Opsins are known as proteins that bind retinal to form light-sensing rhodopsins, which mediate diverse forms of photoreception and are widely used for optogenetic control of cellular activity. Yet many opsins lack canonical retinal-binding features or, in their native biological contexts, are expressed where chromophore availability is limited, raising the question whether retinal-free opsins are silent. Here we show that retinal-free ion-transporting microbial opsins are not inert but adopt intrinsic, light-independent functional states alongside their canonical light-driven activity. Using developmental phenotyping in transgenic Drosophila melanogaster, electrophysiology in Xenopus oocytes and mammalian cells, together with structural and computational analyses, we demonstrate robust light-independent ion conductance in multiple channel-type opsins and an ion-pumping opsin. Structural modeling and molecular dynamics simulations reveal conformational rearrangements in transmembrane helices and internal water networks that are consistent with stabilization of apo-conductive states. Together, these findings establish apo-opsin activity as a defined functional state of microbial opsins, expand the current framework of rhodopsin biology, and highlight the need to consider chromophore-independent activity in both basic research and optogenetic applications.

Chemically inducible expression systems enable transgene expression regulation in response to external small molecules. Tetracycline repressor (TetR)-based gene switches work in plants, but antibiotics are neither approved nor advisable for crop use. Here we report engineering of TetR mutants that respond to approved sulfonylurea (SU) herbicides instead of antibiotics. Designed variants show low-nanomolar EC values for ethametsulfuron-methyl (Es) or chlorsulfuron and tightly bind the Tet operator sequence, but only in the absence of corresponding SUs. Crystal structures of two repressors in complex with their respective SU ligands reveal extensive interactions explaining their strong binding. The Es repressor-based gene switch is introduced into tobacco, soybean, maize, rice, and Arabidopsis, and robust reporter gene activation is observed upon herbicide application. Addition of a repressor-regulated siRNA targeting the repressor transcript increases the magnitude and spatial distribution of the response following herbicide treatment and results in a partially bistable gene switch. The SU repressors also function well in mammalian cell culture and may enable regulation of additional genes in conjunction with TetR.

From insects to mammals, essential brain functions, such as forming long-term memories (LTMs), increase metabolic activity in stimulated neurons to meet the energetic demand associated with brain activation. However, while impairing neuronal metabolism limits brain performance, whether expanding the metabolic capacity of neurons boosts brain function remains poorly understood. Here, we show that LTM formation of flies and mice can be enhanced by increasing mitochondrial metabolism in central memory circuits. By knocking down the mitochondrial Ca exporter Letm1, we favour Ca retention in the mitochondrial matrix of neurons due to reduction of mitochondrial H/Ca exchange. The resulting increase in mitochondrial Ca over-activates mitochondrial metabolism in neurons of central memory circuits, leading to improved LTM storage in training paradigms in which wild-type counterparts of both species fail to remember. Our findings unveil an evolutionarily conserved mechanism that controls mitochondrial metabolism in neurons and indicate its involvement in shaping higher brain functions, such as LTM.

Creating artificial organelles that sequester and release specific proteins in response to a small molecule in mammalian cells is an attractive approach for regulating protein function. In this work, by combining phase-separated condensates formed by the tandem fusion of two oligomeric proteins with a trimethoprim (TMP)-responsive nanobody switch for GFP (LAMA; ligand-modulated antibody fragment), we developed a synthetic condensate system that initially sequesters GFP-tagged proteins within condensates and rapidly releases them into the cytoplasm upon TMP treatment. The released proteins can then be resequestered by washing out the TMP. This system enabled user-defined, temporal, rapid, and reversible control of cellular processes, including membrane ruffling mediated by exogenously expressed GFP-Vav2 and modulation of the cellular localization of endogenous ERK2-GFP generated by genome knock-in. Our results highlight the utility of the LAMA-based synthetic condensate platform as a novel, chemically switchable tool for regulating protein function through controlled protein sequestration and release in mammalian cells.

The use of fluorescent sensors for functional imaging has revolutionized the study of organellar Ca2+ signaling. However, understanding the dynamic interplay between intracellular Ca2+ sinks and sources has been hindered by the lack of bright, photostable, and multiplexed measurements in different organelles, limiting our ability to define how Ca2+ shapes cell physiology across fields of biology. Here we introduce a new toolkit of chemigenetic organellar Ca2+ indicators whose color is tunable by reconstituting their fluorescence with different exogenous rhodamine dye-ligands, which significantly expand the capacity for multiplexing organellar Ca2+ measurements. These sensors, which we named ER-HaloCaMP and Mito-HaloCaMP, are optimized to report Ca2+dynamics in the endoplasmic reticulum (ER) and mitochondria of mammalian cells and neurons, and show significantly improved brightness, photostability and responsiveness when compared to current best-in-class alternatives. Using either red or far-red dye-ligands, both ER-HaloCaMP and Mito-HaloCaMP enable visualizing ER and mitochondrial Ca2+ dynamics in neuronal axons, a subcellular location that only contains a few ER tubules and small mitochondria, structural limitations that have impaired measurements with previous red sensors. To show the expanded multiplexing capacities of our toolkit, we measured interorganellar Ca2+ fluxes simultaneously in three different subcellular compartments in live cells, revealing that the amplitude of ER Ca2+release controls the efficacy of ER-mitochondria Ca2+ coupling in a cooperative manner. Organellar HaloCaMPs enable also measuring Ca2+ dynamics in intact brain tissue from flies and rodents, demonstrating their versatility across biological models. Our new toolkit provides an expanded palette of bright, photostable and responsive organellar Ca2+ sensors, which will facilitate future studies of intracellular Ca2+ signaling across fields of biology in health and disease.

