Protein-Protein Interactions Visualized by Bimolecular Fluorescence Complementation in Arabidopsis thaliana Protoplasts from Leaf.

Bimolecular fluorescence complementation (BiFC) is a powerful tool for studying protein-protein interactions in living cells. By fusing interacting proteins to fluorescent protein fragments, BiFC allows visualization of spatial localization patterns of protein complexes. This method has been adapted to a variety of expression systems in different organisms and is widely used to study protein interactions in plant cells. The Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana (N. benthamiana) leaf cells is widely used, but in this chapter, a method for BiFC assay using Arabidopsis thaliana protoplasts is presented.

Expression, Purification, and Determination of Sensitivity to Calcium Ions of Ctenophore Photoproteins.

Light-sensitive Ca2+-regulated photoproteins of ctenophores are single-chain polypeptide proteins of 206-208 amino acids in length comprising three canonical EF-hand Ca2+-binding sites, each of 12 contiguous residues. These photoproteins are a stable complex of apoprotein and 2-hydroperoxy adduct of coelenterazine. Addition of calcium ions to photoprotein is only required to trigger bright bioluminescence. However, in contrast to the related Ca2+-regulated photoproteins of jellyfish their capacity to bioluminescence disappears on exposure to light over the entire absorption spectral range of ctenophore photoproteins. Here, we describe protocols for expression of gene encoding ctenophore photoprotein in Escherichia coli cells, obtaining of the recombinant apoprotein of high purity and its conversion into active photoprotein with synthetic coelenterazine as well as determination of its sensitivity to calcium ions using light-sensitive Ca2+-regulated photoprotein berovin from ctenophore Beroe abyssicola as an illustrative case.

UFObow: A single-wavelength excitable Brainbow for simultaneous multicolor ex-vivo and in-vivo imaging of mammalian cells.

Brainbow is a genetic cell-labeling technique that allows random colorization of multiple cells and real-time visualization of cell fate within a tissue, providing valuable insights into understanding complex biological processes. However, fluorescent proteins (FPs) in Brainbow have distinct excitation spectra with peak difference greater than 35 nm, which requires sequential imaging under multiple excitations and thus leads to long acquisition times. In addition, they are not easily used together with other fluorophores due to severe spectral bleed-through. Here, we report the development of a single-wavelength excitable Brainbow, UFObow, incorporating three newly developed blue-excitable FPs. We have demonstrated that UFObow enables not only tracking the growth dynamics of tumor cells in vivo but also mapping spatial distribution of immune cells within a sub-cubic centimeter tissue, revealing cell heterogeneity. This provides a powerful means to explore complex biology in a simultaneous imaging manner at a single-cell resolution in organs or in vivo.

Simultaneously Monitoring Multiple Autophagic Processes and Assessing Autophagic Flux in Single Cells by In Situ Fluorescence Cross-Correlation Spectroscopy.

Autophagy is a widely conserved and multistep cellular catabolic process and maintains cellular homeostasis and normal cellular functions via the degradation of some harmful intracellular components. It was reported that high basal autophagic activity may be closely related to tumorigenesis. So far, the fluorescence imaging technique has been widely used to study autophagic processes, but this method is only suitable for distinguishing autophagosomes and autolysosomes. Simultaneously monitoring multiple autophagic processes remains a significant challenge due to the lack of an efficient detection method. Here, we demonstrated a new method for simultaneously monitoring multiple autophagic processes and assessing autophagic flux in single cells based on in situ fluorescence cross-correlation spectroscopy (FCCS). In this study, microtubule-associated protein 1A/1B-light chain 3B (LC3B) was fused with two tandem fluorescent proteins [mCherry red fluorescent protein (mCherry) and enhanced green fluorescent protein (EGFP)] to achieve the simultaneous labeling and distinguishing of multiple autophagic structures based on the differences in characteristic diffusion time (τD). Furthermore, we proposed a new parameter "delivery efficiency of autophagosome (DEAP)" to assess autophagic flux based on the cross correlation (CC) value. Our results demonstrate that FCCS can efficiently distinguish three autophagic structures, assess the induced autophagic flux, and discriminate different autophagy regulators. Compared with the commonly used fluorescence imaging technique, the resolution of FCCS remains unaffected by Brownian motion and fluorescent monomers in the cytoplasm and is well suitable to distinguishing differently colored autophagic structures and monitoring autophagy.

