A molecular rotor FLIM probe reveals dynamic coupling between mitochondrial inner membrane fluidity and cellular respiration.

The inner mitochondrial membrane (IMM), housing components of the electron transport chain (ETC), is the site for respiration. The ETC relies on mobile carriers; therefore, it has long been argued that the fluidity of the densely packed IMM can potentially influence ETC flux and cell physiology. However, it is unclear if cells temporally modulate IMM fluidity upon metabolic or other stimulation. Using a photostable, red-shifted, cell-permeable molecular-rotor, Mitorotor-1, we present a multiplexed approach for quantitatively mapping IMM fluidity in living cells. This reveals IMM fluidity to be linked to cellular-respiration and responsive to stimuli. Multiple approaches combining in vitro experiments and live-cell fluorescence (FLIM) lifetime imaging microscopy (FLIM) show Mitorotor-1 to robustly report IMM 'microviscosity'/fluidity through changes in molecular free volume. Interestingly, external osmotic stimuli cause controlled swelling/compaction of mitochondria, thereby revealing a graded Mitorotor-1 response to IMM microviscosity. Lateral diffusion measurements of IMM correlate with microviscosity reported via Mitorotor-1 FLIM-lifetime, showing convergence of independent approaches for measuring IMM local-order. Mitorotor-1 FLIM reveals mitochondrial heterogeneity in IMM fluidity; between-and-within cells and across single mitochondrion. Multiplexed FLIM lifetime imaging of Mitorotor-1 and NADH autofluorescence reveals that IMM fluidity positively correlates with respiration, across individual cells. Remarkably, we find that stimulating respiration, through nutrient deprivation or chemically, also leads to increase in IMM fluidity. These data suggest that modulating IMM fluidity supports enhanced respiratory flux. Our study presents a robust method for measuring IMM fluidity and suggests a dynamic regulatory paradigm of modulating IMM local order on changing metabolic demand.

A Tunable Palette of Molecular Rotors Allows Multicolor, Ratiometric Fluorescence Imaging and Direct Mapping of Mitochondrial Heterogeneity.

Environment-sensitive molecular probes offer the potential for a comprehensive mapping of the complex cellular milieu. We present here a radically new strategy of multiplexing highly sensitive, spectrally tuned fluorescent dyes for sensing cellular microenvironment. To achieve this multicolor, ratiometric cellular imaging, we first developed a series of highly sensitive, tunable molecular rotors for mitochondrial imaging, with emission wavelengths spanning the visible spectrum. These fluorogenic merocyanine dyes are all sensitive to solvent viscosity despite distinctive photophysical features. Our results show that merocyanine dyes can show a rotor-like behavior despite significant changes to the conventional donor-acceptor or push-pull scaffolds, thereby revealing conserved features of rotor dye chemistry. Developing closely related but spectrally separated dyes that have distinct response functions allows us to do ″two-color, two-dye″ imaging of the mitochondrial microenvironment. Our results with multidye, combinatorial imaging provide a direct visualization of the intrinsic heterogeneity of the mitochondrial microenvironment. The overall mitochondrial microenvironment (including contributions from local membrane order) as reported through two-color fluorescence ″ratio″ changes of multiplexed rotor dyes shows dynamic heterogeneity with distinct spatiotemporal signatures that evolve over time and respond to chemical perturbations. Our results offer a powerful illustration of how multiplexed dye imaging allows the quantitative imaging of mitochondrial membrane order and cellular microenvironment.