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/.
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.
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.
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.
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.
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.
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.
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.
Summary
The rod‐shaped bacterium Escherichia coli grows by insertion of peptidoglycan into the lateral wall during cell elongation and synthesis of new poles during cell division. The monofunctional ...transpeptidases PBP2 and PBP3 are part of specialized protein complexes called elongasome and divisome, respectively, which catalyse peptidoglycan extension and maturation. Endogenous immunolabelled PBP2 localized in the cylindrical part of the cell as well as transiently at midcell. Using the novel image analysis tool Coli‐Inspector to analyse protein localization as function of the bacterial cell age, we compared PBP2 localization with that of other E. coli cell elongation and division proteins including PBP3. Interestingly, the midcell localization of the two transpeptidases overlaps in time during the early period of divisome maturation. Försters Resonance Energy Transfer (FRET) experiments revealed an interaction between PBP2 and PBP3 when both are present at midcell. A decrease in the midcell diameter is visible after 40% of the division cycle indicating that the onset of new cell pole synthesis starts much earlier than previously identified by visual inspection. The data support a new model of the division cycle in which the elongasome and divisome interact to prepare for cell division.
Phototransduction in microvillar photoreceptors is mediated via G protein-coupled phospholipase C (PLC), but how PLC activation leads to the opening of the light-sensitive TRPC channels (TRP and ...TRPL) remains unresolved. In
Drosophila, InsP
3 appears not to be involved, and recent studies have implicated lipid products of PLC activity, e.g., diacylglycerol, its metabolites, or the reduction in PIP
2. The fact that hydrolysis of the phosphodiester bond in PIP
2 by PLC also releases a proton is seldom recognized and has neither been measured in vivo nor implicated previously in a signaling context.
Following depletion of PIP
2 and other phosphoinositides by a variety of experimental manipulations, the light-sensitive channels in
Drosophila photoreceptors become remarkably sensitive to rapid and reversible activation by the lipophilic protonophore 2-4 dinitrophenol in a pH-dependent manner. We further show that light induces a rapid (<10 ms) acidification originating in the microvilli, which is eliminated in mutants of PLC, and that heterologously expressed TRPL channels are activated by acidification of the cytosolic surface of inside-out patches.
Our results indicate that a combination of phosphoinositide depletion and acidification of the membrane/boundary layer is sufficient to activate the light-sensitive channels. Together with the demonstration of light-induced, PLC-dependent acidification, this suggests that excitation in
Drosophila photoreceptors may be mediated by PLC's dual action of phosphoinositide depletion and proton release.
► Light induces rapid phospholipase C-mediated acidification in
Drosophila photoreceptors ► The light-sensitive channels (TRP/TRPL) can be rapidly activated by protonophores ► But only after PIP
2 and other phosphoinositides (PI) have been depleted by PLC ► Combinatorial activation of TRP channels by PI depletion and proton release
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