Single-Molecule Analysis of Transcription Dynamics to Understand the Relationship Between Epigenetic Alterations and Transcriptional Variability.

Heterogeneity in gene expression largely stems from the discontinuous nature of transcription, with transcripts being produced in bursts with defined frequencies. This cell-to-cell variability in transcription within isogenic cell populations is a known phenomenon across numerous genes. Multiple gene regulatory and epigenetic factors have been identified as key contributors to this pulsatile gene activity. Understanding the effects of epigenetic modulation on transcriptional cell-to-cell variability and kinetics of transcriptional activity is crucial for interpreting changes in treatment responsiveness. We present a detailed protocol that guides the assessment of fluctuations in gene expression induced by epigenetic modulation using single-molecule RNA in situ hybridization (smRNA FISH) combined with confocal microscopy imaging, data analysis, and quantification in breast cancer cells. Through smRNA FISH labeling, both mature and nascent transcripts are identified. Subsequently, the number of mature transcripts and the intensity and frequency of nascent transcripts are quantified, and these measurements are used to calculate the burst size and frequency for the labeled gene. By following this step-by-step methodology, insights are obtained into the intricate relationship between epigenetic alterations and the dynamic nature of gene expression in breast cancer cells.

A turquoise fluorescence lifetime-based biosensor for quantitative imaging of intracellular calcium.

The most successful genetically encoded calcium indicators (GECIs) employ an intensity or ratiometric readout. Despite a large calcium-dependent change in fluorescence intensity, the quantification of calcium concentrations with GECIs is problematic, which is further complicated by the sensitivity of all GECIs to changes in the pH in the biological range. Here, we report on a sensing strategy in which a conformational change directly modifies the fluorescence quantum yield and fluorescence lifetime of a circular permutated turquoise fluorescent protein. The fluorescence lifetime is an absolute parameter that enables straightforward quantification, eliminating intensity-related artifacts. An engineering strategy that optimizes lifetime contrast led to a biosensor that shows a 3-fold change in the calcium-dependent quantum yield and a fluorescence lifetime change of 1.3 ns. We dub the biosensor Turquoise Calcium Fluorescence LIfeTime Sensor (Tq-Ca-FLITS). The response of the calcium sensor is insensitive to pH between 6.2-9. As a result, Tq-Ca-FLITS enables robust measurements of intracellular calcium concentrations by fluorescence lifetime imaging. We demonstrate quantitative imaging of calcium concentrations with the turquoise GECI in single endothelial cells and human-derived organoids.

Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration.

Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, alhough it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.

Identification of guanine nucleotide exchange factors that increase Cdc42 activity in primary human endothelial cells.

The Rho GTPase family is involved in actin dynamics and regulates the barrier function of the endothelium. One of the main barrier-promoting Rho GTPases is Cdc42, also known as cell division control protein 42 homolog. Currently, regulation of Cdc42-based signalling networks in endothelial cells (ECs) lack molecular details. To examine these, we focused on a subset of 15 Rho guanine nucleotide exchange factors (GEFs), which are expressed in the endothelium. By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1. A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the overexpressed full-length GEFs. Our data reveal GEF dependent activation of Cdc42, with the most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and PREX1 and the highest selectivity for FGD1. Additionally, we generated truncated GEF constructs that comprise only the catalytic dbl homology (DH) domain or together with the adjacent pleckstrin homology domain (DHPH). The DH domain by itself did not activate Cdc42, whereas the DHPH domain of ITSN1, ITSN2 and PLEKHG1 showed activity towards Cdc42. Together, our study characterized endothelial GEFs that may directly or indirectly activate Cdc42, which will be of great value for the field of vascular biology.

Temporal tracking of quantum-dot apatite across in vitro mycorrhizal networks shows how host demand can influence fungal nutrient transfer strategies.

Arbuscular mycorrhizal fungi function as conduits for underground nutrient transport. While the fungal partner is dependent on the plant host for its carbon (C) needs, the amount of nutrients that the fungus allocates to hosts can vary with context. Because fungal allocation patterns to hosts can change over time, they have historically been difficult to quantify accurately. We developed a technique to tag rock phosphorus (P) apatite with fluorescent quantum-dot (QD) nanoparticles of three different colors, allowing us to study nutrient transfer in an in vitro fungal network formed between two host roots of different ages and different P demands over a 3-week period. Using confocal microscopy and raster image correlation spectroscopy, we could distinguish between P transfer from the hyphae to the roots and P retention in the hyphae. By tracking QD-apatite from its point of origin, we found that the P demands of the younger root influenced both: (1) how the fungus distributed nutrients among different root hosts and (2) the storage patterns in the fungus itself. Our work highlights that fungal trade strategies are highly dynamic over time to local conditions, and stresses the need for precise measurements of symbiotic nutrient transfer across both space and time.

