Division of Mitochondrial Nucleoids Visualized by Biplane FPALM/dSTORM

Abstract

Mitochondrial DNA (mtDNA) is contained in nucleoids with accessory proteins and gene expression machinery proteins. To visualize them and screen sizes, 3D superresolution fluorescent photoactivable localization microscopy (FPALM) with Eos conjugated to the mitochondrial (mt) transcription factor-A (TFAM) [1], was employed in HepG2 cells. Also, 3D-immunocytochemistry of TFAM, mt-single-stranded-DNA-binding protein (mtSSB), or DNA, was used with the direct stochastic optical reconstruction microscopy (dSTORM). The localized points of FPALM/dSTORM data were remodeled using Delaunay tetrahedron modeling or by principal component analysis (PCA). Alternatively, PCA ellipsoid nucleoid images were normalized by Delaunay volume to obtain symmetrical ellipsoid (DVSE) models. For TFAM FPALM average DVSE of 80×80×159 nm dimensions were yielded, confirmed by TFAM dSTORM giving 78×78×170 nm; whereas mtDNA- and mtSSB-contoured ellipsoids were on average 65×65×152 nm and 44×44×133 nm, reflecting the nucleoid core and a space of unfolded mtDNA (or third-strand structures, such as D-loop), respectively. Due to the 2-fold lower axial vs. lateral resolution, only bulky DVSE models with a high aspect ratio and tilted towards the xy-plane were considered as two proximal nucleoids and suspicious to reflect a state immediately after the nucleoid division following mtDNA replication. Such nucleoid dividing pairs were visualized. The existence of twin nucleoids in mtDNA-dSTORM 3D images, representing snapshot of mtDNA “doubling”, i.e., predicted outcome of replication, supports our hypothetical assignments of similar nucleoids twins, when visualized via TFAM. The visualized nucleoid pairs (bulky, exhibiting high aspect ratios along the mt tubular axis) might represent direct observations of mt nucleoid division after mtDNA replication. Supported by grant 13-02033 (GACR).[1] M.J. Mlodzianoski et al. “Sample drift correction in 3D fluorescence photoactivation localization microscopy”. Opt. Express19, 15009-15019 (2011).