Fluorescent Fusion Research Articles

The development of extracellular vesicle markers for the fungal phytopathogen Colletotrichum higginsianum.

Fungal phytopathogens secrete extracellular vesicles (EVs) associated with enzymes and phytotoxic metabolites. While these vesicles are thought to promote infection, defining the true contents and functions of fungal EVs, as well as suitable protein markers, is an ongoing process. To expand our understanding of fungal EVs and their possible roles during infection, we purified EVs from the hemibiotrophic phytopathogen Colletotrichum higginsianum, the causative agent of anthracnose disease in multiple plant species, including Arabidopsis thaliana. EVs were purified in large numbers from the supernatant of protoplasts but not the supernatant of intact mycelial cultures. We purified two separate populations of EVs, each associated with over 700 detected proteins, including proteins involved in vesicle transport, cell wall biogenesis and the synthesis of secondary metabolites. We selected two SNARE proteins (Snc1 and Sso2) and one 14‐3‐3 protein (Bmh1) as potential EV markers and generated transgenic strains expressing fluorescent fusions. Each marker was confirmed to be protected inside EVs. Fluorescence microscopy was used to examine the localization of each marker during infection on Arabidopsis leaves. These findings further our understanding of EVs in fungal phytopathogens and will help build an experimental system to study EV interkingdom communication between plants and fungi.

A Framework to Assess Liquid‐Liquid‐Phase‐Separation in Bacterial Cells

Liquid Liquid Phase Separation (LLPS) has emerged as a mechanism for the assembly of membraneless organelles in eukaryotes, but little is known about this process in bacteria. LLPS refers to the ability of macromolecules to demix into a dilute phase and a dense phase, called a ‘biomolecular condensate’, which can be observed as clusters or foci in the cell. The major challenge for the study of LLPS in bacteria is the poor spatial resolution of foci in such tiny cells. As a result, it is difficult to demonstrate the liquid‐like nature of a focus in bacterial cells using the conventional approaches for studying large condensates in eukaryotic cells. Here, we developed a rigorous experimental framework for the characterization of LLPS in bacteria, using Escherichia coli as the host organism and the intrinsically disordered protein McdB, which robustly forms liquid‐like droplets in vitro. McdB is a protein that coats a bacterial organelle called the carboxysome. This coating demarcates the carboxysome as cargo for its positioning system, which equally distributes carboxysomes along the cell length of rod‐shaped cyanobacteria. We developed a suite of experiments to investigate the LLPS activity of McdB in vivo, based on the ability of biomolecular condensates to tune their size and shape, fuse, dissolve, and transition between phase states. We used both overexpression and tunable promoters to express fluorescent fusions of McdB and cIEP8, a well‐known aggregator protein. We found that fluorescent fusions of McdB formed nucleoid‐excluded foci in E. coli, but also maintained a soluble phase in the cytoplasm, consistent with LLPS theory. The aggregator protein cIEP8, on the other hand, lacked a soluble fraction in the cytoplasm. Condensates form at a saturation concentration threshold, called Csat. A hallmark of LLPS is that condensates will dissolve if the concentration drops below Csat, while insoluble aggregates should remain as stable foci even after dilution. We decreased protein concentration in vivo by increasing cell volume and by generational dilution via cell division. In both methods, McdB foci dissolved while cIEP8 foci remained intact as insoluble aggregates in response to decreased concentration in the cell. Finally, we also discovered that a well‐established marker for insoluble protein aggregates in vivo, IbpA, does not colocalize with McdB foci. The result suggests that the colocalization of IbpA foci can be used as a broad‐use sensor for the material state of protein complexes in bacterial cells. Our results provide multiple lines of evidence in support of LLPS of McdB in vivo. More broadly, our experimental framework for studying LLPS in bacteria overcomes current limitations in the field and can be used to assess the LLPS activity of other proteins of interest in bacterial cells.

