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The ability to label proteins by fusion with genetically encoded fluorescent proteins is a powerful tool for understanding dynamic biological processes. However, current approaches for expressing fluorescent protein fusions possess drawbacks, especially at the whole organism level. Expression by transgenesis risks potential overexpression artifacts while fluorescent protein insertion at endogenous loci is technically difficult and, more importantly, does not allow for tissue-specific study of broadly expressed proteins. To overcome these limitations, we have adopted the split fluorescent protein system mNeonGreen21-10/11 (split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG21-10 is expressed under a tissue-specific promoter using standard transgenesis while mNG211 is inserted into protein-coding genes of interest using CRISPR/Cas-directed gene editing. Each mNG2 fragment on its own is not fluorescent, but when co-expressed the fragments self-assemble into a fluorescent complex. Here, we report successful use of split-mNG2 to achieve differential labeling of the cytoskeleton genes tubb4b and krt8 in various tissues. We also demonstrate that by anchoring the mNG21-10 component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein localization. Our approach should be broadly useful for a wide range of applications.

We describe a binary expression aleatory mosaic (BEAM) system, which relies on DNA delivery by transfection or viral transduction along with nested recombinase activity to generate two genetically distinct, non-overlapping populations of cells for comparative analysis. Control cells labeled with red fluorescent protein (RFP) can be directly compared with experimental cells manipulated by genetic gain or loss of function and labeled with GFP. Importantly, BEAM incorporates recombinase-dependent signal amplification and delayed reporter expression to enable sharper delineation of control and experimental cells and to improve reliability relative to existing methods. We applied BEAM to a variety of known phenotypes to illustrate its advantages for identifying temporally or spatially aberrant phenotypes, for revealing changes in cell proliferation or death, and for controlling for procedural variability. In addition, we used BEAM to test the cortical protomap hypothesis at the individual radial unit level, revealing that area identity is cell autonomously specified in adjacent radial units.

Tobacco mild green mosaic virus (TMGMV)-like nanocarriers were designed for gene delivery to plant cells. High aspect ratio TMGMVs were coated with a polycationic biopolymer, poly(allylamine) hydrochloride (PAH), to generate highly charged nanomaterials (TMGMV-PAH; 56.20 ± 4.7 mV) that efficiently load (1:6 TMGMV:DNA mass ratio) and deliver single-stranded and plasmid DNA to plant cells. The TMGMV-PAH were taken up through energy-independent mechanisms in Arabidopsis protoplasts. TMGMV-PAH delivered a plasmid DNA encoding a green fluorescent protein (GFP) to the protoplast nucleus (70% viability), as evidenced by GFP expression using confocal microscopy and Western blot analysis. TMGMV-PAH were inactivated (iTMGMV-PAH) using UV cross-linking to prevent systemic infection in intact plants. Inactivated iTMGMV-PAH-mediated pDNA delivery and gene expression of GFP in vivo was determined using confocal microscopy and RT-qPCR. Virus-like nanocarrier-mediated gene delivery can act as a facile and biocompatible tool for advancing genetic engineering in plants.

Synthetic biology constitutes a scientific domain focused on intentional redesign of organisms to confer novel functionalities or create new products through strategic engineering of their genetic makeup. Leveraging the inherent capabilities of nature, one may address challenges across diverse sectors including medicine. Inspired by this concept, we have developed an innovative bioengineering platform, enabling high-yield and large-scale production of biological small interfering RNA (BioRNA/siRNA) agents via bacterial fermentation. Herein, we show that with the use of a new tRNA fused pre-miRNA carrier, we can produce various forms of BioRNA/siRNA agents within living host cells. We report a high-level overexpression of nine target BioRNA/siRNA molecules at 100% success rate, yielding 3-10 mg of BioRNA/siRNA per 0.25 L of bacterial culture with high purity (>98%) and low endotoxin (

The polymerization of myosin molecules into thick filaments in muscle sarcomeres is essential for cardiac contractility, with the attenuation of interactions between the heads of myosin molecules within the filaments being proposed to result in hypercontractility, as observed in hypertrophic cardiomyopathy (HCM). However, experimental evidence demonstrates that the structure of these giant macromolecular complexes is highly dynamic, with molecules exchanging between the filaments and a pool of soluble molecules on the minute timescale. Therefore, we sought to test the hypothesis that the enhancement of interactions between the heads of myosin molecules within thick filaments limits the mobility of myosin by taking advantage of mavacamten, a small molecule approved for the treatment of HCM. Myosin molecules were labeled in vivo with a green fluorescent protein (GFP) and imaged in intact hearts using multiphoton microscopy. Treatment of the intact hearts with mavacamten resulted in an unexpected > 5-fold enhancement in GFP-myosin mobility within the sarcomere. In vitro biochemical assays suggested that mavacamten enhanced the mobility of GFP-myosin by increasing the solubility of myosin molecules, through the stabilization of a compact/folded conformation of the molecules, once disassociated from the thick filaments. These findings provide alternative insight into the mechanisms by which molecules exchange into and out of thick filaments and have implications for how mavacamten may affect cardiac contractility.

Fluorescent proteins (FPs) are ubiquitous tools in research, yet their endogenous functions in nature are poorly understood. In this work, we describe a combination of functions for FPs in a clade of intertidal sea anemones whose FPs control a genetic color polymorphism together with the ability to combat oxidative stress. Focusing on the underlying genetics of a fluorescent green "Neon" color morph, we show that allelic differences in a single FP gene generate its strong and vibrant color, by increasing both molecular brightness and FP gene expression level. Natural variation in FP sequences also produces differences in antioxidant capacity. We demonstrate that these FPs are strong antioxidants that can protect live cells against oxidative stress. Finally, based on structural modeling of the responsible amino acids, we propose a model for FP antioxidant function that is driven by molecular surface charge. Together, our findings shed light on the multifaceted functions that can co-occur within a single FP and provide a framework for studying the evolution of fluorescence as it balances spectral and physiological functions in nature.

