Magic-angle phosphorus NMR of functional mitochondria: in situ monitoring of lipid response under apoptotic-like stress.

Using a noninvasive, solid-state magic-angle spinning nuclear magnetic resonance (MAS NMR) approach, we track ex vivo the behavior of individual membrane components in isolated, active mitochondria (model system: potato tubers) during physiological processes. The individual phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL) membrane constituents were identified as distinct lines by applying MAS (31)P NMR on extracted lipid membranes. However, the CL NMR signal appeared to be very broad in functional mitochondria, indicating a tight complex formation with membrane protein. Calcium stress induced severe membrane degradation without recovery of a single CL NMR resonance. This suggests that calcium overload destroys the outer mitochondrial membrane and does not modify strongly the CL protein complexes in the inner membrane; a conclusion confirmed by respiratory controls. Conversely, mitochondrial membrane disruption on time degradation or mechanical stress generates clearly visible identical CL NMR signals, similar to those observed in rehydrated lipid extracts. Similarly, noninvasive based NMR tracking of lipids in response to diverse physiological stimuli can easily be used for other organelles and whole living cells.