Engineered HaloTag variants for fluorescence lifetime multiplexing.

Self-labeling protein tags such as HaloTag are powerful tools that can label fusion proteins with synthetic fluorophores for use in fluorescence microscopy. Here we introduce HaloTag variants with either increased or decreased brightness and fluorescence lifetime compared with HaloTag7 when labeled with rhodamines. Combining these HaloTag variants enabled live-cell fluorescence lifetime multiplexing of three cellular targets in one spectral channel using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. Additionally, the brightest HaloTag variant showed up to 40% higher brightness in live-cell imaging applications.

Probing coenzyme A homeostasis with semisynthetic biosensors.

Coenzyme A (CoA) is one of the central cofactors of metabolism, yet a method for measuring its concentration in living cells is missing. Here we introduce the first biosensor for measuring CoA levels in different organelles of mammalian cells. The semisynthetic biosensor is generated through the specific labeling of an engineered GFP-HaloTag fusion protein with a fluorescent ligand. Its readout is based on CoA-dependent changes in Förster resonance energy transfer efficiency between GFP and the fluorescent ligand. Using this biosensor, we probe the role of numerous proteins involved in CoA biosynthesis and transport in mammalian cells. On the basis of these studies, we propose a cellular map of CoA biosynthesis that suggests how pools of cytosolic and mitochondrial CoA are maintained.

A general method for the development of multicolor biosensors with large dynamic ranges.

Fluorescent biosensors enable the study of cell physiology with spatiotemporal resolution; yet, most biosensors suffer from relatively low dynamic ranges. Here, we introduce a family of designed Förster resonance energy transfer (FRET) pairs with near-quantitative FRET efficiencies based on the reversible interaction of fluorescent proteins with a fluorescently labeled HaloTag. These FRET pairs enabled the straightforward design of biosensors for calcium, ATP and NAD+ with unprecedented dynamic ranges. The color of each of these biosensors can be readily tuned by changing either the fluorescent protein or the synthetic fluorophore, which enables simultaneous monitoring of free NAD+ in different subcellular compartments following genotoxic stress. Minimal modifications of these biosensors furthermore allow their readout to be switched to fluorescence intensity, fluorescence lifetime or bioluminescence. These FRET pairs thus establish a new concept for the development of highly sensitive and tunable biosensors.

Exchangeable HaloTag Ligands for Super-Resolution Fluorescence Microscopy.

The specific and covalent labeling of the protein HaloTag with fluorescent probes in living cells makes it a powerful tool for bioimaging. However, the irreversible attachment of the probe to HaloTag precludes imaging applications that require transient binding of the probe and comes with the risk of irreversible photobleaching. Here, we introduce exchangeable ligands for fluorescence labeling of HaloTag (xHTLs) that reversibly bind to HaloTag and that can be coupled to rhodamines of different colors. In stimulated emission depletion (STED) microscopy, probe exchange of xHTLs allows imaging with reduced photobleaching as compared to covalent HaloTag labeling. Transient binding of fluorogenic xHTLs to HaloTag fusion proteins enables points accumulation for imaging in nanoscale topography (PAINT) and MINFLUX microscopy. We furthermore introduce pairs of xHTLs and HaloTag mutants for dual-color PAINT and STED microscopy. xHTLs thus open up new possibilities in imaging across microscopy platforms for a widely used labeling approach.

Chemogenetic Control of Nanobodies.

We introduce an engineered nanobody whose affinity to green fluorescent protein (GFP) can be switched on and off with small molecules. By controlling the cellular localization of GFP fusion proteins, the engineered nanobody allows interrogation of their roles in basic biological processes, an approach that should be applicable to numerous previously described GFP fusions. We also outline how the binding affinities of other nanobodies can be controlled by small molecules.

Fluorescent and Bioluminescent Calcium Indicators with Tuneable Colors and Affinities.

