-tagged histone-H2B. This single-cell approach allowed us to clearly reveal characteristic apoptotic phenotypes, whose patterns reflected progressive states of programmed cell death due to JNK down-regulation. Our results strongly suggest a role for JNK in the survival of Theileria-infected B cells, and demonstrate the powerful utility of a new and unique 3-dimensional imaging method for living cells in suspension.

Keywords

TheileriaapoptosisB-lymphocytechromatinJNKmicro-rotation imaging
Type
Research Article
Information
Parasitology , Volume 130 , Issue 6 , June 2005 , pp. 629 - 635
Copyright
© 2005 Cambridge University Press

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M1B Supplementary movie

Movie 1B. MR-imaging of H2B-GFP fluorescence signal inside individual JNKN-APF co-transfected B cell.

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M2 Supplementary movie

Movie 2. MR-imaging of H2B-GFP and anexin V-PE labelling inside apoptotic Theileria-infected B cell.

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Video 6.4 MB