PdpC, a Type Six Secretion System Substrate, is Required for Erythrocyte Invasion in Francisella tularensis LVS

Francisella tularensis is an intracellular pathogen and the causative agent of tularemia. The F. tularensis type six secretion system (T6SS) is required for phagolysosomal escape and invasion of erythrocytes. An effector of the T6SS, PdpC, is required for phagosomal escape and we wanted to test if PdpC was also required for erythrocyte invasion. We constructed a pdpC-null mutant in the live vaccine strain, F. tularensis LVS. The pdpC-null strain is required for invasion of both human and sheep erythrocytes and reintroduction of a copy of pdpC, in trans, rescues this phenotype. Differential Immuno-Fluorescence Microscopy (DIFM) showed that the pdpC-null strain is affected in attachment as well as invasion. Further, a fluorescently labelled pdpC-null strain of F. tularensis LVS was unable to proliferate in THP-1 human peripheral blood monocyte cells. Finally, we constructed a fluorescent fusion of pdpC to emgfp and the resulting PdpC-EmGFP fusion protein localizes as discrete foci in a subset of broth cultured F. tularensis LVS cells. Our results confirm previous observations that PdpC is required for infection and virulence in phagocytic host cells and are the first description of an effector of the Type Six Secretion System that is required for erythrocyte invasion. The pdpC-emgfp strain will be a useful tool to further characterize the role of PdpC in both macrophage infection and red blood cell invasion. (This research was made possible by NASA West Virginia Space Grant Consortium Training Grant #NNX15A101H and by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence).

Direct visualization of glucagon‐like peptide‐1 secretion by fluorescent fusion proteins

Live‐cell imaging with fluorescent proteins (FPs) is a powerful tool for investigating the exocytosis processes of hormones. However, the secretion process of glucagon‐like peptide‐1 (GLP‐1) has not been visualized by FPs, which might be because tagging FPs inhibits GLP‐1 synthesis through the post‐translational processing from proglucagon. Here, we have developed FP‐tagged GLP‐1 by inserting FPs into the middle of GLP‐1 and adding the proglucagon signal peptide. Confocal imaging confirmed that GLP‐1 fused to FPs with high folding efficiency showed granular structure, in which secretory vesicle markers colocalized. The fluorescence intensity of FP in the culture supernatant from cells treated with KCl or forskolin was significantly increased compared with those from untreated cells. Furthermore, FP‐tagged GLP‐1 enables direct visualization of stimulation‐dependent exocytosis of GLP‐1 at a single granule resolution with total internal reflection fluorescence microscopy. FP‐tagged GLP‐1 might facilitate the screening of GLP‐1 secretagogues and the discovery of new antidiabetic drugs.

Imaging Unique DNA Sequences in Individual Cells Using a CRISPR-Cas9-Based, Split Luciferase Biosensor.

An extensive arsenal of biosensing tools has been developed based on the clustered regularly interspaced short palindromic repeat (CRISPR) platform, including those that detect specific DNA sequences both in vitro and in live cells. To date, DNA imaging approaches have traditionally used full fluorescent reporter-based fusion probes. Such “always-on” probes differentiate poorly between bound and unbound probe and are unable to sensitively detect unique copies of a target sequence in individual cells. Herein we describe a DNA biosensor that provides a sensitive readout for such low-copy DNA sequences through proximity-mediated reassembly of two independently optimized fragments of NanoLuc luciferase (NLuc), a small, bright luminescent reporter. Applying this “turn-on” probe in live cells, we demonstrate an application not easily achieved by fluorescent reporter-based probes, detection of individual endogenous genomic loci using standard epifluorescence microscopy. This approach could enable detection of gene edits during ex vivo editing procedures and should be a useful platform for many other live cell DNA biosensing applications.

The ER Is a Common Mediator for the Behavior and Interactions of Other Organelles.

Optimal functioning of a plant cell depends upon the efficient exchange of genetic information, ions, proteins and metabolites between the different organelles. Intuitively, increased proximity between organelles would be expected to play an important role in facilitating exchanges between them. However, it remains to be seen whether under normal, relatively non-stressed conditions organelles maintain close proximity at all. Moreover, does interactivity involve direct and frequent physical contact between the different organelles? Further, many organelles transition between spherical and tubular forms or sporadically produce thin tubular extensions, but it remains unclear whether changes in organelle morphology play a role in increasing their interactivity. Here, using targeted multicolored fluorescent fusion proteins, we report observations on the spatiotemporal relationship between plastids, mitochondria, peroxisomes and the endoplasmic reticulum in living plant cells. Under normal conditions of growth, we observe that the smaller organelles do not establish direct, physical contacts with each other but, irrespective of their individual form they all maintain intimate connectivity with the ER. Proximity between organelles does increase in response to stress through concomitant alterations in ER dynamics. Significantly, even under increased proximity the ER still remains sandwiched between the different organelles. Our observations provide strong live-imaging-based evidence for the ER acting as a common mediator in interactions between other organelles.

