Fluorescent indicators are indispensable imaging tools for visualizing the spatiotemporal dynamics of biological processes. Red fluorescent indicators are in particularly high demand because they offer compatibility with existing green fluorescent indicators or optogenetic tools, and longer wavelength fluorescence has inherent advantages for biological applications. We previously described a chemigenetic indicator design that combines a green fluorescent protein and a synthetic chelator in an effort to combine the advantages of conventional protein-based biosensors and synthetic chemosensors. We now demonstrate that this chemigenetic design can be extended to red fluorescent proteins. Through screening of variants with a range of chromophore-chelator orientations, followed by directed evolution, we developed a red fluorescent calcium ion (Ca2+) indicator with 5.4-fold fluorescence intensity change when going from 0 to 39 μM Ca2+ with purified proteins. Although the functionality of the current version is lost when expressed in mammalian cells and the selectivity is low, these results establish this chemigenetic design as a strategy that can be extended to other fluorescent protein color variants.

Genetically encoded calcium (Ca2+) indicators (GECIs) are essential tools for monitoring neuronal activity, but the performance of red fluorescent GECIs has remained limited. In particular, many red indicators are relatively dim, produce low signal-to-noise ratios and can undergo unwanted photoswitching when exposed to blue light, restricting their use in all-optical experiments that combine imaging with optogenetics or multicolor imaging. Here we show the development of PinkyCaMP, a Ca2+ sensor based on the bright red fluorescent protein mScarlet. PinkyCaMP exhibits markedly improved brightness, photostability and signal-to-noise ratio compared to existing red GECIs, while remaining fully compatible with blue-light-based optogenetic and dual-color imaging approaches. PinkyCaMP is well-tolerated by neurons, showing no detectable toxicity or aggregation, both in vitro and in vivo. PinkyCaMP enables a broad spectrum of imaging modalities, including single-photon methods, such as fiber photometry, widefield imaging and miniature microscopy imaging, as well as two-photon imaging in awake mice.

Abstract Citrate is a central intermediate metabolite linking the tricarboxylic acid cycle and lipid biosynthesis. Tools for monitoring of spatiotemporal citrate dynamics are critical for getting a better understanding of cellular metabolism. Here, we develope genetically encoded excitation ratiometric biosensors for citrate, based on our previous intensiometric green fluorescence protein-based citrate biosensor, Citron1. We find that a single mutation in the Citron1 chromophore-forming tripeptide provided an excitation ratiometric response. Further rounds of directed evolution yield highly responsive variants, exhibiting citrate-dependent fluorescence changes between two excitation peaks. When expressed in mammalian cells, these biosensors enable citrate dynamics to be monitored in both the cytosol and mitochondria. Comparative analysis across multiple human breast cancer cell lines uncovers cell line-specific differences in citrate levels and their heterogeneity, which could be linked to their malignancy. Furthermore, flow cytometry-based measurements in mouse embryonic stem cells demonstrate the proteomics signatures underlying the population-level variability in citrate concentrations and citrate rewiring during stem cell differentiation. Together, these results show that these excitation ratiometric citrate biosensors enable quantitative, compartment-resolved, and population-scale analysis of cellular metabolism.

The objective of this work was to overcome some of the long-standing limitations of ratiometric fluorescent protein (FP)-based Ca2+ biosensors, which typically rely on Förster resonance energy transfer (FRET) between two FPs and generally exhibit only relatively modest Ca2+-dependent changes in emission ratio. To develop biosensors with substantially greater ratiometric changes, we explored an alternative biosensor design strategy in which two independently optimized intensiometric single FP-based Ca2+ biosensors were hybridized into a single protein construct such that they employed a shared calmodulin (CaM) and CaM-binding peptide (CBP). By hybridizing a direct-response red FP-based biosensor with an inverse-response green FP-based biosensor, we created SuiCa, a single-polypeptide Ca2+ biosensor that exhibits exceptionally large red-to-green ratiometric fluorescence changes as purified protein (∼60-fold) and when expressed in immortalized cell cultures (∼80-fold) and primary neurons (∼37-fold). Relative to co-expression of two spectrally distinct FP-based Ca2+ biosensors, SuiCa provides the advantages of a smaller gene size, a fixed fluorophore stoichiometry, and a ratiometric response that depends on Ca2+ binding to a single, shared, CaM plus CBP domain. With these advantages, along with its bright fluorescence and large ratiometric change, SuiCa represents a new addition to the Ca2+ imaging toolbox.