The field of hybrid engineered living materials seeks to pair living organisms with synthetic materials to generate biocomposite materials with augmented function since living systems can provide highly-programmable and complex behavior. Engineered living materials have typically been fabricated using techniques in benign aqueous environments, limiting their application. In this work, biocomposite fabrication is demonstrated in which spores from polymer-degrading bacteria are incorporated into a thermoplastic polyurethane using high-temperature melt extrusion. Bacteria are engineered using adaptive laboratory evolution to improve their heat tolerance to ensure nearly complete cell survivability during manufacturing at 135 °C. Furthermore, the overall tensile properties of spore-filled thermoplastic polyurethanes are substantially improved, resulting in a significant improvement in toughness. The biocomposites facilitate disintegration in compost in the absence of a microbe-rich environment. Finally, embedded spores demonstrate a rationally programmed function, expressing green fluorescent protein. This research provides a scalable method to fabricate advanced biocomposite materials in industrially-compatible processes.

Introduction Peripheral nerve injury (PNI) occurs in approximately 3% of all trauma patients and can be challenging to treat, particularly when injury is severe such as with a long-segmental gap. Although peripheral nerves can regenerate after injury, functional recovery is often insufficient, leading to deficits in the quality of life of patients with PNI. Although nerve autografts are the gold standard of care, there are several disadvantages to their use, namely a lack of autologous nerve material for repair. This has led to the pursuit of alternative treatment methods such as axon guidance channels (AGCs). Second-generation AGCs have been shown to be able to deliver growth-enhancing substrates for nerve repair directly to the injury site. Although our laboratory has had success with second-generation AGCs filled with Schwann cells (SCs), SCs have their own set of issues clinically. Because of this, we have begun to utilize SC-derived exosomes as an alternative, as they have the appropriate protein markers, associate to axons in high concentrations, and are able to improve nerve regeneration. However, it is unknown how SC-derived exosomes may react within second-generation AGCs; thus, the aim of the present study was to assess the ability of SC-derived exosomes to be loaded into a second-generation AGC and how they would distribute within it. Materials and Methods A total of 4 dry second-generation AGCs were loaded with SC-derived exosomes that were derived from green fluorescent protein (GFP)-labeled SCs. They were subsequently frozen and sliced before imaging. Results Here, we present findings that SC-derived exosomes can be loaded into second-generation AGCs through our established loading method utilizing negative pressure and are able to survive and equally distribute along the length of the AGC. Conclusions Although only 4 second-generation AGCs were utilized, these findings indicate a potential use for SC-derived exosomes within second-generation AGCs to treat severe PNI. Future research should focus on exploring this in greater detail and in different contexts to assess the ability of SC-derived exosomes to survive at the site of injury and treat PNI. [ABSTRACT FROM AUTHOR]

Disruption of either the auxin transporter PIN-FORMED 1 (PIN1) or the protein kinase PINOID (PID) leads to the development of pin-like inflorescences. Previous studies have shown that phosphoregulation of PIN1 by AGC kinases including PID directs auxin flux to drive organ initiation. Here, we report unexpected findings on the genetic interactions between these two genes. We deleted the first 2/3 of the PIN1 coding sequence using CRISPR/Cas9, and the resulting pin1 mutant (pin1-27) was a strong allele. Surprisingly, heterozygous pin1-27 suppressed two independent pid null mutants, whereas homozygous pin1-27 enhanced the phenotypes of the pid mutants during embryogenesis. Furthermore, we show that deletion of either the hydrophilic loop or the second half of PIN1 also abolished PIN1 function, yet those heterozygous pin1 mutants were also capable of rescuing pid nulls. Moreover, we inserted green fluorescent protein (GFP) into the hydrophilic loop of PIN1 through CRISPR-mediated homology-directed repair (HDR). The GFP signal and pattern in the PIN1-GFPHDR line are similar to those in the previously reported PIN1-GFP transgenic lines. Interestingly, the PIN1-GFPHDR line also rescued various pid null mutant alleles in a semidominant fashion. We conclude that decreasing the number of functional PIN1 copies is sufficient to suppress the pid mutant phenotype, suggesting that PIN1 is likely part of a larger protein complex required for organogenesis.

Antimicrobial resistance has emerged as a global public health threat, and development of novel therapeutics for treating infections caused by multi-drug resistant bacteria is urgent. Staphylococcus aureus is a major human and animal pathogen, responsible for high levels of morbidity and mortality worldwide. The intracellular survival of S. aureus in macrophages contributes to immune evasion, dissemination, and resilience to antibiotic treatment. Here, we present a confocal fluorescence imaging assay for monitoring macrophage infection by green fluorescent protein (GFP)-tagged S. aureus as a front-line tool to identify antibiotic leads. The assay was employed in combination with nanoscaled chemical analyses to facilitate the discovery of a new, active rifamycin analogue. Our findings indicate a promising new approach for the identification of antimicrobial compounds with macrophage intracellular activity. The antibiotic identified here may represent a useful addition to our armory in tackling the silent pandemic of antimicrobial resistance.

CRISPR/Cas9-based genome engineering has revolutionized our ability to manipulate biological systems, particularly in higher organisms. Here, we designed a set of homology-directed repair donor templates that enable efficient tagging of endogenous proteins with affinity tags by transient transfection and selection of genome-edited cells in various human cell lines. Combined with technological advancements in single-particle cryogenic electron microscopy, this strategy allows efficient structural studies of endogenous proteins captured in their native cellular environment and during different cellular processes. We demonstrated this strategy by tagging six different human proteins in both HEK293T and Jurkat cells. Moreover, analysis of endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in HEK293T cells allowed us to follow its behavior spatially and temporally in response to prolonged oxidative stress, correlating the increased number of oxidation-induced inactive catalytic sites in GAPDH with its translocation from cytosol to nucleus.

