Bioorthogonal, Fluorogenic Targeting of Voltage-Sensitive Fluorophores for Visualizing Membrane Potential Dynamics in Cellular Organelles

Abstract

Electrical potential differences across lipid bilayers play foundational roles in cellular physiology. Plasma membrane voltage is the most widely studied; however, the bilayers of organelles like mitochondria, lysosomes, nuclei, and the endoplasmic reticulum (ER) also provide opportunities for ionic compartmentalization and the generation of transmembrane potentials. Unlike plasma membranes, organellar bilayers, cloistered within the cell, remain recalcitrant to traditional approaches like patch-clamp electrophysiology. To address the challenge of monitoring changes in organelle membrane potential, we describe the design, synthesis, and application of the LUnAR RhoVR (Ligation Unquenched for Activation and Redistribution Rhodamine-based Voltage Reporter) for optically monitoring membrane potential changes in the ER of living cells. We pair a tetrazine-quenched RhoVR for voltage sensing with a transcyclooctene (TCO)-conjugated ceramide (Cer-TCO) for targeting to the ER. Bright fluorescence is observed only at the coincidence of the LUnAR RhoVR and TCO in the ER, minimizing non-specific, off-target fluorescence. We show that the product of the LUnAR RhoVR and Cer-TCO is voltage-sensitive and that the LUnAR RhoVR can be targeted to an intact ER in living cells. Using the LUnAR RhoVR, we use two-color, ER-localized, fast voltage imaging coupled with cytosolic Ca2+ imaging to validate the electroneutrality of Ca2+ release from internal stores. Finally, we use the LUnAR RhoVR to directly visualize functional coupling between the plasma–ER membranes in patch clamped cell lines, providing the first direct evidence of the sign of the ER potential response to plasma membrane potential changes. We envision that the LUnAR RhoVR, along with other existing organelle-targeting TCO probes, could be applied widely for exploring organelle physiology.

The significance of bioelectricity on all levels of organization of an organism. Part 1: From the subcellular level to cells

“…Application of the GEVI showed that there is a functional coupling between the endoplasmic reticulum membrane potential and the plasma membrane potential (Fig. 8(b) (Klier et al, 2022)). It is foreseeable that in the near future GEVIs for each cellular organelle will be developed and the electrical functional coupling between the organelles as well as the organelles and the whole cell (i.e.…”

“…It sees that the endoplasmic reticulum "rectifies, or opposes, plasma membrane potential commands at values more positive than 0 mV" (Klier et al, 2022). Reprinted and modified from Klier et al (Klier et al, 2022), with permission from the publisher. the endoplasmic reticulum membrane potential was developed (Klier et al, 2022).…”

“…Reprinted and modified from Klier et al (Klier et al, 2022), with permission from the publisher. the endoplasmic reticulum membrane potential was developed (Klier et al, 2022). Application of the GEVI showed that there is a functional coupling between the endoplasmic reticulum membrane potential and the plasma membrane potential (Fig.…”

“…Shown are time-series of the patch-clamped HeLa cells and the fluorescence associated with the endoplasmic reticulum membrane potential.The relationship between the two electrical membrane potentials follow a non-linear relationship. It sees that the endoplasmic reticulum "rectifies, or opposes, plasma membrane potential commands at values more positive than 0 mV"(Klier et al, 2022). Reprinted and modified from Klier et al(Klier et al, 2022), with permission from the publisher.…”

Molecular design of dual-emission rhodamine analogs

“…[20][21][22][23][24] These colourful, bright, and photostable rhodamines have been deployed in many critical bioimaging experiments. [25][26][27][28][29][30] Additionally, modulating the equilibrium between the closed and the open forms of rhodamines has been exploited to develop fluorescent turn-on probes that detect various species. 31,32 Optimizing this equilibrium also endows several hydroxymethyl rhodamines with spontaneously blinking properties, enabling long-lasting super-resolution imaging based on the single-molecule localization technique.…”