All-in-one: a robust fluorescent fusion protein vector toolbox for protein localization and BiFC analyses in plants.

SummaryFluorescent tagging protein localization (FTPL) and bimolecular fluorescence complementation (BiFC) are popular tools for in vivo analyses of the subcellular localizations of proteins and protein–protein interactions in plant cells. The efficiency of fluorescent fusion protein (FFP) expression analyses is typically impaired when the FFP genes are co‐transformed on separate plasmids compared to when all are cloned and transformed in a single vector. Functional genomics applications using FFPs such as a gene family studies also often require the generation of multiple plasmids. Here, to address these needs, we developed an efficient, modular all‐in‐one (Aio) FFP (AioFFP) vector toolbox, including a set of fluorescently labelled organelle markers, FTPL and BiFC plasmids and associated binary vectors. This toolbox uses Gibson assembly (GA) and incorporates multiple unique nucleotide sequences (UNSs) to facilitate efficient gene cloning. In brief, this system enables convenient cloning of a target gene into various FFP vectors or the insertion of two or more target genes into the same FFP vector in a single‐tube GA reaction. This system also enables integration of organelle marker genes or fluorescently fused target gene expression units into a single transient expression plasmid or binary vector. We validated the AioFFP system by testing genes encoding proteins known to be functional in FTPL and BiFC assays. In addition, we performed a high‐throughput assessment of the accurate subcellular localizations of an uncharacterized rice CBSX protein subfamily. This modular UNS‐guided GA‐mediated AioFFP vector toolkit is cost‐effective, easy to use and will promote functional genomics research in plants.

CRISPR/Cas9-mediated genome editing assists protein dynamics studies in live cells

Spatial and temporal regulation of molecular reactions dictates cell fate. Thus, studying molecular dynamics is essential to understand how cells decide what to do and the fundamental perturbations causing disease. Classically, molecular dynamics has been studied by protocols based in the overexpression of fluorescent fusion proteins. However, overexpression is associated to altered stoichiometry, molecular dynamics and subcellular distribution. We here discuss the necessity to study molecular dynamics of fluorescent fusion proteins expressed under physiological mechanisms in the cell, pointing to CRISPR/Cas9-mediated genome editing as the ideal means to do so. Current genome editing protocols enable us to study molecular dynamics while avoiding drawbacks associated to overexpression.

The Androgen Hormone-Induced Increase in Androgen Receptor Protein Expression Is Caused by the Autoinduction of the Androgen Receptor Translational Activity

The androgen receptor (AR) plays a central role in prostate, muscle, bone and adipose tissue. Moreover, dysregulated AR activity is a driving force in prostate cancer (PCa) initiation and progression. Consequently, antagonizing AR signalling cascades via antiandrogenic therapy is a crucial treatment option in PCa management. Besides, very high androgen levels also inhibit PCa cells’ growth, so this effect could also be applied in PCa therapy. However, on the molecular and cellular level, these mechanisms have hardly been investigated so far. Therefore, the present study describes the effects of varying androgen concentrations on the viability of PCa cells as well as localization, transactivation, and protein stability of the AR. For this purpose, cell viability was determined via WST1 assay. Alterations in AR transactivity were detected by qPCR analysis of AR target genes. A fluorescent AR fusion protein was used to analyse AR localization microscopically. Changes in AR protein expression were detected by Western blot. Our results showed that high androgen concentrations reduce the cell viability in LNCaP and C4-2 cell lines. In addition, androgens have been reported to increase AR transactivity, AR localization, and AR protein expression levels. However, high androgen levels did not reduce these parameters. Furthermore, this study revealed an androgen-induced increase in AR protein synthesis. In conclusion, inhibitory effects on cell viability by high androgen levels are due to AR downstream signalling or non-genomic AR activity. Moreover, hormonal activation of the AR leads to a self-induced stabilization of the receptor, resulting in increased AR activity. Therefore, in clinical use, a therapeutic reduction in androgen levels represents a clinical target and would lead to a decrease in AR activity and, thus, AR-driven PCa progression.

High-throughput and high-content bioassay enables tuning of polyester nanoparticles for cellular uptake, endosomal escape, and systemic in vivo delivery of mRNA.

