Scientists Map Individual Mitochondrial mRNAs at Nanometer Resolution for the First Time

Mitochondria carry their own circular genome (~16.6 kb) encoding 13 proteins critical for oxidative phosphorylation (OXPHOS), yet the spatial organization of mitochondrial mRNAs within these organelles has remained almost entirely unexplored. The core challenge is physical: mitochondria are so small — often near or below the diffraction limit of conventional light microscopy — that standard imaging cannot resolve individual transcripts. This study addresses that gap by establishing a robust smFISH pipeline compatible with STED and MINFLUX nanoscopy, enabling single-molecule visualization of mitochondrial mRNAs at nanometer precision.

The team designed branched DNA (bDNA) smFISH probe sets targeting all 11 human mitochondrial mRNAs, using 3–22 probe pairs per transcript depending on length, with STED-compatible fluorophores assembled into amplification 'trees.' In U-2 OS cells, three mRNAs (MT-ND1, MT-CO3, MT-CYB) were simultaneously imaged in three colors. STED imaging resolved individual mRNA spots with an average diameter (FWHM) of approximately 85 nm — far smaller than the theoretical maximum size of the assembled probe tree — indicating that mitochondrial mRNAs are highly compacted in situ. Pairwise minimum distances between distinct mRNA species had a median of ~200 nm, with similar distributions regardless of whether same-species or different-species pairs were compared.

Co-labeling with GRSF1, a canonical RNA granule marker, revealed that a subset of mRNAs co-localizes with or is in close proximity to RNA granules, consistent with co-transcriptional processing models. In cells treated with chloramphenicol (a mitochondrial translation inhibitor) or ethidium bromide (which depletes mtDNA), mRNA quantities and distributions changed measurably, demonstrating the system's sensitivity to perturbations of mitochondrial gene expression. Critically, the approach was also applied to primary fibroblasts from patients carrying a pathogenic mitochondrial tRNA mutation (m.3243A>G, associated with MELAS syndrome), where mRNA distribution and abundance differed from healthy controls — suggesting the method can detect disease-relevant alterations in mitochondrial transcriptomics.

A particularly striking finding emerged during apoptosis experiments: STED-smFISH showed that mitochondrial mRNAs are released from mitochondria into the cytoplasm during programmed cell death, a phenomenon not previously visualized at this resolution. This release may have implications for innate immune signaling, as cytoplasmic mitochondrial RNA can activate pattern recognition receptors. MINFLUX nanoscopy — which achieves single-digit nanometer localization precision — further revealed that individual mRNA molecules adopt variable folded shapes within mitochondria and are spatially proximate to mitochondrial ribosomes, providing the first direct visual evidence consistent with mitochondrial polysome-like translation complexes in human cells.

The protocols were validated across multiple human cell lines and extended to rat primary neurons, demonstrating broad transferability. The authors note that the bDNA amplification strategy provides high specificity (only paired probes trigger signal) and sufficient brightness for STED imaging without requiring genetic manipulation of cells. Together, these tools establish a new experimental framework for studying mitochondrial gene regulation at the single-molecule level in health, aging, and mitochondrial disease — with direct relevance to conditions ranging from neurodegeneration to cancer.