Red fluorescent proteins engineered from green fluorescent proteins.

Fluorescent proteins (FPs) form a fluorophore through autocatalysis from three consecutive amino acid residues within a polypeptide chain. The two major groups, green FPs (GFPs) and red FPs (RFPs), have distinct fluorophore structures; RFPs have an extended π-conjugation system with an additional double bond. However, due to the low sequence homology between the two groups, amino acid residues essential for determining the different fluorophore structures were unclear. Therefore, engineering a GFP into an RFP has been challenging, and the exact mechanism of how GFPs and RFPs achieve different autocatalytic reactions remained elucidated. Here, we show the conversion of two coral GFPs, AzamiGreen (AG) and mcavGFP, into RFPs by defined mutations. Structural comparison of AG and AzamiRed1.0, an AG-derived RFP, revealed that the mutations triggered drastic rearrangements in the interaction networks between amino acid residues around the fluorophore, suggesting that coordinated multisite mutations are required for the green-to-red conversion. As a result of the structural rearrangements, a cavity suitable for the entry of an oxygen molecule, which is necessary for the double bond formation of the red fluorophores, is created in the proximity of the fluorophore. We also show that a monomeric variant of AzamiRed1.0 can be used for labeling organelles and proteins in mammalian cells. Our results provide a structural basis for understanding the red fluorophore formation mechanism and demonstrate that protein engineering of GFPs is a promising way to create RFPs suitable for fluorescent tags.

Detecting Protein-Protein Interactions Using Bimolecular Fluorescence Complementation (BiFC) and Luciferase Complementation Assays (LCA).

In multicellular organisms, establishing the full body plane involves cell-cell signaling where protein associations are important for the diverse cellular functions within the cells. For the study of protein-protein interactions (PPI), bimolecular fluorescence complementation (BiFC) and luciferase complementation assays (LCA) have proven to be reliable tools that can be used to confirm the physical association of two proteins in a semi-in vivo environment. This chapter provides a detailed description of these two techniques using Nicotiana benthamiana as a semi-in vivo transient expression system. As an example, we will use the interaction of the two well-described transcription factors SHORT-ROOT (SHR) and SCARECROW (SCR), which are known as regulators of asymmetric cell division and stem cell specification in the root meristem of the model plant Arabidopsis thaliana. While the BiFC assay provides subcellular information by displaying a fluorescence signal, nuclear in this case, resulting from the reconstituted fluorophore, the LCA generates a quantitative readout of the SCR-SHR interaction. The combination of both assays provides information on the localization and strength of the PPI.

mScarlet3: a brilliant and fast-maturing red fluorescent protein.

We report the evolution of mScarlet3, a cysteine-free monomeric red fluorescent protein with fast and complete maturation, as well as record brightness, quantum yield (75%) and fluorescence lifetime (4.0 ns). The mScarlet3 crystal structure reveals a barrel rigidified at one of its heads by a large hydrophobic patch of internal residues. mScarlet3 behaves well as a fusion tag, displays no apparent cytotoxicity and it surpasses existing red fluorescent proteins as a Förster resonance energy transfer acceptor and as a reporter in transient expression systems.

Chemically stable fluorescent proteins for advanced microscopy.

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.

Impact of Double Covalent Binding of BV in NIR FPs on Their Spectral and Physicochemical Properties.

Understanding the photophysical properties and stability of near-infrared fluorescent proteins (NIR FPs) based on bacterial phytochromes is of great importance for the design of efficient fluorescent probes for use in cells and in vivo. Previously, the natural ligand of NIR FPs biliverdin (BV) has been revealed to be capable of covalent binding to the inherent cysteine residue in the PAS domain (Cys15), and to the cysteine residue introduced into the GAF domain (Cys256), as well as simultaneously with these two residues. Here, based on the spectroscopic analysis of several NIR FPs with both cysteine residues in PAS and GAF domains, we show that the covalent binding of BV simultaneously with two domains is the reason for the higher quantum yield of BV fluorescence in these proteins as a result of rigid fixation of the chromophore in their chromophore-binding pocket. We demonstrate that since the attachment sites are located in different regions of the polypeptide chain forming a figure-of-eight knot, their binding to BV leads to shielding of many sites of proteolytic degradation due to additional stabilization of the entire protein structure. This makes NIR FPs with both cysteine residues in PAS and GAF domains less susceptible to cleavage by intracellular proteases.