Lysosomes are an important organelle required for the degradation of a range of cellular components. Lysosome function is critical for development and homeostasis as dysfunction can lead to inherited genetic disorders, cancer, and neurodegenerative and metabolic diseases. The acidic and protease-rich environment of lysosomes poses experimental challenges. Many fluorescent proteins are quenched or degraded, while specific red fluorescent proteins can be cleaved from translational fusion partners and accumulate. While studying MLT-11, a Caenorhabditis elegans molting factor that localizes to lysosomes and the cuticle, we sought to optimize several experimental parameters. We found that, in contrast to mNeonGreen fusions, mScarlet fusions to MLT-11 missed cuticular and rectal epithelial localization. Rapid sample lysis and denaturation were critical for preventing MLT-11 fragmentation while preparing lysates for western blots. Using a model lysosomal substrate (NUC-1), we found that rigid polyproline linkers and truncated mCherry constructs do not prevent cleavage of mCherry from NUC-1. We provide evidence that extended localization in lysosomal environments prevents the detection of FLAG epitopes in western blots. Finally, we optimize an acid-tolerant green fluorescent protein (Gamillus) for use in C. elegans. These experiments provide important experimental considerations and new reagents for the study of C. elegans lysosomal proteins.

The kinase ataxia telangiectasia mutated (ATM) plays a key role in the DNA damage response (DDR). It is thus essential to visualize spatiotemporal dynamics of ATM activity during DDR. Here, we designed a robust ATM activity reporter based on phosphorylation-inducible green fluorescent protein phase separation, dubbed ATM-SPARK (separation of phases-based activity reporter of kinase). Upon ATM activation, it undergoes phase separation via multivalent interactions, forming intensely bright droplets. The reporter visualizes spatiotemporal dynamics of endogenous ATM activity in living cells, and its signal is proportional to the amount of DNA damage. ATM-SPARK also enables high-throughput screening of biological and small-molecule regulators. We identified the protein phosphatase 4 that blocks ATM activity. We also identified BGT226 as a potent ATM inhibitor with a median inhibitory concentration of ~3.8 nanomolars. Furthermore, BGT226 sensitizes cancer cells to the radiomimetic drug neocarzinostatin, suggesting that BGT226 might be combined with radiotherapeutic treatment. ATM-SPARK achieves large dynamic range, bright fluorescence, and simple signal pattern.

Fluorescent proteins (FPs) stand as pivotal tools extensively employed across diverse biological research endeavors in various model systems. However, long-standing concerns surround their use due to the numerous side effects associated with their expression. Recent investigations have brought to light the significance of hydrogen peroxide (H2O2) that is associated with the maturation process of green fluorescent protein (GFP) fluorophores. The structural and functional impairments associated with GFP expression are possibly linked to this amount of H2O2. In this study, we assess the impact of the GFP-based HyPer7 biosensor on cellular homeostasis and proteome changes, aiming to identify potential risks related to oxidative stress responses that potentially risks the application of such tools. Cells expressing genome-integrated HyPer7 demonstrated altered mitochondrial membrane potential (MMP), which was alleviated by the addition of antioxidants or culturing cells at physiological normoxia (5 kPa O2). Additionally, HyPer7-expressing cells also exhibited significant impairment in mitochondrial oxidative respiration, suggesting broader mitochondrial dysfunction. Through untargeted proteomics analysis, we identified 26 proteins exhibiting differential expression in HyPer7-expressing cells compared to respective control cells. Functional annotation analysis showed that the list of the delineated proteins is associated with cellular responses to stress and the regulation of antioxidant mechanisms. Our findings underscore the significance of caution and validation in ensuring a thorough comprehension of cellular responses when using fluorescent protein-based tools, thereby enhancing the reliability of the results.

We report the rational engineering of a remarkably stable yellow fluorescent protein (YFP), 'hyperfolder YFP' (hfYFP), that withstands chaotropic conditions that denature most biological structures within seconds, including superfolder green fluorescent protein (GFP). hfYFP contains no cysteines, is chloride insensitive and tolerates aldehyde and osmium tetroxide fixation better than common fluorescent proteins, enabling its use in expansion and electron microscopies. We solved crystal structures of hfYFP (to 1.7-Å resolution), a monomeric variant, monomeric hyperfolder YFP (1.6 Å) and an mGreenLantern mutant (1.2 Å), and then rationally engineered highly stable 405-nm-excitable GFPs, large Stokes shift (LSS) monomeric GFP (LSSmGFP) and LSSA12 from these structures. Lastly, we directly exploited the chemical stability of hfYFP and LSSmGFP by devising a fluorescence-assisted protein purification strategy enabling all steps of denaturing affinity chromatography to be visualized using ultraviolet or blue light. hfYFP and LSSmGFP represent a new generation of robustly stable fluorescent proteins developed for advanced biotechnological applications.

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

Akanes are fluorescent proteins that have several fluorescence maxima. In this report, Akane1 and Akane3 from Scleronephthya gracillima were selected, successfully overexpressed in Escherichia coli and purified by affinity chromatography. Fluorescence spectra of the recombinant Akanes matured in darkness, or ambient light were found to have several fluorescence peaks. SDS-PAGE analysis revealed that Akanes matured in ambient light have two fragments. MS/MS analysis of Akanes digested with trypsin showed that the cleavage site is the same as observed for the photoconvertible fluorescent protein Kaede. The differences between the calculated masses from the amino acid sequence of Akane1 and the measured masses of Akane1 fragments obtained under ambient light coincided with those of Kaede. In contrast, a mass difference between the measured N-terminal Akane3 fragment and the calculated mass indicated that Akane3 is modified in the N-terminal region. These results indicate that numerous peaks in the fluorescent spectra of Akanes partly arise from isoproteins of Akanes and photoconversion. Photoconversion of Akane1 caused a fluorescence change from green to red, which was also observed for Akane3; however, the fluorescent intensity decreased dramatically when compared with that of Akane3. [ABSTRACT FROM AUTHOR]

Transgenic expression of bacterial nitroreductase (NTR) enzymes sensitizes eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell-ablation paradigms that have expanded studies of cell function and regeneration in vertebrates. However, first-generation NTRs required confoundingly toxic prodrug treatments to achieve effective cell ablation, and some cell types have proven resistant. Here we used rational engineering and cross-species screening to develop an NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy, eliminating the need for near-toxic prodrug treatment regimens. NTR 2.0 therefore enables sustained cell-loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and novel modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic reporter/effector resources to facilitate dissemination of NTR 2.0 to the research community.