We introduce a family of bright, rhodamine-based calcium indicators with tuneable affinities and colors. The indicators can be specifically localized to different cellular compartments and are compatible with both fluorescence and bioluminescence readouts through conjugation to HaloTag fusion proteins. Importantly, their increase in fluorescence upon localization enables no-wash live-cell imaging, which greatly facilitates their use in biological assays. Applications as fluorescent indicators in rat hippocampal neurons include the detection of single action potentials and of calcium fluxes in the endoplasmic reticulum. Applications as bioluminescent indicators include the recording of the pharmacological modulation of nuclear calcium in high-throughput compatible assays. The versatility and remarkable ease of use of these indicators make them powerful tools for bioimaging and bioassays.

Kinetic and Structural Characterization of the Self-Labeling Protein Tags HaloTag7, SNAP-tag, and CLIP-tag.

The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag, and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study of the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag, and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rate constants with certain rhodamine substrates, which are more than 2 orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rate constants, however, are less affected by the structure of the label than those of HaloTag7, which vary over 6 orders of magnitude for commonly employed substrates. Determining the crystal structures of HaloTag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics.

Environmentally Sensitive Color-Shifting Fluorophores for Bioimaging.

We introduce color-shifting fluorophores that reversibly switch between a green and red fluorescent form through intramolecular spirocyclization. The equilibrium of the spirocyclization is environmentally sensitive and can be directly measured by determining the ratio of red to green fluorescence, thereby enabling the generation of ratiometric fluorescent probes and biosensors. Specifically, we developed a ratiometric biosensor for imaging calcium ions (Ca2+ ) in living cells, ratiometric probes for different proteins, and a bioassay for the quantification of nicotinamide adenine dinucleotide phosphate.

Imaging and manipulating proteins in live cells through covalent labeling.

The past 20 years have witnessed the advent of numerous technologies to specifically and covalently label proteins in cellulo and in vivo with synthetic probes. These technologies range from self-labeling proteins tags to non-natural amino acids, and the question is no longer how we can specifically label a given protein but rather with what additional functionality we wish to equip it. In addition, progress in fields such as super-resolution microscopy and genome editing have either provided additional motivation to label proteins with advanced synthetic probes or removed some of the difficulties of conducting such experiments. By focusing on two particular applications, live-cell imaging and the generation of reversible protein switches, we outline the opportunities and challenges of the field and how the synergy between synthetic chemistry and protein engineering will make it possible to conduct experiments that are not feasible with conventional approaches.

Luciferases with Tunable Emission Wavelengths.

We introduce luciferases whose emission maxima can be tuned to different wavelengths by chemical labeling. The luciferases are chimeras of NanoLuc with either SNAP-tag or HaloTag7. Labeling of the self-labeling tag with a fluorophore shifts the emission maximum of NanoLuc to that of the fluorophore. Luciferases with tunable colors have applications as reporter genes, for the construction of biosensors and in bioimaging.

A New Inovirus from the Human Blood Encodes Proteins with Nuclear Subcellular Localization.

Viruses infecting bacteria (bacteriophages) represent the most abundant viral particles in the human body. They participate in the control of the human-associated bacterial communities and play an important role in the dissemination of virulence genes. Here, we present the identification of a new filamentous single-stranded DNA phage of the family Inoviridae, named Ralstonia Inoviridae Phage 1 (RIP1), in the human blood. Metagenomics and PCR analyses detected the RIP1 genome in blood serum, in the absence of concomitant bacterial infection or contamination, suggesting inovirus persistence in the human blood. Finally, we have experimentally demonstrated that the RIP1-encoded rolling circle replication initiation protein and serine integrase have functional nuclear localization signals and upon expression in eukaryotic cells both proteins were translocated into the nucleus. This observation adds to the growing body of data suggesting that phages could have an overlooked impact on the evolution of eukaryotic cells.

AspSnFR: A genetically encoded biosensor for real-time monitoring of aspartate in live cells.

Aspartate is crucial for nucleotide synthesis, ammonia detoxification, and maintaining redox balance via the malate-aspartate-shuttle (MAS). To disentangle these multiple roles of aspartate metabolism, tools are required that measure aspartate concentrations in real time and in live cells. We introduce AspSnFR, a genetically encoded green fluorescent biosensor for intracellular aspartate, engineered through displaying and screening biosensor libraries on mammalian cells. In live cells, AspSnFR is able to precisely and quantitatively measure cytosolic aspartate concentrations and dissect its production from glutamine. Combining high-content imaging of AspSnFR with pharmacological perturbations exposes differences in metabolic vulnerabilities of aspartate levels based on nutrient availability. Further, AspSnFR facilitates tracking of aspartate export from mitochondria through SLC25A12, the MAS' key transporter. We show that SLC25A12 is a rapidly responding and direct route to couple Ca2+ signaling with mitochondrial aspartate export. This establishes SLC25A12 as a crucial link between cellular signaling, mitochondrial respiration, and metabolism.

