Mitochondrial DNA Nucleoid Distribution at Simulated Pathologies as Visualized by 3D Super-Resolution Biplane FPALM / dSTORM Microscopy

Abstract

Upon certain insults and/or pathological states fragmented mitochondrial (mt) network persists in cells, with spheroid mt remnants containing clustered nucleoids of mtDNA (1).To image nucleoid distribution within mt network, we employed 3D super-resolution fluorescent photoactivable localization microscopy (FPALM)(2), in conjunction with photoconvertible fluorescent protein conjugates of marker proteins such as mitochondrial (mt) transcription factor A (TFAM) or mt single-stranded-DNA-binding protein (mtSSB). We have shown that nucleoids in hepatocellular HepG2 cells exhibit a rather wide size distribution ranging between 50 to 300 nm. This was confirmed using 3D TFAM/mtSSB immunocytochemistry in conjunction with direct stochastic optical reconstruction microscopy (dSTORM). Positioning in TIM23 dSTORM visualized membranes have confirmed our previous model for nucleoid clustering in mt network fragments (1), which were induced by mtDNA replication/transcription inhibition or other pathogenic simulations.Supported by grants grant 13-02033S, P305/12/P388 and P305/12/1247 of GACR.(1) Tauber J, Dlaskova A, Santorova J, Smolkova K, Alan L, Spacek T, Plecita-Hlavata L, Jabůrek M, Ježek, P Distribution of mitochondrial nucleoids upon mitochondrial network fragmentation and network reintegration in HEPG2 cells. Int J Biochem Cell Biol 45:593-603, 2013.(2) Mlodzianoski MJ, Schreiner JM, Callahan SP, Smolkova K, Dlaskova A, Santorova J, Ježek P, Bewersdorf J. Opt Express 2011; 19:15009-19.