Multiparameter screening method for developing optimized red-fluorescent proteins.

Genetically encoded fluorescent proteins (FPs) are highly utilized in cell biology research to study proteins of interest or signal processes using biosensors. To perform well in specific applications, these FPs require a multitude of tailored properties. It is for this reason that they need to be optimized by using mutagenesis. The optimization process through screening is often based solely on bacterial colony brightness, but multiple parameters ultimately determine the performance of an optimal FP. Instead of characterizing other properties after selection, we developed a multiparameter screening method based on four critical parametersscreened simultaneously: fluorescence lifetime, cellular brightness, maturation efficiency, and photostability. First, a high-throughput primary screen (based on fluorescence lifetime and cellular brightness using a mutated FP library) is performed in bacterial colonies. A secondary multiparameter screen based on all four parameters, using a novel bacterial-mammalian dual-expression vector enables expression of the best FP variants in mammalian cell lines. A newly developed automated multiparameter acquisition and cell-based analysis approach for 96-well plates further increased workflow efficiency. We used this protocol to yield the record-bright mScarlet, a fast-maturating mScarlet-I, and a photostable mScarlet-H. This protocol can also be applied to other FP classes or Förster resonance energy transfer (FRET)-based biosensors with minor adaptations. With an available mutant library of a template FP and a complete and tested laboratory setup, a single round of multiparameter screening (including the primary bacterial screen, secondary mammalian cell screen, sequencing, and data processing) can be performed within 2 weeks.

Mycorrhizal Fungi Respond to Resource Inequality by Moving Phosphorus from Rich to Poor Patches across Networks.

The world's ecosystems are characterized by an unequal distribution of resources [1]. Trade partnerships between organisms of different species-mutualisms-can help individuals cope with such resource inequality [2-4]. Trade allows individuals to exchange commodities they can provide at low cost for resources that are otherwise impossible or more difficult to access [5, 6]. However, as resources become increasingly patchy in time or space, it is unknown how organisms alter their trading strategies [7, 8]. Here, we show how a symbiotic fungus mediates trade with a host root in response to different levels of resource inequality across its network. We developed a quantum-dot-tracking technique to quantify phosphorus-trading strategies of arbuscular mycorrhizal fungi simultaneously exposed to rich and poor resource patches. By following fluorescent nanoparticles of different colors across fungal networks, we determined where phosphorus was hoarded, relocated, and transferred to plant hosts. We found that increasing exposure to inequality stimulated trade. Fungi responded to high resource variation by (1) increasing the total amount of phosphorus distributed to host roots, (2) decreasing allocation to storage, and (3) differentially moving resources within the network from rich to poor patches. Using single-particle tracking and high-resolution video, we show how dynamic resource movement may help the fungus capitalize on value differences across the trade network, physically moving resources to areas of high demand to gain better returns. Such translocation strategies can help symbiotic organisms cope with exposure to resource inequality.

PlotsOfData-A web app for visualizing data together with their summaries.

Reporting of the actual data in graphs and plots increases transparency and enables independent evaluation. On the other hand, data summaries are often used in graphs because they aid interpretation. To democratize state-of-the-art data visualization of raw data with a selection of statistical summaries, a freely available, open-source web app was written using R/shiny that uses the ggplot2 package for generating plots. Users can to choose how to display the data and which of the data summaries to add. In addition, the 95% confidence intervals (95CIs) can be added for visual inferences. By adjusting the visibility of the layers, the visualization of the raw data and their summaries can be tuned for optimal presentation and interpretation. The app is dubbed PlotsOfData and is available at https://huygens.science.uva.nl/PlotsOfData/.

Characterization of a spectrally diverse set of fluorescent proteins as FRET acceptors for mTurquoise2.

