Imaging Reversible Mitochondrial Membrane Potential Dynamics with a Masked Rhodamine Voltage Reporter

Abstract

Mitochondria are the site of aerobic respiration, producing ATP via oxidative phosphorylation as protons flow down their electrochemical gradient through ATP synthase. This negative membrane potential across the inner mitochondrial membrane (ΔΨm) represents a fundamental biophysical parameter central to cellular life. Traditional, electrode-based methods for recording membrane potential are impossible to implement on mitochondria within intact cells. Fluorescent ΔΨm indicators based on cationic, lipophilic dyes are a common alternative, but these indicators are complicated by concentration-dependent artifacts and the requirement to maintain dye in the extracellular solution to visualize reversible ΔΨm dynamics. Here, we report the first example of a fluorescent ΔΨm reporter that does not rely on ΔΨm-dependent accumulation. We redirected the localization of a photoinduced electron transfer (PeT)-based indicator, Rhodamine Voltage Reporter (RhoVR), to mitochondria by masking the carboxylate of RhoVR 1 as an acetoxymethyl (AM) ester. Once within mitochondria, esterases remove the AM ester, trapping RhoVR inside of the mitochondrial matrix, where it can incorporate within the inner membrane and reversibly report on changes in ΔΨm. We show that this Small molecule, Permeable, Internally Redistributing for Inner membrane Targeting Rhodamine Voltage Reporter, or SPIRIT RhoVR, localizes to mitochondria across a number of different cell lines and responds reversibly to changes in ΔΨm induced by exceptionally low concentrations of the uncoupler FCCP without the need for exogenous pools of dye (unlike traditional, accumulation-based rhodamine esters). SPIRIT RhoVR is compatible with multi-color imaging, enabling simultaneous, real-time observation of cytosolic Ca2+, plasma membrane potential, and reversible ΔΨm dynamics.

Bioorthogonal, fluorogenic targeting of voltage-sensitive fluorophores for visualizing membrane potential dynamics in cellular organelles

“…This supports localization of LUnAR RhoVR 1 to the ER, rather than mitochondria, and corroborates colocalization data (Figure 2, Figure S6-8), since we previously established that low doses of FCCP (500 nM) cause a change in the mitochondrial membrane potential. 9 It further corroborates that the fluorescence changes we observe upon switching [K + ] result from membrane potential changes in the ER, rather than off-target pH sensing, since the pH of the ER is similar to the cytosol, 1 while the pH gradient between the cytosol and other organelles like mitochondria and lysosome is much steeper. 1,42 Taken together, these results demonstrate that LUnAR RhoVR 1 is able to sense voltage transients in the ER.…”

“…8 However, we recently found that modifications to a rhodamine-based voltage reporter (RhoVR) enabled passage through cell membranes and localization to mitochondria. 9 Fluorescent dyes can be targeted to subcellular locations either through charge, [10][11][12] covalently attached groups specific to a subcellular location, 13 genetic targeting of protein constructs, [14][15][16][17][18] or bioorthogonal chemistry in which one reaction partner is attached to a targeting group and another 578 nm (Figure 1, Figure S2). The fluorescence of LU-nAR RhoVR 1 increases 7-fold upon reaction with TCO-ol (25); LUnAR RhoVR 0 increases 10-fold (Figure 1, Table 1).…”

Low Polarity-Triggered Basic Hydrolysis of Coumarin as an AND Logic Gate for Broad-Spectrum Cancer Diagnosis

“…In addition, recent studies have shown that mitochondrial polarity levels in cancer cells (Δ f ∼ 0.287) are lower than in normal cells (Δ f ∼ 0.295) . While any of these parameters may not separately necessarily indicate cancer, − combining these inputs (MMP, pH, and polarity) via an AND logic gate could improve the accuracy of detection. Herein, we introduce a novel fluorescent probe that simultaneously utilizes MMP, pH, and polarity in mitochondria as inputs to achieve cancer detection (Scheme A).…”

Voltage-induced fluorescence lifetime imaging of a BODIPY derivative in giant unilamellar vesicles as potential neuron membrane mimics

“…2 Generally speaking there are two types of VSDs according to their response speed; one type is termed a fast-response probe and the other one is a slow-response probe. 3 The disparate response mechanisms by which the two types of VSDs operate offer different levels of information, with fast responsive affording a low signal-tonoise (S/N) ratio but more critical detail of frequent spikes. 4 Slow responsive probes offer a superior S/N but miss subtle details.…”