Tracking Gene Expression of Single Mitochondria in Live Neurons Using Nanotweezers

High Resolution Image Download MS PowerPoint Slide Neurons are highly polarized cells that depend on mitochondria for energy and signaling homeostasis. Importantly, energy and signaling requirements vary considerably across individual neurons both spatially and temporally. Therefore, to fully understand neuronal mitochondria, methods are needed to analyze mitochondria in live cells over time. The nanotweezer, a minimally invasive single-cell sampling technique, enables precise extraction of individual mitochondria from defined subcellular locations. Here, we combine single-mitochondrial extraction from live neurons with targeted mitochondrial gene expression tracking and mtDNA profiling to develop a platform for live-cell single-mitochondrion tracking and analysis. By tracking the expression of specific mitochondrially encoded genes in the same neurons over time, we reveal preliminary data showing a downregulation of mitochondrial genes MT-ND1 and MT-ATP6 following exposure to α-synuclein aggregates, independent of the proximity of the aggregates to the sampled mitochondria. Our approach provides a proof-of-concept for precise, temporal measurements of mitochondrial composition and targeted gene expression in vitro at single-organelle resolution, opening opportunities for single-cell and single-organelle studies of neuronal mitochondrial heterogeneity and its perturbation in models of neurodegeneration.