Coupled aggregation of mitochondrial single-strand DNA-binding protein tagged with Eos fluorescent protein visualizes synchronized activity of mitochondrial nucleoids

  • Authors:
    • Tomáš Olejár
    • David Pajuelo‑Reguera
    • Lukáš Alán
    • Andrea Dlasková
    • Petr Ježek
  • View Affiliations

  • Published online on: July 16, 2015     https://doi.org/10.3892/mmr.2015.4085
  • Pages: 5185-5190
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Oligomer aggregation of green-to-red photoconvertible fluorescent protein Eos (EosFP) is a natural feature of the wild‑type variant. The aim of the present study was to follow up mitochondrial nucleoid behavior under natural conditions of living cells transfected with mitochondrial single‑strand DNA‑binding protein (mtSSB) conjugated with EosFP. HEPG2 and SH‑SY5Y cells were subjected to lentiviral transfection and subsequently immunostained with anti‑DNA, anti‑transcription factor A, mitochondrial (TFAM) or anti‑translocase of the inner membrane 23 antibodies. Fluorescent microscopy, conventional confocal microscopy, superresolution biplane fluorescence photo-activation localization microscopy and direct stochastic optical reconstruction microscopy were used for imaging. In the two cell types, apparent couples of equally‑sized mtSSB‑EosFP‑visualized dots were observed. During the time course of the ongoing transfection procedure, however, a small limited number of large aggregates of mtSSB‑EosFP‑tagged protein started to form in the cells, which exhibited a great co‑localization with the noted coupled positions. Antibody staining and 3D immunocytochemistry confirmed that nucleoid components such as TFAM and DNA were co‑localized with these aggregates. Furthermore, the observed reduction of the mtDNA copy number in mtSSB‑EosFP‑transfected cells suggested a possible impairment of nucleoid function. In conclusion, the present study demonstrated that coupled nucleoids are synchronized by mtSSB‑EosFP overexpression and visualized through their equal binding capacity to mtSSB‑EosFP‑tagged protein. This observation suggested parallel replication and transcription activity of nucleoid couples native from a parental one. Preserved coupling in late stages of artificial EosFP‑mediated aggregation of tagged proteins suggested a rational manner of mitochondrial branching that may be cell-type specifically dependent on hierarchical nucleoid replication.
View Figures
View References

Related Articles

Journal Cover

October 2015
Volume 12 Issue 4

Print ISSN: 1791-2997
Online ISSN:1791-3004

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
APA
Olejár, T., Pajuelo‑Reguera, D., Alán, L., Dlasková, A., & Ježek, P. (2015). Coupled aggregation of mitochondrial single-strand DNA-binding protein tagged with Eos fluorescent protein visualizes synchronized activity of mitochondrial nucleoids. Molecular Medicine Reports, 12, 5185-5190. https://doi.org/10.3892/mmr.2015.4085
MLA
Olejár, T., Pajuelo‑Reguera, D., Alán, L., Dlasková, A., Ježek, P."Coupled aggregation of mitochondrial single-strand DNA-binding protein tagged with Eos fluorescent protein visualizes synchronized activity of mitochondrial nucleoids". Molecular Medicine Reports 12.4 (2015): 5185-5190.
Chicago
Olejár, T., Pajuelo‑Reguera, D., Alán, L., Dlasková, A., Ježek, P."Coupled aggregation of mitochondrial single-strand DNA-binding protein tagged with Eos fluorescent protein visualizes synchronized activity of mitochondrial nucleoids". Molecular Medicine Reports 12, no. 4 (2015): 5185-5190. https://doi.org/10.3892/mmr.2015.4085