Riboswitches are structured non-coding RNAs often located upstream of essential genes in bacterial messenger RNAs. Such RNAs regulate expression of downstream genes by recognizing a specific cellular effector. Although nearly 50 riboswitch classes are known, only a handful recognize multiple effectors. Here, we report the 2.60-Å resolution co-crystal structure of a class I type I preQ1-sensing riboswitch that reveals two effectors stacked atop one another in a single binding pocket. These effectors bind with positive cooperativity in vitro and both molecules are necessary for gene regulation in bacterial cells. Stacked effector recognition appears to be a hallmark of the largest subgroup of preQ1 riboswitches, including those from pathogens such as Neisseria gonorrhoeae. We postulate that binding to stacked effectors arose in the RNA World to closely position two substrates for RNA-mediated catalysis. These findings expand known effector recognition capabilities of riboswitches and have implications for antimicrobial development.

Pigments homologous to the green fluorescent protein (GFP) have been proposed to fine-tune the internal light microclimate of corals, facilitating photoacclimation of photosynthetic coral symbionts (Symbiodiniaceae) to life in different reef habitats and environmental conditions. However, direct measurements of the in vivo light conditions inside the coral tissue supporting this conclusion are lacking. Here, we quantified the intra-tissue spectral light environment of corals expressing GFP-like proteins from widely different light regimes. We focus on: (1) photoconvertible red fluorescent proteins (pcRFPs), thought to enhance photosynthesis in mesophotic habitats via wavelength conversion, and (2) chromoproteins (CPs), which provide photoprotection to the symbionts in shallow water via light absorption. Optical microsensor measurements indicated that both pigment groups strongly alter the coral intra-tissue light environment. Estimates derived from light spectra measured in pcRFP-containing corals showed that fluorescence emission can contribute to >50% of orange-red light available to the photosynthetic symbionts at mesophotic depths. We further show that upregulation of pink CPs in shallow-water corals during bleaching leads to a reduction of orange light by 10-20% compared to low-CP tissue. Thus, screening by CPs has an important role in mitigating the light-enhancing effect of coral tissue scattering and skeletal reflection during bleaching. Our results provide the first experimental quantification of the importance of GFP-like proteins in fine-tuning the light microclimate of corals during photoacclimation.

In eukaryotes, chromatin binding to the inner nuclear membrane (INM) and nuclear pore complexes (NPCs) contributes to spatial organization of the genome and epigenetic programs important for gene expression. In mitosis, chromatin-nuclear envelope (NE) interactions are lost and then formed again as sister chromosomes segregate to postmitotic nuclei. Investigating these processes in S. cerevisiae, we identified temporally and spatially controlled phosphorylation-dependent SUMOylation events that positively regulate postmetaphase chromatin association with the NE. Our work establishes a phosphorylation-mediated targeting mechanism of the SUMO ligase Siz2 to the INM during mitosis, where Siz2 binds to and SUMOylates the VAP protein Scs2. The recruitment of Siz2 through Scs2 is further responsible for a wave of SUMOylation along the INM that supports the assembly and anchorage of subtelomeric chromatin at the INM and localization of an active gene (INO1) to NPCs during the later stages of mitosis and into G1-phase.

Persistent over-activation of CaMKII (Calcium/Calmodulin-dependent protein Kinase II) in the heart is implicated in arrhythmias, heart failure, pathological remodeling, and other cardiovascular diseases. Several post-translational modifications (PTMs)-including autophosphorylation, oxidation, S-nitrosylation, and O-GlcNAcylation-have been shown to trap CaMKII in an autonomously active state. The molecular mechanisms by which these PTMs regulate calmodulin (CaM) binding to CaMKIIδ-the primary cardiac isoform-has not been well-studied particularly in its native myocyte environment. Typically, CaMKII activates upon Ca-CaM binding during locally elevated [Ca]free and deactivates upon Ca-CaM dissociation when [Ca]free returns to basal levels. To assess the effects of CaMKIIδ PTMs on CaM binding, we developed a novel FRET (Förster resonance energy transfer) approach to directly measure CaM binding to and dissociation from CaMKIIδ in live cardiac myocytes. We demonstrate that autophosphorylation of CaMKIIδ increases affinity for CaM in its native environment and that this increase is dependent on [Ca]free. This leads to a 3-fold slowing of CaM dissociation from CaMKIIδ (time constant slows from ~0.5 to 1.5 s) when [Ca]free is reduced with physiological kinetics. Moreover, oxidation further slows CaM dissociation from CaMKIIδ T287D (phosphomimetic) upon rapid [Ca]free chelation and increases FRET between CaM and CaMKIIδ T287A (phosphoresistant). The CaM dissociation kinetics-measured here in myocytes-are similar to the interval between heartbeats, and integrative memory would be expected as a function of heart rate. Furthermore, the PTM-induced slowing of dissociation between beats would greatly promote persistent CaMKIIδ activity in the heart. Together, these findings suggest a significant role of PTM-induced changes in CaMKIIδ affinity for CaM and memory under physiological and pathophysiological processes in the heart.

Understanding complex biological systems requires visualizing structures and processes deep within living organisms. We developed a compact adaptive optics module and incorporated it into two- and three-photon fluorescence microscopes, to measure and correct tissue-induced aberrations. We resolved synaptic structures in deep cortical and subcortical areas of the mouse brain, and demonstrated high-resolution imaging of neuronal structures and somatosensory-evoked calcium responses in the mouse spinal cord at great depths in vivo.

Nonstructural protein 1 (nsp1) is a coronavirus (CoV) virulence factor that restricts cellular gene expression by inhibiting translation through blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We perform a detailed structure-guided mutational analysis of severe acute respiratory syndrome (SARS)-CoV-2 nsp1, revealing insights into how it coordinates these activities against host but not viral mRNA. We find that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, both enhancing its restriction of host gene expression and enabling mRNA containing the SARS-CoV-2 leader sequence to escape translational repression. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in SARS-CoV-2 nsp1 could attenuate the virus.

Super-resolution activity imaging maps the biochemical architecture of living cells yet currently overlooks the locations of collaborating regulators/effectors. Building on the fluorescence fluctuation increase by contact (FLINC) principle, here we devise Dronpa-chromophore-removed FLINC (DrFLINC), where the nonfluorescent Dronpa can nevertheless enhance TagRFP-T fluorescence fluctuations. Exploiting DrFLINC, we develop a superior red label and a next-generation activity sensor for context-rich super-resolution biosensing.