Nanoparticle-based mRNA therapeutics hold great promise, but cellular internalization and endosomal escape remain key barriers for cytosolic delivery. We developed a dual nanoparticle uptake and endosomal disruption assay using high-throughput and high-content image-based screening. Using a genetically encoded Galectin 8 fluorescent fusion protein sensor, endosomal disruption could be detected via sensor clustering on damaged endosomal membranes. Simultaneously, nucleic acid endocytosis was quantified using fluorescently tagged mRNA. We used an array of biodegradable poly(beta-amino ester)s as well as Lipofectamine and PEI to demonstrate that this assay has higher predictive capacity for mRNA delivery compared to conventional polymer and nanoparticle physiochemical characteristics. Top nanoparticle formulations enabled safe and efficacious mRNA expression in multiple tissues following intravenous injection, demonstrating that the in vitro screening method is also predictive of in vivo performance. Efficacious nonviral systemic delivery of mRNA with biodegradable particles opens up new avenues for genetic medicine and human health.

Materials for thermally activated delayed fluorescence and/or triplet fusion upconversion

Guest editors Malika Jeffries-EL, Nobuhiro Yanai and Eli Zysman-Colman introduce this themed issue on thermally activated delayed fluorescence, and triplet–triplet annihilation-based photon upconversion, materials.

Amount of Cas9 protein introduced into mouse embryos via electroporation affects the genome-editing rate.

Genetically engineered animals can be produced quickly using genome editing technology. A new electroporation technique, technique for animal knockout system by electroporation (TAKE), aids in the production of genome-edited animals by introducing nucleases into intact embryos using electroporation instead of microinjection. It is difficult to confirm nuclease delivery into embryos after electroporation using the conventional TAKE method. We previously reported the successful visualization of fluorescently-labeled tracrRNA in embryos after electroporation Cas9 paired with the crRNA:tracrRNA-ATTO550 duplex. However, the amount of fluorescence signal from labeled tracrRNA in embryos did not correlate with the genome editing rate of the offspring. This study examined the visualization of Cas9 protein in embryos after electroporation and its correlation with the genome editing rate of the offspring using a fluorescent Cas9 fusion protein. The fluorescent Cas9 protein was observed in all embryos that survived following electroporation. We found that the efficiency of Cas9 protein delivery into embryos via electroporation depended on the pulse length. Furthermore, we demonstrated that the amount of fluorescent Cas9 protein detected in the embryos correlated with the genome editing efficiency of the embryos. These data indicate that the TAKE method using fluorescently-labeled nucleases can be used to optimize the delivery conditions and verify nuclease delivery into individual embryos prior to embryo transfer for the efficient production of genome-edited animals.

The characterization of transmembrane protein 59-like (TMEM59L) reveals its role in the regulating the level of the GDI protein family

The characterization and functions of transmembrane protein 59-like (TMEM59L), a type I transmembrane protein, are not clearly understood until now. Some TMEM59L and fluorescent fusion proteins constructs were transfected in cell lines and liposomes, and their localization was observed. The effects of protein constructs were studied by fluorescence microscopy and western blotting. This study reports a novel function of human TMEM59L (hTMEM59L) related to the expression and location of some proteins. In addition, we report two novel splice variants of human TMEM59L (hTMEM59L). The localization of mutants of this protein, lacking a middle region, and a C-terminal deletion, markedly differed from that of full-length hTMEM59L. Intracellular movement assessment in living cells showed the localization of TMEM59L to vesicular structures in the Golgi bodies, and the cell membrane was observed in living cells. Overexpression of TMEM59L markedly increased the level of amyloid precursor protein because of TMEM59L-mediated inhibition of cell membrane transport but did not affect the expression of beta-secretase 2. TMEM59L overexpression also significantly increased the levels of Rab GDP dissociation inhibitor alpha and Rab GDP dissociation inhibitor beta proteins. These results suggest that TMEM59L is involved in the packaging of acidic vesicles and thereby functions in the trafficking and processing of intracellular proteins.

Phospholipid scrambling by a TMEM16 homolog of Arabidopsis thaliana.