Genetically encoded calcium indicators (GECIs) are vital tools for fluorescence-based visualization of neuronal activity with high spatial and temporal resolution. However, current highest-performance GECIs are predominantly green or red fluorescent, limiting multiplexing options and efficient excitation with fixed-wavelength femtosecond lasers operating at 1030 nm. In an effort to overcome these limitations, we developed OCaMP, an orange fluorescent GECI engineered from O-GECO1 through targeted substitutions to improve calcium affinity while retaining the favorable photophysical properties of mOrange2. OCaMP exhibits improved two-photon cross-section, responsiveness, photostability, and calcium affinity relative to O-GECO1. In cultured neurons, zebrafish, and mouse cortex, OCaMP outperforms the red GECIs jRCaMP1a and jRGECO1a in sensitivity, photostability, and signal-to-noise ratio. Here we show that OCaMP, an orange fluorescent GECI, is a robust tool for high-fidelity neural imaging optimized for wavelengths above 1000 nm and a practical option within the spectral gap between existing green and red GECIs.

Variable domains of new antigen receptors (VNARs) derived from shark immunoglobulin new antigen receptors (IgNARs) have gained attention as promising single-domain antibody modalities owing to their compact structures, structural stability, and unique binding capabilities. This study aimed to demonstrate the feasibility of screening a synthetic library based on VNARs derived from the brownbanded bamboo shark (Chiloscyllium punctatum) using a complementary DNA (cDNA) display system. A synthetic VNAR library was constructed by randomizing the CDR1 and CDR3 loops, resulting in a genetic diversity of approximately 1012 sequences in the cDNA display format. Using fluorescent proteins as model antigens, in vitro selection was performed, followed by next-generation sequencing and biochemical validation. VNAR-E1 and VNAR-N1 were identified as binding to EGFP and mNeonGreen, respectively. This work represents the first demonstration of applying a cDNA display system to VNAR selection.

Fluorescent proteins (FPs) that are pH-sensitive play a crucial role in investigating pH-related cellular processes, such as endocytosis and exocytosis. Existing pH-sensitive FPs generated from Aequorea victoria green fluorescent protein (GFP), such as superecliptic pHluorin (SEP) and Lime, have been widely employed to study these processes, but suffer from low photostability. Here, we report the development and characteristics of serapH, a genetically encodable pH biosensor with improved photostability compared to GFP analogues, which we generated using mStayGold as a scaffold. To aid in the development of serapH, we developed a method for screening pH-sensitive FP variants by directly evaluating both brightness and pH sensitivity in bacterial colonies on agar. This significantly increased the number of colonies that could be screened per round and reduced the time needed per round. The photostability of serapH should improve spatiotemporal resolution by increasing tolerance to higher excitation intensities and longer imaging durations, thereby expanding the range of applications of pH-sensitive FPs.

With this status report, we aim to provide a timely snapshot of the protein engineering field as a broad and rapidly advancing discipline that integrates computational, molecular biology, structure-guided, evolutionary, and synthetic approaches to create new and improved proteins with tailored structures and useful functions. The report is organized into eight thematic areas spanning core methodologies and major application domains, including enzymes, therapeutics, detection, synthetic biology, and materials. Contributions from experts across these areas highlight both the historical foundations and recent advances in their respective fields, with particular emphasis on the growing influence of machine learning and artificial intelligence-based methods. Emerging from this broad overview is a central message: protein engineering appears to be entering a golden age, defined by a rapidly accelerating pace of progress, even as significant challenges in design, screening, and real-world application remain. Looking ahead, the continued integration of computational and experimental strategies is poised to further accelerate the impact of protein engineering across an expanding range of economically and societally important sectors, from therapeutics and molecular imaging to diagnostics, plastic recycling, and industrial chemistry.

L-Lactate, once considered a metabolic waste product of glycolysis, is now recognized as a vitally important metabolite and signaling molecule in multiple biological pathways. However, exploring L-lactate's emerging intra- and extra-cellular roles is hindered by a lack of tools to perturb L-lactate concentration intracellularly and extracellularly. Photocaged compounds are a powerful way to introduce bioactive molecules with spatiotemporal precision using illumination. Here, we report the development of a photocaged derivative of L-lactate, 4-methoxy-7-nitroindolinyl-L-lactate (MNI-L-lac), that releases L-lactate upon illumination. We validated MNI-L-lac in cell culture by demonstrating that the photorelease of L-lactate elicits a response from genetically encoded extra- and intracellular L-lactate biosensors (eLACCO1, eLACCO2.1, R-iLACCO1.2). To demonstrate the utility of MNI-L-lac, we employed the photorelease of L-lactate to activate G protein-coupled receptor 81 (GPR81), as revealed by the inhibition of adenylyl cyclase activity and concomitant decrease of cAMP. These results indicate that MNI-L-lac may be useful for perturbing the concentration of endogenous L-lactate in order to investigate L-lactate's roles in metabolic and signaling pathways.