Cells inherit two centrioles, the older of which is uniquely capable of generating a cilium. Using proteomics and superresolved imaging, we identify a module that we term DISCO (distal centriole complex). The DISCO components CEP90, MNR, and OFD1 underlie human ciliopathies. This complex localizes to both distal centrioles and centriolar satellites, proteinaceous granules surrounding centrioles. Cells and mice lacking CEP90 or MNR do not generate cilia, fail to assemble distal appendages, and do not transduce Hedgehog signals. Disrupting the satellite pools does not affect distal appendage assembly, indicating that it is the centriolar populations of MNR and CEP90 that are critical for ciliogenesis. CEP90 recruits the most proximal known distal appendage component, CEP83, to root distal appendage formation, an early step in ciliogenesis. In addition, MNR, but not CEP90, restricts centriolar length by recruiting OFD1. We conclude that DISCO acts at the distal centriole to support ciliogenesis by restraining centriole length and assembling distal appendages, defects in which cause human ciliopathies.

R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA-DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA-DNA hybrids. GFP-dRNH1 binds strongly to RNA-DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA-DNA hybrids under a wide range of conditions.

Cell death is a critical process that occurs normally in health and disease. However, its study is limited due to available technologies that only detect very late stages in the process or specific death mechanisms. Here, we report the development of a family of fluorescent biosensors called genetically encoded death indicators (GEDIs). GEDIs specifically detect an intracellular Ca2+ level that cells achieve early in the cell death process and that marks a stage at which cells are irreversibly committed to die. The time-resolved nature of a GEDI delineates a binary demarcation of cell life and death in real time, reformulating the definition of cell death. We demonstrate that GEDIs acutely and accurately report death of rodent and human neurons in vitro, and show that GEDIs enable an automated imaging platform for single cell detection of neuronal death in vivo in zebrafish larvae. With a quantitative pseudo-ratiometric signal, GEDIs facilitate high-throughput analysis of cell death in time-lapse imaging analysis, providing the necessary resolution and scale to identify early factors leading to cell death in studies of neurodegeneration.

The clinical utility of emulsions as delivery vehicles is hindered by a dependence on passive release. Stimuli-responsive emulsions overcome this limitation but rely on external triggers or are composed of nanoparticle-stabilized droplets that preclude sizes necessary for biomedical applications. Here, we employ cleavable poly(2-oxazoline) diblock copolymer surfactants to form perfluorocarbon (PFC) nanoemulsions that release cargo upon exposure to glutathione. These surfactants allow for the first example of redox-responsive nanoemulsions in cellulo. A noncovalent fluorous tagging strategy is leveraged to solubilize a GFP plasmid inside the PFC nanoemulsions, whereupon protein expression is achieved selectively when employing a stimuli-responsive surfactant. This work contributes a methodology for non-viral gene delivery and represents a general approach to nanoemulsions that respond to endogenous stimuli.

Genetically introducing novel chemical bonds into proteins provides innovative avenues for biochemical research, protein engineering, and biotherapeutic applications. Recently, latent bioreactive unnatural amino acids (Uaas) have been incorporated into proteins to covalently target natural residues through proximity-enabled reactivity. Aryl fluorosulfate is particularly attractive due to its exceptional biocompatibility and multitargeting capability via sulfur(VI) fluoride exchange (SuFEx) reaction. Thus far, fluorosulfate-l-tyrosine (FSY) is the only aryl fluorosulfate-containing Uaa that has been genetically encoded. FSY has a relatively rigid and short side chain, which restricts the diversity of proteins targetable and the scope of applications. Here we designed and genetically encoded a new latent bioreactive Uaa, fluorosulfonyloxybenzoyl-l-lysine (FSK), in E. coli and mammalian cells. Due to its long and flexible aryl fluorosulfate-containing side chain, FSK was particularly useful in covalently linking protein sites that are unreachable with FSY, both intra- and intermolecularly, in vitro and in live cells. In addition, we created covalent nanobodies that irreversibly bound to epidermal growth factor receptors (EGFR) on cells, with FSK and FSY targeting distinct positions on EGFR to counter potential mutational resistance. Moreover, we established the use of FSK and FSY for genetically encoded chemical cross-linking to capture elusive enzyme-substrate interactions in live cells, allowing us to target residues aside from Cys and to cross-link at the binding periphery. FSK complements FSY to expand target diversity and versatility. Together, they provide a powerful, genetically encoded, latent bioreactive SuFEx system for creating covalent bonds in diverse proteins in vitro and in vivo, which will be widely useful for biological research and applications.

Foxp3+ T regulatory (Treg) cells promote immunological tumor tolerance, but how their immune-suppressive function is regulated in the tumor microenvironment (TME) remains unknown. Here, we used intravital microscopy to characterize the cellular interactions that provide tumor-infiltrating Treg cells with critical activation signals. We found that the polyclonal Treg cell repertoire is pre-enriched to recognize antigens presented by tumor-associated conventional dendritic cells (cDCs). Unstable cDC contacts sufficed to sustain Treg cell function, whereas T helper cells were activated during stable interactions. Contact instability resulted from CTLA-4-dependent downregulation of co-stimulatory B7-family proteins on cDCs, mediated by Treg cells themselves. CTLA-4-blockade triggered CD28-dependent Treg cell hyper-proliferation in the TME, and concomitant Treg cell inactivation was required to achieve tumor rejection. Therefore, Treg cells self-regulate through a CTLA-4- and CD28-dependent feedback loop that adjusts their population size to the amount of local co-stimulation. Its disruption through CTLA-4-blockade may off-set therapeutic benefits in cancer patients.