The performance of Förster Resonance Energy Transfer (FRET) biosensors depends on brightness and photostability, which are dependent on the characteristics of the fluorescent proteins that are employed. Yellow fluorescent protein (YFP) is often used as an acceptor but YFP is prone to photobleaching and pH changes. In this study, we evaluated the properties of a diverse set of acceptor fluorescent proteins in combination with the optimized CFP variant mTurquoise2 as the donor. To determine the theoretical performance of acceptors, the Förster radius was determined. The practical performance was determined by measuring FRET efficiency and photostability of tandem fusion proteins in mammalian cells. Our results show that mNeonGreen is the most efficient acceptor for mTurquoise2 and that the photostability is better than SYFP2. The non-fluorescent YFP variant sREACh is an efficient acceptor, which is useful in lifetime-based FRET experiments. Among the orange and red fluorescent proteins, mCherry and mScarlet-I are the best performing acceptors. Several new pairs were applied in a multimolecular FRET based sensor for detecting activation of a heterotrimeric G-protein by G-protein coupled receptors. Overall, the sensor with mNeonGreen as acceptor and mTurquoise2 as donor showed the highest dynamic range in ratiometric FRET imaging experiments with the G-protein sensor.

In vivo FRET-FLIM reveals cell-type-specific protein interactions in Arabidopsis roots.

During multicellular development, specification of distinct cell fates is often regulated by the same transcription factors operating differently in distinct cis-regulatory modules, either through different protein complexes, conformational modification of protein complexes, or combinations of both. Direct visualization of different transcription factor complex states guiding specific gene expression programs has been challenging. Here we use in vivo FRET-FLIM (Förster resonance energy transfer measured by fluorescence lifetime microscopy) to reveal spatial partitioning of protein interactions in relation to specification of cell fate. We show that, in Arabidopsis roots, three fully functional fluorescently tagged cell fate regulators establish cell-type-specific interactions at endogenous expression levels and can form higher order complexes. We reveal that cell-type-specific in vivo FRET-FLIM distributions reflect conformational changes of these complexes to differentially regulate target genes and specify distinct cell fates.

mScarlet: a bright monomeric red fluorescent protein for cellular imaging.

We report the engineering of mScarlet, a truly monomeric red fluorescent protein with record brightness, quantum yield (70%) and fluorescence lifetime (3.9 ns). We developed mScarlet starting with a consensus synthetic template and using improved spectroscopic screening techniques; mScarlet's crystal structure reveals a planar and rigidified chromophore. mScarlet outperforms existing red fluorescent proteins as a fusion tag, and it is especially useful as a Förster resonance energy transfer (FRET) acceptor in ratiometric imaging.

Quantitative Single-Cell Analysis of Signaling Pathways Activated Immediately Downstream of Histamine Receptor Subtypes.

Genetically encoded biosensors based on Förster resonance energy transfer (FRET) can visualize responses of individual cells in real time. Here, we evaluated whether FRET-based biosensors provide sufficient contrast and specificity to measure activity of G-protein-coupled receptors. The four histamine receptor subtypes (H1R, H2R, H3R, and H4R) respond to the ligand histamine by activating three canonical heterotrimeric G-protein-mediated signaling pathways with a reported high degree of specificity. Using FRET-based biosensors, we demonstrate that H1R activates Gαq. We also observed that H1R activates Gαi, albeit at a 10-fold lower potency. In addition to increasing cAMP levels, most likely via Gαs, we found that the H2R induces Gαq-mediated calcium release. The H3R and H4R activated Gαi with high specificity and a high potency. We demonstrate that a number of FRET sensors provide sufficient contrast to: 1) analyze the specificity of the histamine receptor subtypes for different heterotrimeric G-protein families with single-cell resolution, 2) probe for antagonist specificity, and 3) allow the measurement of single-cell concentration-response curves.

Mathematics of Experimentally Generated Chemoattractant Gradients.

Many eukaryotic cells move in the direction of a chemical gradient. Several assays have been developed to measure this chemotactic response, but no complete mathematical models of the spatial and temporal gradients are available to describe the fundamental principles of chemotaxis. Here we provide analytical solutions for the gradients formed by release of chemoattractant from a point source by passive diffusion or forced flow (micropipettes) and gradients formed by laminar diffusion in a Zigmond chamber. The results show that gradients delivered with a micropipette are formed nearly instantaneously, are very steep close to the pipette, and have a steepness that is strongly dependent on the distance from the pipette. In contrast, gradients in a Zigmond chamber are formed more slowly, are nearly independent of the distance from the source, and resemble the temporal and spatial properties of the natural cAMP wave that Dictyostelium cells experience during cell aggregation.