Membrane asymmetry is important for cellular physiology and established by energy‐dependent unidirectional lipid translocases, which have diverse physiological functions in plants. By contrast, the role of phospholipid scrambling (PLS), the passive bidirectional lipid transfer leading to the break‐down of membrane asymmetry, is currently still unexplored. The Arabidopsis thaliana genome contains a single gene (At1g73020) with homology to the eukaryotic TMEM16 family of Ca2+‐activated phospholipid scramblases. Here, we investigated the protein function of this Arabidopsis homolog. Fluorescent AtTMEM16 fusions localized to the ER both in transiently expressing Arabidopsis protoplasts and HEK293 cells. A putative scrambling domain (SCRD) was identified on the basis of sequence conservation and conferred PLS to transfected HEK293 cells, when grafted into the backbone of the non‐scrambling plasma membrane‐localized TMEM16A chloride channel. Finally, AtTMEM16 ‘gain‐of‐function’ variants gave rise to cellular phenotypes typical of aberrant scramblase activity, which were reversed by the additional introduction of a ‘loss‐of‐function’ mutation into the SCRD. In conclusion, our data suggest AtTMEM16 works as an ER‐resident lipid scramblase in Arabidopsis.

Imaging of CPP Delivery Mechanisms of Oligonucleotides.

Cationic cell-penetrating peptides spontaneously associate with negatively charged oligonucleotides to form submicron nanoparticles, so-called polyplexes. Contact with cells leads to endosomal uptake of these nanoparticles. Oligonucleotide activity critically depends on endosomal release and finally dissociation of polyplexes. Fluorescence provides a highly powerful means to follow the spatial dynamics of oligonucleotide uptake, trafficking and decomplexation, in particular when combined with markers of subcellular compartments that enable a quantitative analysis of colocalization and thereby mapping of trafficking routes. In this chapter, we describe protocols for a highly defined formation of polyplexes. We then point out the use of fluorescent fusion proteins to identify subcellular trafficking compartments and image analysis protocols to obtain quantitative information on trafficking routes and endosomal release.

Oligomerization of yeast α-factor receptor detected by fluorescent energy transfer between ligands

G-protein-coupled receptors (GPCRs) comprise a large superfamily of transmembrane receptors responsible for transducing responses to the binding of a wide variety of hormones, neurotransmitters, ions, and other small molecules. There is extensive evidence that GPCRs exist as homo-and hetero-oligomeric complexes; however, in many cases, the role of oligomerization and the extent to which it occurs at low physiological levels of receptor expression in cells remain unclear. We report here the use of flow cytometry to detect receptor-receptor interactions based on fluorescence resonance energy transfer between fluorescently labeled cell-impermeant ligands bound to yeast α-mating pheromone receptors that are members of the GPCR superfamily. A novel, to our knowledge, procedure was used to analyze energy transfer as a function of receptor occupancy by donor and acceptor ligands. Measurements of loss of donor fluorescence due to energy transfer in cells expressing high levels of receptors were used to calibrate measurements of enhanced acceptor emission due to energy transfer in cells expressing low levels of receptors. The procedure allows determination of energy transfer efficiencies over a 50-fold range of expression of full-length receptors at the surface of living cells without the need to create fluorescent or bioluminescent fusion proteins. Energy transfer efficiencies for fluorescently labeled derivatives of the receptor agonist α-factor do not depend on receptor expression level and are unaffected by C-terminal truncation of receptors. Fluorescently labeled derivatives of α-factor that act as receptor antagonists exhibit higher transfer efficiencies than those for labeled agonists. Although the approach cannot determine the number of receptors per oligomer, these results demonstrate that ligand-bound, native α-factor receptors exist as stable oligomers in the cell membranes of intact yeast cells at normal physiological expression levels and that the extent of oligomer formation is not dependent on the concentration of receptors in the membrane.

Characterization of the Bifunctional Enzyme BioDA Involved in Biotin Synthesis and Pathogenicity in Aspergillus flavus.