Synchronous neuronal ensembles play a pivotal role in the consolidation of long-term memory in the hippocampus. However, their organization during the acquisition of spatial memory remains less clear. In this study, we used neuronal population voltage imaging to investigate the synchronization patterns of CA1 pyramidal neuronal ensembles during the exploration of a new environment, a critical phase for spatial memory acquisition. We found synchronous ensembles comprising approximately 40% of CA1 pyramidal neurons, firing simultaneously in brief windows (∼25ms) during immobility and locomotion in novel exploration. Notably, these synchronous ensembles were not associated with contralateral ripple oscillations but were instead phase-locked to theta waves recorded in the contralateral CA1 region. Moreover, the subthreshold membrane potentials of neurons exhibited coherent intracellular theta oscillations with a depolarizing peak at the moment of synchrony. Among newly formed place cells, pairs with more robust synchronization during locomotion displayed more distinct place-specific activities. These findings underscore the role of synchronous ensembles in coordinating place cells of different place fields.

Neurochemical signals like dopamine (DA) play a crucial role in a variety of brain functions through intricate interactions with other neuromodulators and intracellular signaling pathways. However, studying these complex networks has been hindered by the challenge of detecting multiple neurochemicals in vivo simultaneously. To overcome this limitation, we developed a single-protein chemigenetic DA sensor, HaloDA1.0, which combines a cpHaloTag-chemical dye approach with the G protein-coupled receptor activation-based (GRAB) strategy, providing high sensitivity for DA, sub-second response kinetics, and an extensive spectral range from far-red to near-infrared. When used together with existing green and red fluorescent neuromodulator sensors, Ca2+ indicators, cAMP sensors, and optogenetic tools, HaloDA1.0 provides high versatility for multiplex imaging in cultured neurons, brain slices, and behaving animals, facilitating in-depth studies of dynamic neurochemical networks.

Synchronous neuronal ensembles play a pivotal role in the consolidation of long-term memory in the hippocampus. However, their organization during the acquisition of spatial memory remains less clear. In this study, we used neuronal population voltage imaging to investigate the synchronization patterns of CA1 pyramidal neuronal ensembles during the exploration of a new environment, a critical phase for spatial memory acquisition. We found synchronous ensembles comprising approximately 40% of CA1 pyramidal neurons, firing simultaneously in brief windows (∼25ms) during immobility and locomotion in novel exploration. Notably, these synchronous ensembles were not associated with ripple oscillations but were instead phase-locked to local field potential theta waves. Specifically, the subthreshold membrane potentials of neurons exhibited coherent theta oscillations with a depolarizing peak at the moment of synchrony. Among newly formed place cells, pairs with more robust synchronization during locomotion displayed more distinct place-specific activities. These findings underscore the role of synchronous ensembles in coordinating place cells of different place fields.

Genetically encoded voltage indicators (GEVIs) allow optical recording of membrane potential from targeted cells in vivo. However, red GEVIs that are compatible with two-photon microscopy and that can be multiplexed in vivo with green reporters like GCaMP, are currently lacking. To address this gap, we explored diverse rhodopsin proteins as GEVIs and engineered a novel GEVI, 2Photron, based on a rhodopsin from the green algae Klebsormidium nitens. 2Photron, combined with two photon ultrafast local volume excitation (ULoVE), enabled multiplexed readout of spiking and subthreshold voltage simultaneously with GCaMP calcium signals in visual cortical neurons of awake, behaving mice. These recordings revealed the cell-specific relationship of spiking and subthreshold voltage dynamics with GCaMP responses, highlighting the challenges of extracting underlying spike trains from calcium imaging.

Ionic driving forces provide the net electromotive force for ion movement across receptors, channels, and transporters, and are a fundamental property of all cells. In the nervous system, fast synaptic inhibition is mediated by chloride permeable GABA and glycine receptors, and single-cell intracellular recordings have been the only method for estimating driving forces across these receptors (DF). Here we present a tool for quantifying inhibitory receptor driving force named ORCHID: all-Optical Reporting of CHloride Ion Driving force. We demonstrate ORCHID's ability to provide accurate, high-throughput measurements of resting and dynamic DF from genetically targeted cell types over multiple timescales. ORCHID confirms theoretical predictions about the biophysical mechanisms that establish DF, reveals differences in DF between neurons and astrocytes, and affords the first in vivo measurements of intact DF. This work extends our understanding of inhibitory synaptic transmission and demonstrates the potential for all-optical methods to assess ionic driving forces.