| Autor: |
Klier PEZ; Department of Chemistry, University of California, Berkeley, California 94720, United States., Gest AMM; Department of Chemistry, University of California, Berkeley, California 94720, United States., Martin JG; Department of Chemistry, University of California, Berkeley, California 94720, United States., Roo R; Department of Chemistry, University of California, Berkeley, California 94720, United States., Navarro MX; Department of Chemistry, University of California, Berkeley, California 94720, United States., Lesiak L; Department of Chemistry, University of California, Berkeley, California 94720, United States., Deal PE; Department of Chemistry, University of California, Berkeley, California 94720, United States., Dadina N; Department of Chemistry, University of California, Berkeley, California 94720, United States., Tyson J; Department of Chemistry, University of California, Berkeley, California 94720, United States., Schepartz A; Department of Chemistry, University of California, Berkeley, California 94720, United States.; Department of Molecular & Cell Biology, University of California, Berkeley, California 94720, United States., Miller EW; Department of Chemistry, University of California, Berkeley, California 94720, United States.; Department of Molecular & Cell Biology, University of California, Berkeley, California 94720, United States.; Helen Wills Neuroscience Institute, University of California, Berkeley, California 94720, United States. |
| Abstrakt: |
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 ( L igation Un quenched for A ctivation and R edistribution Rho damine-based V oltage R eporter) 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 Ca 2+ imaging to validate the electroneutrality of Ca 2+ 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. |
