Future of the Search for Life: Workshop Report.

The 2-week, virtual Future of the Search for Life science and engineering workshop brought together more than 100 scientists, engineers, and technologists in March and April 2022 to provide their expert opinion on the interconnections between life-detection science and technology. Participants identified the advances in measurement and sampling technologies they believed to be necessary to perform in situ searches for life elsewhere in our Solar System, 20 years or more in the future. Among suggested measurements for these searches, those pertaining to three potential indicators of life termed "dynamic disequilibrium," "catalysis," and "informational polymers" were identified as particularly promising avenues for further exploration. For these three indicators, small breakout groups of participants identified measurement needs and knowledge gaps, along with corresponding constraints on sample handling (acquisition and processing) approaches for a variety of environments on Enceladus, Europa, Mars, and Titan. Despite the diversity of these environments, sample processing approaches all tend to be more complex than those that have been implemented on missions or envisioned for mission concepts to date. The approaches considered by workshop breakout groups progress from nondestructive to destructive measurement techniques, and most involve the need for fluid (especially liquid) sample processing. Sample processing needs were identified as technology gaps. These gaps include technology and associated sampling strategies that allow the preservation of the thermal, mechanical, and chemical integrity of the samples upon acquisition; and to optimize the sample information obtained by operating suites of instruments on common samples. Crucially, the interplay between science-driven life-detection strategies and their technological implementation highlights the need for an unprecedented level of payload integration and extensive collaboration between scientists and engineers, starting from concept formulation through mission deployment of life-detection instruments and sample processing systems.

Fluorescent indicators for imaging membrane potential of organelles.

Plasma membrane potential is a key driver of the physiology of excitable cells like neurons and cardiomyocytes. Voltage-sensitive fluorescent indicators offer a powerful complement to traditional electrode-based approaches to measuring and monitoring membrane potential. Intracellular organelles can also generate membrane potential, yet the electrode- and fluorescent indicator-based approaches used for plasma membrane potential imaging are difficult to implement on intact organelles in their native environment. Here, we survey recent advances in developing and deploying voltage-sensitive fluorescent indicators to interrogate organelle membrane potential in intact cells.

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.

Imaging Reversible Mitochondrial Membrane Potential Dynamics with a Masked Rhodamine Voltage Reporter.

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 (ΔΨm) 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 ΔΨm 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 ΔΨm dynamics. Here, we report the first example of a fluorescent ΔΨm reporter that does not rely on ΔΨm-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 ΔΨm. 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 ΔΨm 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 Ca2+, plasma membrane potential, and reversible ΔΨm dynamics.

Small Molecule-Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence.

We report a small-molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This quenching bioluminescent voltage indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human-induced pluripotent stem cells (hiPSCs), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule-protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.