Recombinant optogenetic and chemogenetic proteins are potent tools for manipulating neuronal activity and controlling neural circuit function. However, there are few analogous tools for manipulating the structure of neural circuits. Here, we introduce three rationally designed genetically encoded tools that use E3 ligase-dependent mechanisms to trigger the degradation of synaptic scaffolding proteins, leading to functional ablation of synapses. First, we developed a constitutive excitatory synapse ablator, PFE3, analogous to the inhibitory synapse ablator GFE3. PFE3 targets the RING domain of the E3 ligase Mdm2 and the proteasome-interacting region of Protocadherin 10 to the scaffolding protein PSD-95, leading to efficient ablation of excitatory synapses. In addition, we developed a light-inducible version of GFE3, paGFE3, using a novel photoactivatable complex based on the photocleavable protein PhoCl2c. paGFE3 degrades Gephyrin and ablates inhibitory synapses in response to 400 nm light. Finally, we developed a chemically inducible version of GFE3, chGFE3, which degrades inhibitory synapses when combined with the bio-orthogonal dimerizer HaloTag ligand-trimethoprim. Each tool is specific, reversible, and capable of breaking neural circuits at precise locations.

Pyruvate is the end-product of glycolysis and a central metabolite involved in many biochemical pathways. However, a lack of high-performance (i.e., ΔF/F 0 > 10) single fluorescent protein (FP)-based biosensors has hindered efforts to investigate the physiological role of pyruvate. Here, we present the GreenPy1 and ApplePy1 series, which are green FP (GFP)-based and red FP (RFP)-based pyruvate biosensors, respectively. Both series exhibit large fluorescence intensity change (ΔF/F 0 ~ 20 to >40) and a range of affinities (10s of μM to several mM). We demonstrate the utility of these pyruvate biosensors for multicolor imaging of metabolite concentration changes in mammalian cells.

Self-complementing bipartite fluorescent proteins (FPs) are useful tools for the detection of protein-protein proximity and for localizing fluorophores to membrane-membrane contact sites. Here, we report versions of circularly permuted green FP (GFP), red FP (RFP), and mNeonGreen (NG), which are split into a large fragment composed of nine β-strands and a small fragment composed of two β-strands. In each case, the large and small fragments can associate in live cells to form the complete 11-stranded FP β-barrel. We further converted each of these three self-complementing FPs into bipartite calcium ion (Ca2+) biosensors. We demonstrate that appropriately targeted versions of these split FPs, and split FP-based biosensors, can be functionally assembled at membrane-membrane contact sites. We employ the bipartite NG-based Ca2+ biosensor for visualization of pharmacologically induced Ca2+ release at mitochondria-endoplasmic reticulum contact sites (MERCs).

Intense brain activity elevates extracellular potassium, potentially leading to overexcitation and seizures. Astrocytes are crucial for restoring healthy potassium levels, and an emerging focus on their Kir4.1 channels has reopened the quest into the underlying mechanisms. We find that the Kir4.1 level in individual astrocytes sets the kinetics of their potassium and glutamate uptake current. Combining electrophysiology with multiplexed optical sensor imaging and FLIM reveals that rises in extracellular potassium would normally boost presynaptic Ca2+ entry and release probability at excitatory synapses unless such synapses are surrounded by the Kir4.1-overexpressing astrocytes. Inside the territories of Kir4.1-overexpressing astrocytes, high-frequency afferent stimulation fails to induce long-term synaptic potentiation, and the high-potassium waves of cortical spreading depolarization are markedly attenuated. Biophysical exploration explains how astrocytes can regulate local potassium homeostasis by engaging Kir4.1 channels. Our findings thus point to a fundamental astrocytic mechanism that can restrain the activity-driven rise of excitability in brain circuits.

L-Lactate is increasingly recognized as an intercellular energy currency in mammals, but mysteries remain regarding the spatial and temporal dynamics of its release and uptake between cells via the extracellular environment. Here we introduce R-eLACCO2.1, a red fluorescent extracellular L-lactate biosensor that is superior to previously reported green fluorescent biosensors in in vivo sensitivity to increases in extracellular L-lactate and spectral orthogonality. R-eLACCO2.1 exhibits excellent fluorescence response in cultured cells, mouse brain slices, and live mice. R-eLACCO2.1 also serves as an effective fluorescence lifetime-based biosensor. Using R-eLACCO2.1, we monitor whisker stimulation and locomotion-induced changes in endogenous extracellular L-lactate in the somatosensory cortex of awake mice. To highlight the potential insights gained from in vivo measurements with R-eLACCO2.1, we perform dual-color imaging from the somatosensory cortex of actively locomoting mice. This enables us to simultaneously observe the neural activity, reported by a green fluorescent GCaMP calcium ion biosensor, and extracellular L-lactate. As the high-performance tool in the suite of extracellular L-lactate biosensors, R-eLACCO2.1 is ideally suited to delimit the emerging roles of L-lactate in mammalian metabolism.

