Biology subjects

Gadella, T. W. J.

Publications and source records attributed to Gadella, T. W. J..

11 recordsLinked to original sources

bioRxiv2025

mScarlet3-H with low brightness and fluorescence lifetime has potential for cellular lifetime-unmixing and lifetime-based pH-sensing applications

An independent evaluation of the spectroscopic properties, cellular performance, merits and pitfalls of the redfluorescent protein mScarlet3-H as compared to mScarlet3 is reported. mScarlet3-H was generated from mScarlet3 by a single M163H mutation. Purified mScarlet3-H is characterized by a molar coeWicient of 79,040 M-1cm-1, afluorescence quantum yield of 17.8%, molecular brightness of 14.1 and a heterogeneous multiexponential decay with an averagefluorescence lifetime of 1 ns. Evaluation in living mammalian cells revealed a comparable maturation speed and eWiciency of mScarlet3 and mScarlet3-H, but the overall cellular brightness of mScarlet3-H was 5-fold lower than that of mScarlet3. Photobleaching analysis in live cells revealed identical photobleaching kinetics of mScarlet3-H and mScarlet-H. Thefluorescence intensity,fluorescence spectra andfluorescence lifetime of mScarlet3-H were found to be strongly pH-dependent between pH 4-8. Thefluorescence lifetime increased from 1 ns to 3 ns in lowering the pH from 8 to 4 with a pK of [~]6. The much lower lifetime of mScarlet3-H ([~]1 ns) as compared to mScarlet3 ([~]4 ns) allows dualfluorescence lifetime unmixing applications in single channel FLIM recordings in compartments with neutral to slightly alkaline pH. Furthermore, the strongly pH-dependentfluorescence lifetime of mScarlet3-H enablesfluorescence lifetime-based pH sensing in a pH region between pH 5 to 7. With this property autophagy of the cytoplasm can be visualized by the pH-dependentfluorescence lifetime with mScarlet3-H accumulation in lysosomes. Potential useful applications and pitfalls regarding the special properties of mScarlet3-H are discussed.

cell biology↗

bioRxiv2024

Exploration of mScarlet for development of a red lifetime sensor for calcium imaging

The past decades, researchers have worked on the development of genetically encoded biosensors, including over 60 genetically encoded calcium indicators (GECIs) containing a single fluorescent protein (FP). Red fluorescent GECIs provide advantages in terms of imaging depths and reduced cell toxicity. Most of GECIs respond with a fluorescence intensity change, and researchers have strived to improve the sensors in terms of brightness and fold-change. Unfortunately, fluorescence intensity is influenced by many factors other than the desired sensor response. GECIs with a fluorescence lifetime contrast overcome this drawback, but so far, no bright red GECI has been developed that shows a fluorescence lifetime contrast. We tried to tackle this challenge by using the brightest red fluorescent proteins from the mScarlet family to develop a new sensor. We did succeed in creating remarkable bright probes, but the fluorescence lifetime contrast we observed in bacterial lysates was lost in mammalian cells. Based on our results, and the success of others to develop a pH and a voltage sensor of mScarlet, we are confident that a GECI with mScarlet is feasible. To this end, we propose to continue development using a mammalian cell-based screening, instead of screening in bacterial lysates.

cell biology↗

bioRxiv2024

A green lifetime biosensor for calcium that remains bright over its full dynamic range

Fluorescent biosensors toggle between two states and for the vast majority of biosensors one state is bright and the other state is dim. As a consequence, there is a substantial difference in the signal-to-noise ratio (SNR) for the two states. The dim state has a low signal-to-noise ratio, which is problematic when precise, quantitative measurements are needed. During the engineering of a red-shifted variant of an mTurquoise-based calcium sensor, we serendipitously generated a green-emitting sensor that shows high brightness in both the calcium-bound and -unbound state, while still showing a calcium dependent lifetime change of >1 nanosecond. This sensor, named G-Ca-FLITS, is comparable in brightness to the bright state of GCaMP3 and jGCaMP7c in mammalian cells. The calcium induced loss in fluorescence intensity is only around 30% and therefore we observe little variation in the SNR when calcium levels change. G-Ca-FLITS shows negligible sensitivity to pH in the physiological range, like its turquoise parent. Using fluorescence lifetime imaging (FLIM), we measured the calcium concentration with G-Ca-FLITS in various organelles and observed in HeLa cells transient and spatially heterogeneous calcium elevations in mitochondria. Finally, we evaluated the use of G-Ca-FLITS and its turquoise predecessor for two-photon FLIM in Drosophila brains. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/622032v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@1caf016org.highwire.dtl.DTLVardef@586996org.highwire.dtl.DTLVardef@7c15cdorg.highwire.dtl.DTLVardef@7b6f03_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Heterogeneity and dynamics of ERK and Akt activation by G protein-coupled receptors depend on the activated heterotrimeric G proteins

