Mitochondrial DNA Nucleoid Redistribution after Mitochondrial Network Fragmentation as Visualized by 3D Super-Resolution Biplane Fpalm Microscopy

Mitochondrial (mt) network undergoes locally frequent fragmentation and fusion events. When integrated over time, its basic morphology encompasses highly interconnected mt reticulum, a single mitochondrion within the cell (1). Upon certain insults and/or pathological states fragmented network persists. MtDNA is organized in nucleoids containing assessor proteins and recruited proteins of mt replication/ transcription machinery. It is debated on their uniform size and whether a single nucleoid contains a single mtDNA molecule or up to average 6 mtDNA molecules. To image nucleoid distribution within mt network, we employed 3D super-resolution fluorescent photactivable localization microscopy of Biplane schema (1). Mt network of hepatocellular carcinoma HepG2 cells was imaged first by its matrix space using mtEos2 or as outer mitochondrial membrane contour using Eos2-conjugates of truncated FIS1 protein (not inducing massive fission, Eos2-FIS1tr). Resulting 3D images confirm the existence of highly-connected mt network and unlike conventional confocal microscopy, 3D BiplaneFPALM distinguished a hollow character of mt reticulum tubules when visualized by Eos2-FIS1tr. Upon network fragmentation, hollow max ∼2 micrometer spheres occurred. Imaging of mt nucleoids confirmed the existence of ∼1000 nucleoids per cell with size distribution from 50 nm to 300 nm. Optimized dual transfection strategy had to be employed for simultaneous imaging of network (mtEos or Eos2-FIS1tr) and nucleoids (mtSSB-PSCFP2). Images revealed an equidistant nucleoid distribution of an average distance of ∼1 micrometer between nucleoids. Fragmentation by different agents led to observations of clusters of mt nucleoids within the spherical fragmented objects thus formed.Supported by grants P302/10/0346, P305/12/P388 and P305/12/1247 of GACR and ME09029 (Czech Ministry of Education); and 1R01GM091791-02 (NIH).(1) Mlodzianoski MJ, Schreiner JM, Callahan SP, Smolkova K, Dlaskova A, Santorova J, Ježek P, Bewersdorf J. Opt Express 2011;19:15009–19.