Bimolecular Fluorescence Complementation to Test for Protein-Protein Interactions and to Uncover Regulatory Mechanisms During Gametogenesis.

Bimolecular fluorescence complementation (BiFC) assay is one of the sensitive techniques that allows to investigate direct protein-protein interactions (PPI) in vivo and visualize the subcellular localization of interacting proteins. It is based on splitting of a fluorescent protein into two nonfluorescent parts accordingly fused to two putative interacting partners. If interaction between studied proteins is possible, nonfluorescent parts come to close proximity resulting in reconstitution of the functional fluorescent protein and giving fluorescence under certain wavelength. BiFC analysis implies transient or stable expression of the proteins of interest and can be used as a method to test or validate the direct PPI in various biological pathways, including the regulation of gametogenesis, which is the main focus of this book. In our protocol we give detailed information for beginners about three main steps of BiFC analysis of centromeric protein interactions. These steps include (1) generation of appropriate expression clones with the help of Gateway cloning technology, (2) infiltration of Nicotiana benthamiana plants by Agrobacteria containing generated constructs, and (3) microscopic analysis of plants under fluorescence microscope. Also, we discuss appropriate negative controls that can be used for evaluation as well as recommendable vector systems, possible artifacts and measures to avoid artifactual interactions for BiFC assay.

Dynamic assembly of the mRNA m6A methyltransferase complex is regulated by METTL3 phase separation.

m6A methylation is the most abundant and reversible chemical modification on mRNA with approximately one-fourth of eukaryotic mRNAs harboring at least one m6A-modified base. The recruitment of the mRNA m6A methyltransferase writer complex to phase-separated nuclear speckles is likely to be crucial in its regulation; however, control over the activity of the complex remains unclear. Supported by our observation that a core catalytic subunit of the methyltransferase complex, METTL3, is endogenously colocalized within nuclear speckles as well as in noncolocalized puncta, we tracked the components of the complex with a Cry2-METTL3 fusion construct to disentangle key domains and interactions necessary for the phase separation of METTL3. METTL3 is capable of self-interaction and likely provides the multivalency to drive condensation. Condensates in cells necessarily contain myriad components, each with partition coefficients that establish an entropic barrier that can regulate entry into the condensate. In this regard, we found that, in contrast to the constitutive binding of METTL14 to METTL3 in both the diffuse and the dense phase, WTAP only interacts with METTL3 in dense phase and thereby distinguishes METTL3/METTL14 single complexes in the dilute phase from METTL3/METTL14 multicomponent condensates. Finally, control over METTL3/METTL14 condensation is determined by its small molecule cofactor, S-adenosylmethionine (SAM), which regulates conformations of two gate loops, and some cancer-associated mutations near gate loops can impair METTL3 condensation. Therefore, the link between SAM binding and the control of writer complex phase state suggests that the regulation of its phase state is a potentially critical facet of its functional regulation.

Multicolor strategies for investigating clonal expansion and tissue plasticity.

Understanding the generation of complexity in living organisms requires the use of lineage tracing tools at a multicellular scale. In this review, we describe the different multicolor strategies focusing on mouse models expressing several fluorescent reporter proteins, generated by classical (MADM, Brainbow and its multiple derivatives) or acute (StarTrack, CLoNe, MAGIC Markers, iOn, viral vectors) transgenesis. After detailing the multi-reporter genetic strategies that serve as a basis for the establishment of these multicolor mouse models, we briefly mention other animal and cellular models (zebrafish, chicken, drosophila, iPSC) that also rely on these constructs. Then, we highlight practical applications of multicolor mouse models to better understand organogenesis at single progenitor scale (clonal analyses) in the brain and briefly in several other tissues (intestine, skin, vascular, hematopoietic and immune systems). In addition, we detail the critical contribution of multicolor fate mapping strategies in apprehending the fine cellular choreography underlying tissue morphogenesis in several models with a particular focus on brain cytoarchitecture in health and diseases. Finally, we present the latest technological advances in multichannel and in-depth imaging, and automated analyses that enable to better exploit the large amount of data generated from multicolored tissues.

Long-term imaging of living adult zebrafish.