Kinases are fundamental regulators of cellular functions and play key roles in GPCR-mediated signaling pathways. Kinase activities are generally inferred from cell lysates, hiding the heterogeneity of the individual cellular responses to extracellular stimuli. Here, we study the dynamics and heterogeneity of ERK and Akt in genetically identical cells in response to activation of endogenously expressed GPCRs. We use kinase translocation reporters, high-content imaging, automated segmentation and clustering methods to assess cell-to-cell signaling heterogeneity. We observed ligand-concentration dependent response kinetics to histamine, a2-adrenergic, and S1P receptor stimulation that varied between cells. By using G protein inhibitors, we observed that Gq mediated the ERK and Akt responses to histamine. In contrast, Gi was necessary for ERK and Akt activation in response to 2-adrenergic receptor activation. ERK and Akt were also strongly activated by S1P, showing high heterogeneity at the single cell level, especially for ERK. In all cases, the cellular heterogeneity was not explained by distinct pre-stimulation levels or saturation of the measured response. Cluster analysis of time-series derived from 68,000 cells obtained under the different conditions revealed several distinct populations of cells that display similar response dynamics. The single-cell ERK responses to histamine and brimonidine showed remarkably similar dynamics, despite the activation of different heterotrimeric G proteins. In contrast, the ERK response dynamics to S1P showed high heterogeneity, which was reduced by the inhibition of Gi. To conclude, we have set up an imaging and analysis strategy that reveals substantial cell-to-cell heterogeneity in kinase activity driven by GPCRs. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

cell biology↗

bioRxiv2021

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

The most successful genetically encoded calcium indicators (GECIs) employ an intensity or intensiometric 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 novel 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. A new 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. Additionally, the response of the calcium sensor is insensitive to pH between 6.2-9. As a result, the turquoise GECI enables robust measurements of intracellular calcium concentrations by fluorescence lifetime imaging. We demonstrate quantitative imaging of calcium concentration with the turquoise GECI in single endothelial cells and human-derived organoids. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/449214v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@12beb07org.highwire.dtl.DTLVardef@5337f1org.highwire.dtl.DTLVardef@17db68forg.highwire.dtl.DTLVardef@5ca499_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Visualizing endogenous RhoA activity with an improved localization-based, genetically encoded biosensor

Rho GTPases are regulatory proteins, which orchestrate cell features such as morphology, polarity and movement. Therefore, probing Rho GTPase activity is key to understanding processes such as development, cell migration and wound healing. Localization-based reporters for active Rho GTPases are attractive probes to study Rho GTPase-mediated processes, in real time with subcellular resolution in living cells and tissue. Until now, relocation RhoA biosensors seem to only be useful in certain organisms and have not been characterized well. In this paper, we systematically examined the contribution of the fluorescent protein and RhoA binding peptides, on the performance of localization-based sensors. To test the performance, we compared relocation efficiency and specificity in cell-based assays. We identified several improved localization-based, genetically encoded, fluorescent biosensors for detecting endogenous RhoA activity. This enables a broader application of RhoA relocation biosensors, which was demonstrated by using the improved biosensor to visualize RhoA activity, during cell division, during random migration, at the Golgi membrane and induced by G protein-coupled receptor signaling. Due to the improved avidity of the new biosensors for RhoA activity, cellular processes regulated by RhoA can be better understood. O_FIG O_LINKSMALLFIG WIDTH=134 HEIGHT=200 SRC="FIGDIR/small/430250v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1847c24org.highwire.dtl.DTLVardef@f22c92org.highwire.dtl.DTLVardef@14c4aa8org.highwire.dtl.DTLVardef@1b9657d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2019