Plasma membrane restricted RhoGEF activity is sufficient for RhoA-mediated actin polymerization.

The small GTPase RhoA is involved in cell morphology and migration. RhoA activity is tightly regulated in time and space and depends on guanine exchange factors (GEFs). However, the kinetics and subcellular localization of GEF activity towards RhoA are poorly defined. To study the mechanism underlying the spatiotemporal control of RhoA activity by GEFs, we performed single cell imaging with an improved FRET sensor reporting on the nucleotide loading state of RhoA. By employing the FRET sensor we show that a plasma membrane located RhoGEF, p63RhoGEF, can rapidly activate RhoA through endogenous GPCRs and that localized RhoA activity at the cell periphery correlates with actin polymerization. Moreover, synthetic recruitment of the catalytic domain derived from p63RhoGEF to the plasma membrane, but not to the Golgi apparatus, is sufficient to activate RhoA. The synthetic system enables local activation of endogenous RhoA and effectively induces actin polymerization and changes in cellular morphology. Together, our data demonstrate that GEF activity at the plasma membrane is sufficient for actin polymerization via local RhoA signaling.

Domain analysis of the Nematostella vectensis SNAIL ortholog reveals unique nucleolar localization that depends on the zinc-finger domains.

SNAIL transcriptional factors are key regulators during development and disease. They arose early during evolution, and in cnidarians such as Nematostella vectensis, NvSNAILA/B are detected in invaginating tissues during gastrulation. The function of SNAIL proteins is well established in bilaterians but their roles in cnidarians remain unknown. The structure of NvSNAILA and B is similar to the human SNAIL1 and 2, including SNAG and zinc-finger domains. Here, we performed a molecular analysis on localization and mobility of NvSNAILA/B using mammalian cells and Nematostella embryos. NvSNAILA/B display nuclear localization and mobility similar to HsSNAIL1/2. Strikingly, NvSNAILA is highly enriched in the nucleoli and shuttles between the nucleoli and the nucleoplasm. Truncation of the N-terminal SNAG domain, reported to contain Nuclear Localization Signals, markedly reduces nucleolar levels, without effecting nuclear localization or mobility. Truncation of the C-terminal zinc-fingers, involved in DNA binding in higher organisms, significantly affects subcellular localization and mobility. Specifically, the zinc-finger domains are required for nucleolar enrichment of NvSNAILA. Differently from SNAIL transcriptional factors described before, NvSNAILA is specifically enriched in the nucleoli co-localizing with nucleolar markers even after nucleolar disruption. Our findings implicate additional roles for SNAG and zinc-finger domains, suggesting a role for NvSNAILA in the nucleolus.

Cell age dependent concentration of Escherichia coli divisome proteins analyzed with ImageJ and ObjectJ.

The rod-shaped Gram-negative bacterium Escherichia coli multiplies by elongation followed by binary fission. Longitudinal growth of the cell envelope and synthesis of the new poles are organized by two protein complexes called elongasome and divisome, respectively. We have analyzed the spatio-temporal localization patterns of many of these morphogenetic proteins by immunolabeling the wild type strain MC4100 grown to steady state in minimal glucose medium at 28°C. This allowed the direct comparison of morphogenetic protein localization patterns as a function of cell age as imaged by phase contrast and fluorescence wide field microscopy. Under steady state conditions the age distribution of the cells is constant and is directly correlated to cell length. To quantify cell size and protein localization parameters in 1000s of labeled cells, we developed 'Coli-Inspector,' which is a project running under ImageJ with the plugin 'ObjectJ.' ObjectJ organizes image-analysis tasks using an integrated approach with the flexibility to produce different output formats from existing markers such as intensity data and geometrical parameters. ObjectJ supports the combination of automatic and interactive methods giving the user complete control over the method of image analysis and data collection, with visual inspection tools for quick elimination of artifacts. Coli-inspector was used to sort the cells according to division cycle cell age and to analyze the spatio-temporal localization pattern of each protein. A unique dataset has been created on the concentration and position of the proteins during the cell cycle. We show for the first time that a subset of morphogenetic proteins have a constant cellular concentration during the cell division cycle whereas another set exhibits a cell division cycle dependent concentration variation. Using the number of proteins present at midcell, the stoichiometry of the divisome is discussed.

In vivo imaging of Nematostella vectensis embryogenesis and late development using fluorescent probes.