Recording physiological history of cells with chemical labeling.

Recordings of the physiological history of cells provide insights into biological processes, yet obtaining such recordings is a challenge. To address this, we introduce a method to record transient cellular events for later analysis. We designed proteins that become labeled in the presence of both a specific cellular activity and a fluorescent substrate. The recording period is set by the presence of the substrate, whereas the cellular activity controls the degree of the labeling. The use of distinguishable substrates enabled the recording of successive periods of activity. We recorded protein-protein interactions, G protein-coupled receptor activation, and increases in intracellular calcium. Recordings of elevated calcium levels allowed selections of cells from heterogeneous populations for transcriptomic analysis and tracking of neuronal activities in flies and zebrafish.

A clinically applicable connectivity signature for glioblastoma includes the tumor network driver CHI3L1.

Tumor microtubes (TMs) connect glioma cells to a network with considerable relevance for tumor progression and therapy resistance. However, the determination of TM-interconnectivity in individual tumors is challenging and the impact on patient survival unresolved. Here, we establish a connectivity signature from single-cell RNA-sequenced (scRNA-Seq) xenografted primary glioblastoma (GB) cells using a dye uptake methodology, and validate it with recording of cellular calcium epochs and clinical correlations. Astrocyte-like and mesenchymal-like GB cells have the highest connectivity signature scores in scRNA-sequenced patient-derived xenografts and patient samples. In large GB cohorts, TM-network connectivity correlates with the mesenchymal subtype and dismal patient survival. CHI3L1 gene expression serves as a robust molecular marker of connectivity and functionally influences TM networks. The connectivity signature allows insights into brain tumor biology, provides a proof-of-principle that tumor cell TM-connectivity is relevant for patients' prognosis, and serves as a robust prognostic biomarker.

Crystallization and preliminary X-ray diffraction analysis of the organophosphorus hydrolase OPHC2 from Pseudomonas pseudoalcaligenes.

Enzymes that are capable of degrading neurotoxic organophosphorus compounds are of increasing interest because of the lack of efficient and clean methods for their removal. Recently, a novel organophosphorus hydrolase belonging to the metallo-ß-lactamase superfamily was identified and isolated from the mesophilic bacterium Pseudomonas pseudoalcaligenes. This enzyme, named OPHC2, is endowed with significant thermal and pH stability, making it an appealing candidate for engineering studies to develop an efficient organophosphorus biodecontaminant. Combined with biochemical studies, structural information will help decipher the catalytic mechanism of organophosphorus hydrolysis by OPHC2 and identify the residues involved in its substrate specificity. Here, the expression, purification, crystallization and X-ray data collection at 2.1 Šresolution of OPHC2 are presented.

Crystallization and preliminary X-ray diffraction analysis of the lactonase VmoLac from Vulcanisaeta moutnovskia.

Phosphotriesterase-like lactonases (PLLs) are native lactonases that are capable of hydrolyzing lactones such as aliphatic lactones or acyl-homoserine lactones, which are involved in bacterial quorum sensing. Previously characterized PLLs are moreover endowed with a promiscuous phosphotriesterase activity and are therefore able to detoxify organophosphate insecticides. A novel PLL representative, dubbed VmoLac, has been identified from the hyperthermophilic crenarchaeon Vulcanisaeta moutnovskia. Because of its intrinsic high thermal stability, VmoLac may constitute an appealing candidate for engineering studies with the aim of producing an efficient biodecontaminant for organophosphorus compounds and a bacterial antivirulence agent. In combination with biochemical studies, structural information will allow the identification of the residues involved in substrate specificity and an understanding of the enzymatic catalytic mechanisms. Here, the expression, purification, crystallization and X-ray data collection at 2.4 Šresolution of VmoLac are reported.