Genetically encoded calcium ion (Ca 2+ ) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca 2+ dynamics and neuronal activities on a single cell level. Here we report the design and development of two new far-red fluorescent GECIs, FR-GECO1a and FR-GECO1c, based on the monomeric far-red fluorescent protein mKelly. We characterized these far-red GECIs as purified proteins and assessed their performance when expressed in cultured neurons. FR-GECOs have excitation and emission maxima at ~ 596 nm and ~ 644 nm, respectively, display large responses to Ca 2+ (Δ F / F 0 = 6 for FR-GECO1a, 18 for FR-GECO1c), and are bright under both one-photon and two-photon illumination. FR-GECOs also have high affinities (apparent K d = 29 nM for FR-GECO1a, 83 nM for FR-GECO1c) for Ca 2+ , and they enable sensitive and fast detection of single action potentials in neurons.

Genetically-encoded, fluorescent protein (FP)-based biosensors are powerful tools for imaging dynamic cellular activities. Directed evolution is a highly effective method for developing enhanced versions of FP-based biosensors, but the screening process is laborious and time-consuming. Mammalian cell-based screening with electrical stimulation methods has been successful in accurately selecting variants of biosensors for imaging neuronal activities. We introduce an automated mammalian cell screening platform utilizing a fluorescence microscope and a liquid dispenser to enable the screening of biosensor responsiveness to chemical stimulation. We demonstrated the effectiveness of this platform in improving the response of a red fluorescent biosensor for Ca2+, K-GECO, for detection of histamine-induced changes in Ca(2+ )concentration. This method should be applicable to any FP-based biosensor that responds to pharmacological treatment or other exogenous chemical stimulation, simplifying efforts to develop biosensors tailored for specific applications in diverse biological contexts.

We developed a system for optogenetic release of single molecules in live cells. We confined soluble and transmembrane proteins to the Golgi apparatus via a photocleavable protein and released them by short pulses of light. Our method allows for the controlled delivery of functional proteins to cytosol and plasma membrane in amounts compatible with single molecule imaging, greatly simplifying access to single molecule microscopy of any protein in live cells. Furthermore, we could reconstitute cellular functions such as ion conductance by delivering BK and VRAC ion channels to the plasma membrane. Finally, we could induce NF-kB signaling in T-Lymphoblasts stimulated by IL-1 by controlled release of a signaling protein that had been knocked-out in the same cells. We observed light induced formation of functional inflammatory signaling complexes that could trigger IKK phosphorylation in single cells. We thus developed an optogenetic method for the reconstitution and investigation of cellular function at the single molecule level.

Synthetic-based fluorescent chemosensors and protein-based fluorescent biosensors are two well-established classes of tools for visualizing and monitoring biological processes in living tissues. Chemigenetic sensors, created using a combination of both synthetic parts and protein parts, are an emerging class of tools that aims to combine the strengths, and overcome the drawbacks, of traditional chemosensors and biosensors. This review will survey the landscape of strategies used for fluorescent chemigenetic sensor design. These strategies include: attachment of synthetic elements to proteins using in vitro protein conjugation; attachment of synthetic elements to proteins using autonomous protein labeling; and translational incorporation of unnatural amino acids.

ABSTRACTSignificanceGenetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for monitoring intracellular Ca2+concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca2+concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities forin vitroandin vivofluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy.AimWe describe the development and applications of T-GECO1 – a high-performance blue-shifted GECI based on theClavularia sp.-derived mTFP1.ApproachWe used protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performancein vitrousing one-photon excitation in cultured rat hippocampal neurons,in vivousing one-photon excitation fiber photometry in mice, andex vivousing two-photon Ca2+imaging in hippocampal slices.ResultsThe Ca2+-bound state of T-GECO1 has an excitation peak maximum of 468 nm, an emission peak maximum of 500 nm, an extinction coefficient of 49,300 M-1cm-1, a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The Ca2+-dependent fluorescence increase is 15-fold and the apparentKdfor Ca2+is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant.ConclusionT-GECO1 is a high performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants.