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

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.

Water-Soluble BODIPY Photocages with Tunable Cellular Localization.

Photoactivation of bioactive molecules allows manipulation of cellular processes with high spatiotemporal precision. The recent emergence of visible-light excitable photoprotecting groups has the potential to further expand the established utility of the photoactivation strategy in biological applications by offering higher tissue penetration, diminished phototoxicity, and compatibility with other light-dependent techniques. Nevertheless, a critical barrier to such applications remains the significant hydrophobicity of most visible-light excitable photocaging groups. Here, we find that applying the conventional 2,6-sulfonation to meso-methyl BODIPY photocages is incompatible with their photoreaction due to an increase in the excited state barrier for photorelease. We present a simple, remote sulfonation solution to BODIPY photocages that imparts water solubility and provides control over cellular permeability while retaining their favorable spectroscopic and photoreaction properties. Peripherally disulfonated BODIPY photocages are cell impermeable, making them useful for modulation of cell-surface receptors, while monosulfonated BODIPY retains the ability to cross the cellular membrane and can modulate intracellular targets. This new approach is generalizable for controlling BODIPY localization and was validated by sensitization of mammalian cells and neurons by visible-light photoactivation of signaling molecules.

Metabolic Chemical Reporters of Glycans Exhibit Cell-Type-Selective Metabolism and Glycoprotein Labeling.

Since the pioneering work by Reutter and co-workers that demonstrated structural flexibility in the carbohydrate biosynthesis and glycosylation pathways, many different labs have used unnatural monosaccharide analogues to perform glycan engineering on the surface of living cells. A subset of these unnatural monosaccharides contain bioorthogonal groups that enable the selective installation of visualization or enrichment tags. These metabolic chemical reporters (MCRs) have proven to be powerful for the unbiased identification of glycoproteins; however, they do have certain limitations. For example, they are incorporated substoichiometrically into glycans, and most MCRs are not selective for one class (e.g., O-GlcNAcylation) of glycoprotein. Here, we explore the relationship between the biosynthesis of MCR donor sugars in cells and the labeling levels of four different N-acetylglucosamine- and N-acetylgalactosamine-based MCRs. We found that the buildup of the different donor sugars correlated well with the overall labeling levels but less so with intracellular labeling of proteins by O-GlcNAcylation.

The Small Molecule 2-Azido-2-deoxy-glucose Is a Metabolic Chemical Reporter of O-GlcNAc Modifications in Mammalian Cells, Revealing an Unexpected Promiscuity of O-GlcNAc Transferase.

Glycans can be directly labeled using unnatural monosaccharide analogs, termed metabolic chemical reporters (MCRs). These compounds enable the secondary visualization and identification of glycoproteins by taking advantage of bioorthogonal reactions. Most widely used MCRs have azides or alkynes at the 2-N-acetyl position but are not selective for one class of glycoprotein over others. To address this limitation, we are exploring additional MCRs that have bioorthogonal functionality at other positions. Here, we report the characterization of 2-azido-2-deoxy-glucose (2AzGlc). We find that 2AzGlc selectively labels intracellular O-GlcNAc modifications, which further supports a somewhat unexpected, structural flexibility in this pathway. In contrast to the endogenous modification N-acetyl-glucosamine (GlcNAc), we find that 2AzGlc is not dynamically removed from protein substrates and that treatment with higher concentrations of per-acetylated 2AzGlc is toxic to cells. Finally, we demonstrate that this toxicity is an inherent property of the small-molecule, as removal of the 6-acetyl-group renders the corresponding reporter nontoxic but still results in protein labeling.