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Bioinformatic and image analyses of the cellular localization of the apoptotic proteins endonuclease G, AIF, and AMID during apoptosis in human cells

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Abstract

We studied the cellular localization of the apoptotic proteins endonuclease G, AIF, and AMID in silico using three prediction tools and in living cells using both single-cell colocalization image analysis and nuclear translocation analysis. We confirmed the mitochondrial localization of endonuclease G and AIF by prediction analysis and by single-cell colocalization image analysis. We found the AMID protein to be cytoplasmic, most probably incorporated into the cytoplasmic side of the membranes of various organelles. The highest concentration of AMID was observed associated with the Golgi. Colocalization of AMID with lysosomes was also indirectly confirmed by analysis of AMID-rich vesicle velocity using manual tracking analysis. Bioinformatic analysis also detected nuclear localization signals in endonuclease G and AIF, but not in AMID. A novel analysis of time-lapse fluorescence image data during staurosporine-induced apoptosis revealed nuclear translocation only for endonuclease G and AIF.

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Abbreviations

AIF:

apoptosis-inducing factor

AMID:

AIF-homologous mitochondrion-associated inducer of death

endoG:

endonuclease G

EYFP:

enhanced yellow fluorescent protein

MLS:

mitochondrial localization sequence

P :

calculated probability

PDM:

product of the differences from the mean

R :

Mander’s overlap coefficient

ROI:

region of interest

Rr :

Pearson’s correlation coefficient

STS:

staurosporine

tHcRed:

tandem of two connected far-red fluorescent proteins from Heteractis crispa

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Acknowledgments

We thank Prof. Reddel from Children’s Medical Research Institute in Sydney (Australia) for the generous gift of the U-2 OS cell line; Pavel Matula for software optimizations; Vladan Ondřej and Stanislav Kozubek for help and valuable advice. This work was supported by the Grant Agency of the Czech Republic (grant numbers 202/04/0907, 204/03/D031, and 204/05/P090) and by The Ministry of Education, Youth and Sports of the Czech Republic (project numbers MSM0021622419 and LC535).

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Authors and Affiliations

  1. Centre for Biomedical Image Analysis, Faculty of Informatics, Masaryk University, Botanická 68a, Brno, 602 00, Czech Republic

    Miroslav Vařecha, Jana Amrichová, Michal Zimmermann, Vladimír Ulman & Michal Kozubek

  2. Laboratory of Molecular Cytology and Cytometry, Institute of Biophysics of the Academy of Sciences of the Czech Republic, Brno, 612 65, Czech Republic

    Emilie Lukášová

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  1. Miroslav Vařecha
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Correspondence to Miroslav Vařecha.

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Vařecha, M., Amrichová, J., Zimmermann, M. et al. Bioinformatic and image analyses of the cellular localization of the apoptotic proteins endonuclease G, AIF, and AMID during apoptosis in human cells. Apoptosis 12, 1155–1171 (2007). https://doi.org/10.1007/s10495-007-0061-0

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