The Sphere of Influence

Live-cell microscopy of dividing yeast cells (S. cerevisiae). Mitochondria are visualized with a mitochondrial-targeted red fluorescent protein (mKate2). Mitochondrial DNA is labeled with the mitochondrial LacO-LacI system. LacO repeats are integrated upstream of the COX2 gene. A fusion protein consitisint of the lac-repressor (LacI) and the green fluorescent protein mNeonGreen is expressed from a gene integrated into the nuclear genome and targeted to mitochondria. The movie spans two hours. Images were taken every 15 minutes. z-projections are shown. The images were acquired with a Nikon Ti2 widefield microscope equipped with a Photometrix 95B camera.

A high-resolution visualization of mitochondrial genome spatial organization in S. cerevisiae. Using widefield OMX microscopy, we captured 41 Z-planes to resolve the relationship between the mitochondrial network (mt-dsRed, red) and individual mtDNA nucleoids. To track the genome, LacO repeats were integrated upstream of the COX2 gene and labeled via a mitochondrially targeted LacI-3xGFP fusion. Following precise segmentation in Imaris, the volumetric data was rendered in Blender to reveal the 3D architecture and distribution of the mitochondrial DNA within the organelle.

A 5-hour time-lapse capturing the coordinated segregation of the endoplasmic reticulum (ER) and the nuclear genome during Saccharomyces cerevisiae cell division. The ER architecture is visualized using the translocon component Sec63 tagged with mKate2/mCherry (magenta), highlighting the nuclear envelope and peripheral ER distribution. Simultaneously, a specific genomic locus is tracked using 112 LacO repeats integrated at the URA3 locus and labeled via a cyan fluorescent reporter. The movie transitions from a composite overlay of brightfield and fluorescence channels to a high-contrast dual-channel fluorescence view, detailing the precise timing of nuclear migration and ER inheritance into the developing daughter cell.

A 12-hour time-lapse capturing the transition from heteroplasmy to homoplasmy in S. cerevisiae. A heteroplasmic zygote was generated containing two mtDNA variants: one encoding Atp6-mKate2 (visualized in magenta) and another encoding Atp6-mNeonGreen (visualized in cyan). Imaged within a CellASIC ONIX2 microfluidic chamber, the movie illustrates the segregation of mitochondrial genomes during successive budding events. While initial cells exhibit signals from both variants (heteroplasmy), the lineage progressively drifts toward single-color fluorescence, demonstrating the spatial sequestering and inheritance patterns of mtDNA-encoded proteins.