The zebrafish has become a widely used animal model due, in large part, to its accessibility to and usefulness for high-resolution optical imaging. Although zebrafish research has historically focused mostly on early development, in recent years the fish has increasingly been used to study regeneration, cancer metastasis, behavior and other processes taking place in juvenile and adult animals. However, imaging of live adult zebrafish is extremely challenging, with survival of adult fish limited to a few tens of minutes using standard imaging methods developed for zebrafish embryos and larvae. Here, we describe a new method for imaging intubated adult zebrafish using a specially designed 3D printed chamber for long-term imaging of adult zebrafish on inverted microscope systems. We demonstrate the utility of this new system by nearly day-long observation of neutrophil recruitment to a wound area in living double-transgenic adult casper zebrafish with fluorescently labeled neutrophils and lymphatic vessels, as well as intubating and imaging the same fish repeatedly. We also show that Mexican cavefish can be intubated and imaged in the same way, demonstrating this method can be used for long-term imaging of adult animals from diverse aquatic species.

N-extended photoprotein obelin to competitively detect small protein tumor markers.

Two variants of Ca2+-regulated photoprotein obelin, extended from the N-terminus with small tumor markers - melanoma inhibitory activity protein (MIA) and survivin, one of the protein inhibitors of apoptosis, were designed, obtained and studied. Both domains in the obtained hybrid proteins exhibit the properties of the initial molecules: the main features of Ca2+-triggered bioluminescence are close to those of obelin, and the tumor markers' domains are recognized and bound by the corresponding antibodies. The obtained hybrids compete with the corresponding tumor markers for binding with antibodies, immobilized on the surface and their use has been shown to be promising as bioluminescent labels in a one-stage solid-phase competitive immunoassay.

Generation of a NES-mScarlet Red Fluorescent Reporter Human iPSC Line for Live Cell Imaging and Flow Cytometric Analysis and Sorting Using CRISPR-Cas9-Mediated Gene Editing.

Advances in the regenerative stem cell field have propelled the generation of tissue-specific cells in the culture dish for subsequent transplantation, drug screening purposes, or the elucidation of disease mechanisms. One major obstacle is the heterogeneity of these cultures, in which the tissue-specific cells of interest usually represent only a fraction of all generated cells. Direct identification of the cells of interest and the ability to specifically isolate these cells in vitro is, thus, highly desirable for these applications. The type VI intermediate filament protein NESTIN is widely used as a marker for neural stem/progenitor cells (NSCs/NPCs) in the developing and adult central and peripheral nervous systems. Applying CRISPR-Cas9 technology, we have introduced a red fluorescent reporter (mScarlet) into the NESTIN (NES) locus of a human induced pluripotent stem cell (hiPSC) line. We describe the generation and characterization of NES-mScarlet reporter hiPSCs and demonstrate that this line is an accurate reporter of NSCs/NPCs during their directed differentiation into human midbrain dopaminergic (mDA) neurons. Furthermore, NES-mScarlet hiPSCs can be used for direct identification during live cell imaging and for flow cytometric analysis and sorting of red fluorescent NSCs/NPCs in this paradigm.

High Incidence of Lymph-node Metastasis in a Pancreatic-cancer Patient-derived Orthotopic Xenograft (PDOX) NOG-Mouse Model.

BACKGROUND/AIM: Our laboratory pioneered the patient-derived orthotopic xenograft (PDOX) model. An important goal of PDOX-model development is facile visualization of metastasis in live mice. In the present report we evaluated tumor growth and metastasis in pancreatic cancer PDOX NOG [Non-obese diabetes (NOD)/Scid/IL2Rγnull]-and nude-mouse models using red fluorescent protein (RFP)-expressing tumor stroma to visualize the primary tumor and metastasis. MATERIALS AND METHODS: A patient-derived pancreatic cancer was initially implanted in transgenic RFP-expressing nude mice. Then, tumor fragments, which acquired RFP expressing stroma while growing in RFP-expressing nude mice were orthotopically implanted in nude and NOG mice. The primary pancreatic tumor and metastasis were observed 8 weeks after implantation. RESULTS: Lymph-node metastases expressing red fluorescence were detected only in NOG mice. Significantly faster growth of primary pancreatic tumors and a higher incidence of lymph-node metastasis occurred in NOG mice compared to nude mice. CONCLUSION: RFP-expressing tumor stroma, which traffics together with cancer cells to lymph nodes, is useful to observe tumor behavior, such as lymph-node metastasis in a PDOX NOG-mouse model which can be used for evaluation of novel anti-metastatic agents, as well as personalized therapy to identify effective drugs.