Studies of the spatiotemporal, transcriptomic, and morphological diversity of radial glia (RG) have spurred our current models of human corticogenesis. In the developing cortex, neural intermediate progenitor cells (nIPCs) are a neuron-producing transit-amplifying cell type born in the germinal zones of the cortex from RG. The potential diversity of the nIPC population, that produces a significant portion of excitatory cortical neurons, is understudied, particularly in the developing human brain. Here we explore the spatiotemporal, transcriptomic, and morphological variation that exists within the human nIPC population and provide a resource for future studies. We observe that the spatial distribution of nIPCs in the cortex changes abruptly around gestational week (GW) 19/20, marking a distinct shift in cellular distribution and organization during late neurogenesis. We also identify five transcriptomic subtypes, one of which appears at this spatiotemporal transition. Finally, we observe a diversity of nIPC morphologies that do not correlate with specific transcriptomic subtypes. These results provide an analysis of the spatiotemporal, transcriptional, and morphological diversity of nIPCs in developing brain tissue and provide an atlas of nIPC subtypes in the developing human cortex that can benchmark in vitro models of human development such as cerebral organoids and help inform future studies of how nIPCs contribute to cortical neurogenesis.

The Agrobacterium growth pole ring (GPR) protein forms a hexameric ring at the growth pole (GP) that is essential for polar growth. GPR is large (2,115 amino acids) and contains 1,700 amino acids of continuous α-helices. To dissect potential GPR functional domains, we created deletions of regions with similarity to human apolipoprotein A-IV (396 amino acids), itself composed of α-helical domains. We also tested deletions of the GPR C terminus. Deletions were inducibly expressed as green fluorescent protein (GFP) fusion proteins and tested for merodiploid interference with wild-type (WT) GPR function, for partial function in cells lacking GPR, and for formation of paired fluorescent foci (indicative of hexameric rings) at the GP. Deletion of domains similar to human apolipoprotein A-IV in GPR caused defects in cell morphology when expressed in trans to WT GPR and provided only partial complementation to cells lacking GPR. Agrobacterium-specific domains A-IV-1 and A-IV-4 contain predicted coiled coil (CC) regions of 21 amino acids; deletion of CC regions produced severe defects in cell morphology in the interference assay. Mutants that produced the most severe effects on cell shape also failed to form paired polar foci. Modeling of A-IV-1 and A-IV-4 reveals significant similarity to the solved structure of human apolipoprotein A-IV. GPR C-terminal deletions profoundly blocked complementation. Finally, peptidoglycan (PG) synthesis is abnormally localized circumferentially in cells lacking GPR. The results support the hypothesis that GPR plays essential roles as an organizing center for membrane and PG synthesis during polar growth.IMPORTANCE Bacterial growth and division are extensively studied in model systems (Escherichia coli, Bacillus subtilis, and Caulobacter crescentus) that grow by dispersed insertion of new cell wall material along the length of the cell. An alternative growth mode-polar growth-is used by some Actinomycetales and Proteobacteria species. The latter phylum includes the family Rhizobiaceae, in which many species, including Agrobacterium tumefaciens, exhibit polar growth. Current research aims to identify growth pole (GP) factors. The Agrobacterium growth pole ring (GPR) protein is essential for polar growth and forms a striking hexameric ring structure at the GP. GPR is long (2,115 amino acids), and little is known about regions essential for structure or function. Genetic analyses demonstrate that the C terminus of GPR, and two internal regions with homology to human apolipoproteins (that sequester lipids), are essential for GPR function and localization to the GP. We hypothesize that GPR is an organizing center for membrane and cell wall synthesis during polar growth.

Lysosomes are important sites for macromolecular degradation, defined by an acidic lumenal pH of ∼4.5. To better understand lysosomal pH, we designed a novel, genetically encoded, fluorescent protein (FP)-based pH biosensor called Fluorescence Indicator REporting pH in Lysosomes (FIRE-pHLy). This biosensor was targeted to lysosomes with lysosomal-associated membrane protein 1 (LAMP1) and reported lumenal pH between 3.5 and 6.0 with monomeric teal fluorescent protein 1 (mTFP1), a bright cyan pH-sensitive FP variant with a pKa of 4.3. Ratiometric quantification was enabled with cytosolically oriented mCherry using high-content quantitative imaging. We expressed FIRE-pHLy in several cellular models and quantified the alkalinizing response to bafilomycin A1, a specific V-ATPase inhibitor. In summary, we have engineered FIRE-pHLy, a specific, robust, and versatile lysosomal pH biosensor, that has broad applications for investigating pH dynamics in aging- and lysosome-related diseases, as well as in lysosome-based drug discovery.

Growing evidence suggests that many essential intracellular signaling events are compartmentalized within kinetically distinct microdomains in cells. Genetically encoded fluorescent biosensors are powerful tools to dissect compartmentalized signaling, but current approaches to probe these microdomains typically rely on biosensor fusion and overexpression of critical regulatory elements. Here, we present a novel class of biosensors named FluoSTEPs (fluorescent sensors targeted to endogenous proteins) that combine self-complementing split green fluorescent protein, CRISPR-mediated knock-in, and fluorescence resonance energy transfer biosensor technology to probe compartmentalized signaling dynamics in situ. We designed FluoSTEPs for simultaneously highlighting endogenous microdomains and reporting domain-specific, real-time signaling events including kinase activities, guanosine triphosphatase activation, and second messenger dynamics in live cells. A FluoSTEP for 3,5-cyclic adenosine monophosphate (cAMP) revealed distinct cAMP dynamics within clathrin microdomains in response to stimulation of G protein-coupled receptors, showcasing the utility of FluoSTEPs in probing spatiotemporal regulation within endogenous signaling architectures.

CRISPR (clustered regularly interspaced short palindromic repeats)-based gene inactivation provides a powerful means for linking genes to particular cellular phenotypes. CRISPR-based screening typically uses large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited to phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed a microscopy-based approach, which we name optical enrichment, to select cells displaying a particular CRISPR-induced phenotype by automated imaging-based computation, mark them by photoactivation of an expressed photoactivatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). A plugin was developed for the open source software μManager to automate the phenotypic identification and photoactivation of cells, allowing ∼1.5 million individual cells to be screened in 8 h. We used this approach to screen 6,092 sgRNAs targeting 544 genes for their effects on nuclear size regulation and identified 14 bona fide hits. These results present a scalable approach to facilitate imaging-based pooled CRISPR screens.

