| Popis: |
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 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 LUnAR RhoVR (Ligation Unquenched for Activation and Redistribution Rhodamine based Voltage Reporter) for optically monitoring membrane potential changes in the endoplasmic reticulum (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 LUnAR RhoVR and TCO in the ER, minimizing non-specific, off-target fluorescence. We show that the product of LUnAR RhoVR and Cer-TCO is voltage sensitive and that LUnAR RhoVR can be targeted to intact ER in living cells. Using 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 LUnAR RhoVR to directly visualize functional coupling between plasma-ER membrane 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 LUnAR RhoVR, along with other existing organelle-targeting TCO probes, could be applied widely for exploring organelle physiology. |