BACKGROUND: Cnidarians are the closest living relatives to bilaterians and have been instrumental to studying the evolution of bilaterian properties. The cnidarian model, Nematostella vectensis, is a unique system in which embryology and regeneration are both studied, making it an ideal candidate to develop in vivo imaging techniques. Live imaging is the most direct way for quantitative and qualitative assessment of biological phenomena. Actin and tubulin are cytoskeletal proteins universally important for regulating many embryological processes but so far studies in Nematostella primarily focused on the localization of these proteins in fixed embryos. RESULTS: We used fluorescent probes expressed in vivo to investigate the dynamics of Nematostella development. Lifeact-mTurquoise2, a fluorescent cyan F-actin probe, can be visualized within microvilli along the cellular surface throughout embryonic development and is stable for two months after injection. Co-expression of Lifeact-mTurquoise2 with End-Binding protein1 (EB1) fused to mVenus or tdTomato-NLS allows for the visualization of cell-cycle properties in real time. Utilizing fluorescent probes in vivo helped to identify a concentrated 'flash' of Lifeact-mTurquoise2 around the nucleus, immediately prior to cytokinesis in developing embryos. Moreover, Lifeact-mTurquoise2 expression in adult animals allowed the identification of various cell types as well as cellular boundaries. CONCLUSION: The methods developed in this manuscript provide an alternative protocol to investigate Nematostella development through in vivo cellular analysis. This study is the first to utilize the highly photo-stable florescent protein mTurquoise2 as a marker for live imaging. Finally, we present a clear methodology for the visualization of minute temporal events during cnidarian development.

Nod factor receptors form heteromeric complexes and are essential for intracellular infection in medicago nodules.

Rhizobial Nod factors are the key signaling molecules in the legume-rhizobium nodule symbiosis. In this study, the role of the Nod factor receptors NOD FACTOR PERCEPTION (NFP) and LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) in establishing the symbiotic interface in root nodules was investigated. It was found that inside Medicago truncatula nodules, NFP and LYK3 localize at the cell periphery in a narrow zone of about two cell layers at the nodule apex. This restricted accumulation is narrower than the region of promoter activity/mRNA accumulation and might serve to prevent the induction of defense-like responses and/or to restrict the rhizobium release to precise cell layers. The distal cell layer where the receptors accumulate at the cell periphery is part of the meristem, and the proximal layer is part of the infection zone. In these layers, the receptors can most likely perceive the bacterial Nod factors to regulate the formation of symbiotic interface. Furthermore, our Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicates that NFP and LYK3 form heteromeric complexes at the cell periphery in M. truncatula nodules.

The fidelity of stochastic single-molecule super-resolution reconstructions critically depends upon robust background estimation.

The quality of super resolution images obtained by stochastic single-molecule microscopy critically depends on image analysis algorithms. We find that the choice of background estimator is often the most important determinant of reconstruction quality. A variety of techniques have found use, but many have a very narrow range of applicability depending upon the characteristics of the raw data. Importantly, we observe that when using otherwise accurate algorithms, unaccounted background components can give rise to biases on scales defeating the purpose of super-resolution microscopy. We find that a temporal median filter in particular provides a simple yet effective solution to the problem of background estimation, which we demonstrate over a range of imaging modalities and different reconstruction methods.

Richardson-Lucy deconvolution as a general tool for combining images with complementary strengths.

We use Richardson-Lucy (RL) deconvolution to combine multiple images of a simulated object into a single image in the context of modern fluorescence microscopy techniques. RL deconvolution can merge images with very different point-spread functions, such as in multiview light-sheet microscopes,1, 2 while preserving the best resolution information present in each image. We show that RL deconvolution is also easily applied to merge high-resolution, high-noise images with low-resolution, low-noise images, relevant when complementing conventional microscopy with localization microscopy. We also use RL deconvolution to merge images produced by different simulated illumination patterns, relevant to structured illumination microscopy (SIM)3, 4 and image scanning microscopy (ISM). The quality of our ISM reconstructions is at least as good as reconstructions using standard inversion algorithms for ISM data, but our method follows a simpler recipe that requires no mathematical insight. Finally, we apply RL deconvolution to merge a series of ten images with varying signal and resolution levels. This combination is relevant to gated stimulated-emission depletion (STED) microscopy, and shows that merges of high-quality images are possible even in cases for which a non-iterative inversion algorithm is unknown.