Understanding how biological species arise is critical for understanding the evolution of life on Earth. Bioinformatic analyses have recently revealed that viruses, like multicellular life, form reproductively isolated biological species. Viruses are known to share high rates of genetic exchange, so how do they evolve genetic isolation? Here, we evaluate two related bacteriophages and describe three factors that limit genetic exchange between them: 1) A nucleus-like compartment that physically separates replicating phage genomes, thereby limiting inter-phage recombination during co-infection; 2) A tubulin-based spindle that orchestrates phage replication and forms nonfunctional hybrid polymers; and 3) A nuclear incompatibility factor that reduces phage fitness. Together, these traits maintain species differences through Subcellular Genetic Isolation where viral genomes are physically separated during co-infection, and Virogenesis Incompatibility in which the interaction of cross-species components interferes with viral production.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic toxicity associated with prominent lipid accumulation in humans. Here, the authors report that the lysosomal copper transporter SLC46A3 is induced by TCDD and underlies the hepatic lipid accumulation in mice, potentially via effects on mitochondrial function. SLC46A3 was localized to the lysosome where it modulated intracellular copper levels. Forced expression of hepatic SLC46A3 resulted in decreased mitochondrial membrane potential and abnormal mitochondria morphology consistent with lower copper levels. SLC46A3 expression increased hepatic lipid accumulation similar to the known effects of TCDD exposure in mice and humans. The TCDD-induced hepatic triglyceride accumulation was significantly decreased in Slc46a3-/- mice and was more pronounced when these mice were fed a high-fat diet, as compared to wild-type mice. These data are consistent with a model where lysosomal SLC46A3 induction by TCDD leads to cytosolic copper deficiency resulting in mitochondrial dysfunction leading to lower lipid catabolism, thus linking copper status to mitochondrial function, lipid metabolism and TCDD-induced liver toxicity.

Increases in adhesive and invasive commensal bacteria, such as Escherichia coli, and subsequent disruption of the epithelial barrier is implicated in the pathogenesis of inflammatory bowel disease (IBD). However, the protective systems against such barrier disruption are not fully understood. Here, we show that secretion of luminal glycoprotein 2 (GP2) from pancreatic acinar cells is induced in a TNF-dependent manner in mice with chemically induced colitis. Fecal GP2 concentration is also increased in Crohn's diease patients. Furthermore, pancreas-specific GP2-deficient colitis mice have more severe intestinal inflammation and a larger mucosal E. coli population than do intact mice, indicating that digestive-tract GP2 binds commensal E. coli, preventing epithelial attachment and penetration. Thus, the pancreas-intestinal barrier axis and pancreatic GP2 are important as a first line of defense against adhesive and invasive commensal bacteria during intestinal inflammation.

Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of this include flaviviruses, such as dengue virus and Zika virus, which cause millions of yearly infections around the globe, and coronaviruses, such as SARS-CoV-2, the source of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of research aimed at determining methods to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective treatments. Here, we describe the generation and characterization of a reporter system that can be used to visualize and identify cells infected with dengue virus or SARS-CoV-2. This system is based on viral protease activity that mediates cleavage and nuclear translocation of an engineered fluorescent protein stably expressed in cells. We show the suitability of this system for live cell imaging, for visualization of single infected cells, and for screening and testing of antiviral compounds. With the integrated modular building blocks, this system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility.IMPORTANCE Reporter systems are useful tools for fast and quantitative visualization of virus-infected cells within a host cell population. Here, we describe a reporter system that takes advantage of virus-encoded proteases expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the GFP moiety translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.

Through the efforts of many groups, a wide range of fluorescent protein reporters and sensors based on green fluorescent protein and its relatives have been engineered in recent years. Here we explore the incorporation of sensing modalities into de novo designed fluorescence-activating proteins, called mini-fluorescence-activating proteins (mFAPs), that bind and stabilize the fluorescent cis-planar state of the fluorogenic compound DFHBI. We show through further design that the fluorescence intensity and specificity of mFAPs for different chromophores can be tuned, and the fluorescence made sensitive to pH and Ca2+ for real-time fluorescence reporting. Bipartite split mFAPs enable real-time monitoring of protein-protein association and (unlike widely used split GFP reporter systems) are fully reversible, allowing direct readout of association and dissociation events. The relative ease with which sensing modalities can be incorporated and advantages in smaller size and photostability make de novo designed fluorescence-activating proteins attractive candidates for optical sensor engineering.

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control over gene expression timing, location, and amplitude using light as the inducing agent. In this protocol, an optogenetic expression system is used to achieve light-inducible gene expression in zebrafish embryos. This system relies on an engineered transcription factor called TAEL based on a naturally occurring light-activated transcription factor from the bacterium E. litoralis. When illuminated with blue light, TAEL dimerizes, binds to its cognate regulatory element called C120, and activates transcription. This protocol uses transgenic zebrafish embryos that express the TAEL transcription factor under the control of the ubiquitous ubb promoter. At the same time, the C120 regulatory element drives the expression of a fluorescent reporter gene (GFP). Using a simple LED panel to deliver activating blue light, induction of GFP expression can first be detected after 30 min of illumination and reaches a peak of more than 130-fold induction after 3 h of light treatment. Expression induction can be assessed by quantitative real-time PCR (qRT-PCR) and by fluorescence microscopy. This method is a versatile and easy-to-use approach for optogenetic gene expression.

The maintenance of intracellular NAD+/NADH homeostasis across multiple, subcellular compartments requires the presence of NADH-shuttling proteins, which circumvent the lack of permeability of organelle membranes to these cofactors. Very little is known regarding these proteins in the methylotrophic yeast, Pichia pastoris. During the study of the subcellular locations of these shuttling proteins, which often have dual subcellular locations, it became necessary to develop new ways to detect the weak peroxisomal locations of some of these proteins. We have developed a novel variation of the traditional Bimolecular Fluorescence Complementation (BiFC), called divergent BiFC, to detect intraorganellar colocalization of two noninteracting proteins based on their proximity-based protein crowding within a small subcellular compartment, rather than on the traditional protein-protein interactions expected for BiFC. This method is used to demonstrate the partially peroxisomal location of one such P. pastoris NADH-shuttling protein, malate dehydrogenase B, only when cells are grown in oleate, but not when grown in methanol or glucose. We discuss the mode of NADH shuttling in P. pastoris and the physiological basis of the medium-dependent compartmentalization of PpMdhB.