[DING proteins, their biochemical and structural properties, and their ability to inhibit HIV-1 replication]. / Les protéines DING: propriétés biochimiques, structurales, et capacité a inhiber la réplication du virus VIH-1.

DING proteins comprise an intriguing phosphate-binding protein family present in all animal phyla. Five different DING representatives have been described in humans. Eukaryotic DING proteins are mostly involved in cellular processes such as cell cycle regulation, and also in pathological process such as rheumatoid arthritis and kidney stone formation. Although these proteins are ubiquitous in eukaryotes, no relevant locus or ORF has yet been found in sequenced genomes. This lack of sequence information has considerably hampered functional and structural studies of these proteins, and has required the use of novel and original techniques such as ab initio protein sequencing based on a combination of X-ray crystallography and mass spectrometry. Sub-Angstrom structural resolution has elucidated the molecular binding mechanism of phosphate ions by these high-affinity proteins. Immunohistochemical studies show that these proteins are present in a wide variety of mouse tissues. Some DING proteins, particularly human phosphate binding protein (HPBP), can inhibit HIV replication. This inhibition takes place at the transcriptional step, which is not targeted by any current antiretroviral drug. Initial studies suggest that HPBP warrants animal testing. This recent discovery opens new possibilities for the treatment of HIV infection.

Live-Cell Fluorescence Lifetime Multiplexing Using Synthetic Fluorescent Probes.

Fluorescence lifetime multiplexing requires fluorescent probes with distinct fluorescence lifetimes but similar spectral properties. Even though synthetic probes for many cellular targets are available for multicolor live-cell fluorescence microscopy, few of them have been characterized for their use in fluorescence lifetime multiplexing. Here, we demonstrate that, from a panel of 18 synthetic probes, eight pairwise combinations are suitable for fluorescence lifetime multiplexing in living mammalian cell lines. Moreover, combining multiple pairs in different spectral channels enables us to image four and with the help of self-labeling protein tags up to eight different biological targets, effectively doubling the number of observable targets. The combination of synthetic probes with fluorescence lifetime multiplexing is thus a powerful approach for live-cell imaging.

Synergizing Exchangeable Fluorophore Labels for Multitarget STED Microscopy.

Investigating the interplay of cellular proteins with optical microscopy requires multitarget labeling. Spectral multiplexing using high-affinity or covalent labels is limited in the number of fluorophores that can be discriminated in a single imaging experiment. Advanced microscopy methods such as STED microscopy additionally demand balanced excitation, depletion, and emission wavelengths for all fluorophores, further reducing multiplexing capabilities. Noncovalent, weak-affinity labels bypass this "spectral barrier" through label exchange and sequential imaging of different targets. Here, we combine exchangeable HaloTag ligands, weak-affinity DNA hybridization, and hydrophophic and protein-peptide interactions to increase labeling flexibility and demonstrate six-target STED microscopy in single cells. We further show that exchangeable labels reduce photobleaching as well as facilitate long acquisition times and multicolor live-cell and high-fidelity 3D STED microscopy. The synergy of different types of exchangeable labels increases the multiplexing capabilities in fluorescence microscopy, and by that, the information content of microscopy images.

Crystallization and preliminary X-ray diffraction analysis of the hyperthermophilic Sulfolobus islandicus lactonase.

Phosphotriesterase-like lactonases (PLLs) constitute an interesting family of enzymes that are of paramount interest in biotechnology with respect to their catalytic functions. As natural lactonases, they may act against pathogens such as Pseudomonas aeruginosa by shutting down their quorum-sensing system (quorum quenching) and thus decreasing pathogen virulence. Owing to their promiscuous phosphotriesterase activity, which can inactivate toxic organophosphorus compounds such as pesticides and nerve agents, they are equally appealing as potent bioscavengers. A new representative of the PLL family has been identified (SisPox) and its gene was cloned from the hyperthermophilic archeon Sulfolobus islandicus. Owing to its hyperthermostable architecture, SisPox appears to be a good candidate for engineering studies. Here, production, purification, crystallization conditions and data collection to 2.34 Šresolution are reported for this lactonase from the hyperthermophilic S. islandicus.