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Replicative senescence in MSCWJ-1 human umbilical cord mesenchymal stem cells is marked by characteristic changes in motility, cytoskeletal organization, and RhoA localization

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Abstract

Here, we document changes in cell motility and organization of the contractile apparatus of human umbilical cord Wharton's jelly mesenchymal stem cells (MSCWJ-1) in the process of replicative senescence. Colocalization dynamics of F-actin and actin-binding proteins (myosin-9, α-actinin-4, RhoA) were examined in the MSCWJ-1 cell line. The results show that nuclear-cytoplasmic redistribution of RhoA occurs during replicative senescence, with maximal RhoA/nucleus colocalization evident at passage 15. At that time point, decreases in colocalization, namely myosin-9/F-actin and α-actinin-4/F-actin, were seen and myosin-9 was found in cytosolic extracts in the assembly-incompetent form. Using an automated intravital confocal cytometry system and quantitative analysis of MSCWJ-1 movements, we found that changes in cytoskeletal organization correlate with cell motility characteristics over a time period from passages 9 to 38. The factors examined (cytoskeleton structure, cell motility) indicate that the process by which cells transition to replicative senescence is best represented as three stages. The first stage lasts from cell culture isolation to passage 15 and is characterized by: accumulation of actin-binding proteins in assembly-incompetent forms; nuclear RhoA accumulation; and an increase in movement tortuosity. The second stage extends from passages 15 to 28 and is characterized by: an increase in the structural integrity of the actin cytoskeleton; exit of RhoA and alpha-actinin-4 from the nucleus; and a decrease in path tortuosity. The third stage extends from passage 28 to 38 and is marked by: a plateau in actin cytoskeleton structural integrity; significant decreases in nuclear RhoA levels; and decreases in cell speed.

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Acknowledgements

The authors are deeply grateful to their colleagues: Dr. G. Stein and M. Vorobyov, for their expert assistance in confocal microscopy; S. Boykov, for assistance in computational trajectory analysis; I. Kropacheva and Dr. A. Koltsova, for technical help and general support; and E. Ramsay, for his assistance with writing and editing.

Funding

This work was carried out as part of a State Assignment of the Institute of Cytology RAS (No. AAAA-A19-119020190093–9).

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Authors and Affiliations

  1. Collection of Vertebrate Cell Cultures, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Prospekt, St. Petersburg, Russia, 194064

    Danila Bobkov, Anastasia Musorina & Galina Poljanskaya

  2. Laboratory of Intracellular Signaling and Transport, Smorodintsev Research Institute of Influenza, 15/17 Prof. Popova Street, St. Petersburg, Russia, 197376

    Danila Bobkov

  3. Institute of Physics, Nanotechnology and Telecommunications, Peter the Great St. Petersburg Polytechnic University, 29 Polytechnicheskaya Ulitsa, St. Petersburg, Russia, 195251

    Anastasia Polyanskaya

  4. Tumor Cell Molecular Genetics Group, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Prospekt, St. Petersburg, Russia, 194064

    Ekaterina Lomert

  5. Cell Technologies Center, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Prospekt, St. Petersburg, Russia, 194064

    Sergey Shabelnikov

Authors
  1. Danila Bobkov
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  2. Anastasia Polyanskaya
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  3. Anastasia Musorina
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  4. Ekaterina Lomert
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  5. Sergey Shabelnikov
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  6. Galina Poljanskaya
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Contributions

Danila Bobkov and Galina Poljanskaya designed the research and analyzed data. Anastasia Polyanskaya performed immunofluorescence and western blot work. Anastasia Musorina maintained the cell line and performed β-galactosidase analysis. Ekaterina Lomert carried out intravital confocal cytometry. Sergey Shabelnikov performed chromatographic separations. The first draft of the manuscript was written by Danila Bobkov, and all authors commented on previous versions of the manuscript. All authors have read and approved the final manuscript.

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Correspondence to Danila Bobkov.

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Bobkov, D., Polyanskaya, A., Musorina, A. et al. Replicative senescence in MSCWJ-1 human umbilical cord mesenchymal stem cells is marked by characteristic changes in motility, cytoskeletal organization, and RhoA localization. Mol Biol Rep 47, 3867–3883 (2020). https://doi.org/10.1007/s11033-020-05476-6

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