Genetic engineering techniques have contributed to the now widespread use of zebrafish to investigate gene function, but zebrafish-based human disease studies, and particularly for neurological disorders, are limited. Here we used CRISPR-Cas9 to generate 40 single-gene mutant zebrafish lines representing catastrophic childhood epilepsies. We evaluated larval phenotypes using electrophysiological, behavioral, neuro-anatomical, survival and pharmacological assays. Local field potential recordings (LFP) were used to screen ∼3300 larvae. Phenotypes with unprovoked electrographic seizure activity (i.e., epilepsy) were identified in zebrafish lines for 8 genes; ARX, EEF1A, GABRB3, GRIN1, PNPO, SCN1A, STRADA and STXBP1. We also created an open-source database containing sequencing information, survival curves, behavioral profiles and representative electrophysiology data. We offer all zebrafish lines as a resource to the neuroscience community and envision them as a starting point for further functional analysis and/or identification of new therapies.

The respiratory disease COVID-19 is caused by the coronavirus SARS-CoV-2. Here we report the discovery of ethacridine as a potent drug against SARS-CoV-2 (EC50 ~ 0.08 μM). Ethacridine was identified via high-throughput screening of an FDA-approved drug library in living cells using a fluorescence assay. Plaque assays, RT-PCR and immunofluorescence imaging at various stages of viral infection demonstrate that the main mode of action of ethacridine is through inactivation of viral particles, preventing their binding to the host cells. Consistently, ethacridine is effective in various cell types, including primary human nasal epithelial cells that are cultured in an air-liquid interface. Taken together, our work identifies a promising, potent, and new use of the old drug via a distinct mode of action for inhibiting SARS-CoV-2.

Generating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily consisting of ventricular-like CMs. Here we describe the creation of two chamber-specific reporter hiPSC lines by site-directed genomic integration using CRISPR-Cas9 technology. In the MYL2-tdTomato reporter, the red fluorescent tdTomato was inserted upstream of the 3' untranslated region of the Myosin Light Chain 2 (MYL2) gene in order faithfully label hiPSC-derived ventricular-like CMs while avoiding disruption of endogenous gene expression. Similarly, in the SLN-CFP reporter, Cyan Fluorescent Protein (CFP) was integrated downstream of the coding region of the atrial-specific gene, Sarcolipin (SLN). Purification of tdTomato+ and CFP+ CMs using flow cytometry coupled with transcriptional and functional characterization validated these genetic tools for their use in the isolation of bona fide ventricular-like and atrial-like CMs, respectively. Finally, we successfully generated a double reporter system allowing for the isolation of both ventricular and atrial CM subtypes within a single hiPSC line. These tools provide a platform for chamber-specific hiPSC-derived CM purification and analysis in the context of atrial- or ventricular-specific disease and therapeutic opportunities.

Microglia are brain resident macrophages that play vital roles in central nervous system (CNS) development, homeostasis, and pathology. Microglia both remodel synapses and engulf apoptotic cell corpses during development, but whether unique molecular programs regulate these distinct phagocytic functions is unknown. Here we identify a molecularly distinct microglial subset in the synapse rich regions of the zebrafish (Danio rerio) brain. We found that ramified microglia increased in synaptic regions of the midbrain and hindbrain between 7 and 28 days post fertilization. In contrast, microglia in the optic tectum were ameboid and clustered around neurogenic zones. Using single-cell mRNA sequencing combined with metadata from regional bulk sequencing, we identified synaptic-region associated microglia (SAMs) that were highly enriched in the hindbrain and expressed multiple candidate synapse modulating genes, including genes in the complement pathway. In contrast, neurogenic associated microglia (NAMs) were enriched in the optic tectum, had active cathepsin activity, and preferentially engulfed neuronal corpses. These data reveal that molecularly distinct phagocytic programs mediate synaptic remodeling and cell engulfment, and establish the zebrafish hindbrain as a model for investigating microglial-synapse interactions.

It is known that a subpopulation of T cells expresses two T cell receptor (TCR) clonotypes, though the extent and functional significance of this is not established. To definitively evaluate dual TCRα cells, we generated mice with green fluorescent protein and red fluorescent protein reporters linked to TCRα, revealing that ∼16% of T cells express dual TCRs, notably higher than prior estimates. Importantly, dual TCR expression has functional consequences, as dual TCR cells predominated response to lymphocytic choriomeningitis virus infection, comprising up to 60% of virus-specific CD4+ and CD8+ T cells during acute responses. Dual receptor expression selectively influenced immune memory, as postinfection memory CD4+ populations contained significantly increased frequencies of dual TCR cells. These data reveal a previously unappreciated contribution of dual TCR cells to the immune repertoire and highlight their potential effects on immune responses.

ObjectivesSorghum is one of the most recalcitrant species for transformation. Considering the time and effort required for stable transformation in sorghum, establishing a transient system to screen the efficiency and full functionality of vector constructs is highly desirable.ResultsHere, we report an Agrobacterium-mediated transient transformation assay with intact sorghum leaves using green fluorescent protein as marker. It also provides a good monocot alternative to tobacco and protoplast assays with a direct, native and more reliable system for testing single guide RNA (sgRNA) expression construct efficiency. Given the simplicity and ease of transformation, high reproducibility, and ability to test large constructs, this method can be widely adopted to speed up functional genomic and genome editing studies.

This protocol focuses on the cloning and stable integration of sequences of interest by the use of a mosaic analysis with dual recombinases (MADR) plasmid that includes fusion proteins or independent proteins under the control of 2A peptide or IRES elements. Additionally, we describe how to generate a neural stem cell culture from Gt(ROSA)26Sortm4(ACTB-tdTomato, EGFP)Luo/J mice, and validate the MADR plasmids in vitro and in vivo by neonatal mouse brain electroporation. This protocol can be generalized to analyze any transgenic element using MADR technology. For complete details on the use and execution of this protocol, please refer to Kim et al. (2019).