Efficient gene editing through an intronic selection marker in cells.

BACKGROUND: Gene editing technology has provided researchers with the ability to modify genome sequences in almost all eukaryotes. Gene-edited cell lines are being used with increasing frequency in both bench research and targeted therapy. However, despite the great importance and universality of gene editing, the efficiency of homology-directed DNA repair (HDR) is too low, and base editors (BEs) cannot accomplish desired indel editing tasks. RESULTS AND DISCUSSION: Our group has improved HDR gene editing technology to indicate DNA variation with an independent selection marker using an HDR strategy, which we named Gene Editing through an Intronic Selection marker (GEIS). GEIS uses a simple process to avoid nonhomologous end joining (NHEJ)-mediated false-positive effects and achieves a DsRed positive rate as high as 87.5% after two rounds of fluorescence-activated cell sorter (FACS) selection without disturbing endogenous gene splicing and expression. We re-examined the correlation of the conversion tract and efficiency, and our data suggest that GEIS has the potential to edit approximately 97% of gene editing targets in human and mouse cells. The results of further comprehensive analysis suggest that the strategy may be useful for introducing multiple DNA variations in cells.

DogCatcher allows loop-friendly protein-protein ligation.

There are many efficient ways to connect proteins at termini. However, connecting at a loop is difficult because of lower flexibility and variable environment. Here, we have developed DogCatcher, a protein that forms a spontaneous isopeptide bond with DogTag peptide. DogTag/DogCatcher was generated initially by splitting a Streptococcus pneumoniae adhesin. We optimized DogTag/DogCatcher through rational design and evolution, increasing reaction rate by 250-fold and establishing millimolar solubility of DogCatcher. When fused to a protein terminus, DogTag/DogCatcher reacts slower than SpyTag003/SpyCatcher003. However, inserted in loops of a fluorescent protein or enzyme, DogTag reacts much faster than SpyTag003. Like many membrane proteins, the ion channel TRPC5 has no surface-exposed termini. DogTag in a TRPC5 extracellular loop allowed normal calcium flux and specific covalent labeling on cells in 1 min. DogTag/DogCatcher reacts under diverse conditions, at nanomolar concentrations, and to 98% conversion. Loop-friendly ligation should expand the toolbox for creating protein architectures.

Detecting Förster resonance energy transfer in living cells by conventional and spectral flow cytometry.

Assays based on Förster resonance energy transfer (FRET) can be used to study many processes in cell biology. Although this is most often done with microscopy for fluorescence detection, we report two ways to measure FRET in living cells by flow cytometry. Using a conventional flow cytometer and the "3-cube method" for intensity-based calculation of FRET efficiency, we measured the enzymatic activity of specific kinases in cells expressing a genetically-encoded reporter. For both AKT and protein kinase A, the method measured kinase activity in time-course, dose-response, and kinetic assays. Using the Cytek Aurora spectral flow cytometer, which applies linear unmixing to emission measured in multiple wavelength ranges, FRET from the same reporters was measured with greater single-cell precision, in real time and in the presence of other fluorophores. Results from gene-knockout studies suggested that spectral flow cytometry might enable the sorting of cells on the basis of FRET. The methods we present provide convenient and flexible options for using FRET with flow cytometry in studies of cell biology.

Structural and Functional Characterization of a Biliverdin-Binding Near-Infrared Fluorescent Protein From the Serpin Superfamily.

Biliverdin-binding serpins (BBSs) are proteins that are responsible for coloration in amphibians and fluoresce in the near-infrared (NIR) spectral region. Here we produced the first functional recombinant BBS of the polka-dot treefrog Boana punctata (BpBBS), assembled with its biliverdin (BV) chromophore, and report its biochemical and photochemical characterization. We determined the crystal structure of BpBBS at 2.05 Å resolution, which demonstrated its structural homology to the mammalian protease inhibitor alpha-1-antitrypsin. BV interaction with BpBBS was studied and it was found that the N-terminal polypeptide (residues 19-50) plays a critical role in the BV binding. By comparing BpBBS with the available NIR fluorescent proteins and expressing it in mammalian cells, we demonstrated its potential as a NIR imaging probe. These results provide insight into the non-inhibitory function of serpins, provide a basis for improving their performance in mammalian cells, and suggest possible paths for the development of BBS-based fluorescent probes.