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A single-cell analytical approach to quantify activated caspase-3/7 during osteoblast proliferation, differentiation, and apoptosis

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Abstract

The protein heterogeneity at the single-cell level has been recognized to be vital for an understanding of various life processes during animal development. In addition, the knowledge of accurate quantity of relevant proteins at cellular level is essential for appropriate interpretation of diagnostic and therapeutic results. Some low-copy-number proteins are known to play a crucial role during cell proliferation, differentiation, and also in apoptosis. The fate decision is often based on the concentration of these proteins in the individual cells. This is likely to apply also for caspases, cysteine proteases traditionally associated with cell death via apoptosis but recently being discovered also as important factors in cell proliferation and differentiation. The hypothesis was tested in bone-related cells, where modulation of fate from apoptosis to proliferation/differentiation and vice versa is particularly challenging, e.g., towards anti-osteoporotic treatments and anti-cancer strategies. An ultrasensitive and highly selective method based on bioluminescence photon counting was used to quantify activated caspase-3/7 in order to demonstrate protein-level heterogeneity in individual cells within one population and to associate quantitative measurements with different cell fates (proliferation, differentiation, apoptosis). The results indicate a gradual increase of caspase-3/7 activation from the proliferative status to differentiation (more than three times) and towards apoptosis (more than six times). The findings clearly support one of the putative key mechanisms of non-apoptotic functions of pro-apoptotic caspases based on fine-tuning of their activation levels.

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Funding

The research was supported by the Grant Agency of the Czech Republic, project no. 20-00726S. M.K. is Brno Ph.D. Talent Scholarship Holder funded by the Brno City Municipality.

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Authors and Affiliations

  1. Department of Bioanalytical Instrumentation, Institute of Analytical Chemistry, Czech Academy of Sciences, 602 00, Brno, Czech Republic

    Michael Killinger, Markéta Procházková & Karel Klepárník

  2. Department of Chemistry, Faculty of Science, Masaryk University, 602 00, Brno, Czech Republic

    Michael Killinger & Markéta Procházková

  3. Laboratory of Odontogenesis and Osteogenesis, Institute of Animal Physiology and Genetics, Czech Academy of Sciences, 602 00, Brno, Czech Republic

    Barbora Veselá & Eva Matalová

Authors
  1. Michael Killinger
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  2. Barbora Veselá
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  3. Markéta Procházková
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  4. Eva Matalová
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  5. Karel Klepárník
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Contributions

K. Klepárník and E. Matalová conceived and designed the experiments and contributed the reagents and materials. M. Killinger and M. Procházková performed the experiments and analyzed the data. M. Killinger, K. Klepárník, and B. Veselá wrote the paper. All authors read and approved the final manuscript.

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Correspondence to Karel Klepárník.

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Killinger, M., Veselá, B., Procházková, M. et al. A single-cell analytical approach to quantify activated caspase-3/7 during osteoblast proliferation, differentiation, and apoptosis. Anal Bioanal Chem 413, 5085–5093 (2021). https://doi.org/10.1007/s00216-021-03471-9

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  • DOI: https://doi.org/10.1007/s00216-021-03471-9

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