Biotin is an important enzyme cofactor that plays a key role in all three domains. The classical bifunctional enzyme BioDA in eukaryotes (such as Aspergillus flavus and Arabidopsis thaliana) is involved in the antepenultimate and penultimate steps of biotin biosynthesis. In this study, we identified a A. flavus bifunctional gene bioDA which could complement both Escherichia coli ΔEcbioD and ΔEcbioA mutants. Interestingly, the separated domain of AfBioD and AfBioA could, respectively, fuse with EcBioA and EcBioD well and work together. What is more, we found that BioDA was almost localized to the mitochondria in A. flavus, as shown by N-terminal red fluorescent protein tag fusion. Noteworthy, the subcellular localization of AfBioDA is never affected by common environmental stresses (such as hyperosmotic stress or oxidative stress). The knockout strategy demonstrated that the deletion of AfbioDA gene from the chromosome impaired the biotin de novo synthesis pathway in A. flavus. Importantly, this A. flavus mutant blocked biotin production and decreased its pathogenicity to infect peanuts. Based on the structural comparison, we found that two inhibitors (amiclenomycin and gemcitabine) could be candidates for antifungal drugs. Taken together, our findings identified the bifunctional AfbioDA gene and shed light on biotin biosynthesis in A. flavus.

NBS-LRR Gene TaRPS2 is Positively Associated with the High-Temperature Seedling Plant Resistance of Wheat Against Puccinia striiformis f. sp. tritici.

Xiaoyan6 (XY6) is a wheat (Triticum aestivum) cultivar possessing nonrace-specific high-temperature seedling plant (HTSP) resistance against stripe rust, caused by Puccinia striiformis f. sp. tritici. Previously, we identified one particular gene, TaRPS2, for its involvement in the HTSP resistance. To elucidate the role of TaRPS2 in the HTSP resistance, we cloned the full length of TaRPS2 from XY6. The transcriptional expression of TaRPS2 was rapidly upregulated (19.11-fold) under the normal-high-normal temperature treatment that induces the HTSP resistance. The expression level of TaRPS2 in leaves was higher than that in the stems and roots. Quantification of the endogenous hormones in wheat leaves after P. striiformis f. sp. tritici inoculation showed that 1-aminocyclopropane-1-carboxylic acid, salicylic acid (SA), and jasmonic acid were involved in the HTSP resistance. In addition, detection of hydrogen peroxide (H2O2) accumulation indicated that reactive oxygen species burst was also associated with the HTSP resistance. Two hours after exogenous H2O2 treatment or 0.5 h after SA treatment, the expression level of TaRPS2 was increased by 2.66 and 2.35 times, respectively. The subcellular localization of enhanced green fluorescent protein-TaRPS2 fusion protein was in the nuclei and plasma membranes. Virus-induced gene silencing of TaRPS2 reduced the level of HTSP resistance in XY6. Compared with the nonsilenced leaves, the TaRPS2-silenced leaves had the reduction of necrotic cells but a greater number of uredinia. These results indicated that TaRPS2 positively regulates the HTSP resistance of XY6 against P. striiformis f. sp. tritici and is related to the SA and H2O2 signaling pathways.

Tracking single particles for hours via continuous DNA-mediated fluorophore exchange

Monitoring biomolecules in single-particle tracking experiments is typically achieved by employing fixed organic dyes or fluorescent fusion proteins linked to a target of interest. However, photobleaching typically limits observation times to merely a few seconds, restricting downstream statistical analysis and observation of rare biological events. Here, we overcome this inherent limitation via continuous fluorophore exchange using DNA-PAINT, where fluorescently-labeled oligonucleotides reversibly bind to a single-stranded DNA handle attached to the target molecule. Such versatile and facile labeling allows uninterrupted monitoring of single molecules for extended durations. We demonstrate the power of our approach by observing DNA origami on membranes for tens of minutes, providing perspectives for investigating cellular processes on physiologically relevant timescales.

A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress.

Simple Summaryγ-H2AX, a phosphorylated variant of histone H2A, is a widely used biomarker of DNA replication stress. To develop an immunological probe able to detect and track γ-H2AX in live cancer cells, we have isolated single domain antibodies (called nanobodies) that are easily expressed as functional recombinant proteins and here we report the extensive characterization of a novel nanobody that specifically recognizes γ-H2AX. The interaction of this nanobody with the C-terminal end of γ-H2AX was determined by X-ray crystallography. Moreover, the generation of a bivalent nanobody allowed us to precisely detect γ-H2AX foci in drug-treated cells as efficiently as with commercially available conventional antibodies. Furthermore, we tracked γ-H2AX foci in live cells upon intracellular delivery of the bivalent nanobody fused to the red fluorescent protein dTomato, making, consequently, this new cost-effective reagent useful for studying drug-induced replication stress in both fixed and living cancer cells.Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.