I. Rosenhek-Goldian, E.O. Zorikova, S. Chourasia, S.R. Cohen, S.V. Nesterov, A. Gross
Weizmann Institute of Science,
Israel
Keywords: mitochondria, atomic force microscopy (AFM), electron transport chain, Young's modulus, respiration, integrated power, mitochondria membrane potential (ΔΨm), mitochondrial carrier homolog 2 (MTCH2), mitofusin 1 (MFN1), mitofusin 2 (MFN2)
Summary:
Mitochondria govern energy conversion and cell fate, yet label-free methods to assess their functional and physical states at the single-organelle level do not currently exist. We address this need by combining the imaging capabilities of atomic force microscopy (AFM) with functional phenotyping by quantifying nanomechanics and noise spectrum of height signal of isolated mitochondria. Under various respiratory manipulations, a simple scalar of low-frequency height fluctuations closely paralleled changes in mitochondrial membrane potential (ΔΨm). This confirms that the AFM can be used to monitor functional changes at the level of a single mitochondria. In liver mitochondria lacking mitochondrial carrier homolog 2 (MTCH2), AFM revealed a compact, mechanically stiff, fluctuation-elevated state consistent with hyperpolarization and distinct from inhibitor/uncoupler signatures. Extending to mitochondria isolated from mouse embryonic fibroblasts, AFM data can distinguish between genotypes: loss of mitofusin 1 or 2 (MFN1 or MFN2) produced a stiff, low-fluctuation, low-ΔΨm phenotype, whereas loss of MTCH2 yielded a stiff, high-fluctuation, high-ΔΨm profile. By bridging nanomechanics and mitochondrial bioenergetics, this approach provides an orthogonal, dye-free platform for single-organelle functional phenotyping across tissues, energetic state, and disease models.