J Am Chem Soc. 2021 Mar 24;143(11):4095-4099. doi: 10.1021/jacs.0c13110. Epub 2021 Mar 12.
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 (ΔΨ) 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 ΔΨ 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 ΔΨ dynamics. Here, we report the first example of a fluorescent ΔΨ reporter that does not rely on ΔΨ-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 ΔΨ. 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 ΔΨ 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 Ca, plasma membrane potential, and reversible ΔΨ dynamics.
线粒体是有氧呼吸的场所，通过氧化磷酸化产生 ATP，质子沿着电化学梯度流经 ATP 合酶流动。这种跨线粒体内膜的负膜电位（ΔΨ）代表了细胞生命的一个基本生物物理参数。基于电极的传统膜电位记录方法不可能在完整细胞内的线粒体上实施。基于阳离子亲脂染料的荧光 ΔΨ 指示剂是一种常见的替代方法，但这些指示剂由于浓度依赖性假象和需要将染料保持在细胞外溶液中以可视化可逆的 ΔΨ 动力学而变得复杂。在这里，我们报告了第一个不依赖于 ΔΨ 依赖性积累的荧光 ΔΨ 报告器的示例。我们通过将 Rhodamine Voltage Reporter（RhoVR）的定位重定向到线粒体，该指示剂基于光诱导电子转移（PeT），通过将 RhoVR 的羧酸基团掩蔽为乙氧基甲酯（AM）酯。一旦进入线粒体，酯酶就会去除 AM 酯，将 RhoVR 困在基质内，它可以在其中整合到内膜中，并可逆地报告 ΔΨ 的变化。我们表明，这种小分子、可渗透、内部重分布的用于内膜靶向的罗丹明电压报告器，或 SPIRIT RhoVR，在许多不同的细胞系中都能定位于线粒体，并对通过非常低浓度的解偶联剂 FCCP 诱导的 ΔΨ 变化做出可逆反应，而无需外部染料池（与传统的基于罗丹明酯的积累方法不同）。SPIRIT RhoVR 与多色成像兼容，能够同时实时观察细胞质 Ca、质膜电位和可逆的 ΔΨ 动力学。