Selectivity of Guanine Nucleotide Exchange Factor-mediated Cdc42 activation 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 signaling 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 full-length GEFs. Our data reveal a specific GEF dependent activation profile, with 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 activate Cdc42, which will be of great value for the field of vascular biology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/541219v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@96ad9forg.highwire.dtl.DTLVardef@60aa3borg.highwire.dtl.DTLVardef@4811f0org.highwire.dtl.DTLVardef@191cfd1_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

cell biology↗

bioRxiv2017

A FRET based biosensor for measuring Gα13 activation in single cells

Forster Resonance Energy Transfer (FRET) provides a way to directly observe the activation of heterotrimeric G-proteins by G-protein coupled receptors (GPCRs). To this end, FRET based biosensors are made, employing heterotrimeric G-protein subunits tagged with fluorescent proteins. These FRET based biosensors complement existing, indirect, ways to observe GPCR activation. Here we report on the insertion of mTurquoise2 at several sites in the human G13 subunit. Three variants were found to be functional based on i) plasma membrane localization and ii) ability to recruit p115-RhoGEF upon activation of the LPA2 receptor. The tagged G13 subunits were used as FRET donor and combined with cp173Venus fused to the G{gamma}2 subunit as the acceptor. We constructed G13 biosensors by generating a single plasmid that produces G13-mTurquoise2, G{beta}1 and cp173Venus-G{gamma}2. The G13 activation biosensors showed a rapid and robust response when used in primary human endothelial cells that were treated with thrombin, triggering endogenous protease activated receptors (PARs). This response was efficiently inhibited by the RGS domain of p115-RhoGEF and from the biosensor data we inferred that this is due to GAP activity. Finally, we demonstrated that the G13 sensor could be used to dissect heterotrimeric G-protein coupling efficiency in single living cells. We conclude that the G13 biosensor is a valuable tool for live-cell measurements that probe G13 activation.

cell biology↗

bioRxiv2017

Robust and Bright Genetically Encoded Fluorescent Markers for Highlighting Structures and Compartments in Mammalian Cells

To increase our understanding of the inner working of cells, there is a need for specific markers to identify biomolecules, cellular structures and compartments. One type of markers comprises genetically encoded fluorescent probes that are linked with protein domains, peptides and/or signal sequences. These markers are encoded on a plasmid and they allow straightforward, convenient labeling of cultured mammalian cells by introducing the plasmid into the cells. Ideally, the fluorescent marker combines favorable spectroscopic properties (brightness, photostability) with specific labeling of the structure or compartment of interest. Here, we report our ongoing efforts to generate robust and bright genetically encoded fluorescent markers for highlighting structures and compartments in living cells. The plasmids are distributed by addgene: https://www.addgene.org/browse/article/28189953/ O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/160374v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@67f3aaorg.highwire.dtl.DTLVardef@ae2235org.highwire.dtl.DTLVardef@47f729org.highwire.dtl.DTLVardef@b3c9d1_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2017

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

Genetically encoded Forster Resonance Energy Transfer (FRET) based biosensors report on changes in biochemical states in single living cells. The performance of biosensors depends on their brightness and dynamic range, which are dependent on the characteristics of the fluorescent proteins that are employed. Cyan fluorescent protein (CFP) is frequently combined with yellow fluorescent protein (YFP) as FRET pair in biosensors. However, current YFPs are prone to photobleaching and pH changes. In addition, more efficient acceptors may yield biosensors that have higher contrast. 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 Forster 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, mChery 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. The sensor with mScarlet-I as acceptor and mTurquoise2 as donor shows a higher dynamic range in ratiometric FRET imaging experiments and less variability than with mCherry as acceptor, due to the high quantum yield and efficient maturation of mScarlet-I. 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.

cell biology↗

bioRxiv2017

Photorelease of Diacylglycerol Increases the Amplitude and Duration of Protein Kinase C-BetaII Relocation in cyto

Diacylglycerol (DAG) is a lipid second messenger produced by receptor stimulated phospholipase C and is capable of activating several PKC isoforms. Classical PKC isoforms require simultaneous presence of calcium and DAG for activation and relocation to membranes. The aim of this study was to synthesize a photolabile precursor of DAG and examine the effect of an immediate increase of the signaling lipid on PKC relocation. Caged DAG was synthesized using a photoreleasable 7-diethyl-aminocoumarin (DEACM) group. Photolysis was monitored in vitro by an increase in coumarin fluorescence from which an uncaging quantum yield of 1.1% was determined. This quantum yield proved ideal for efficient uncaging at high UV power while allowing localization of the fluorescent compound at low UV power. Taking advantage of the coumarin fluorescence, it was demonstrated that DEACM-DiC8 accumulated inside cells. Effects of DAG photorelease on periodic relocations of PKCbetaII, induced by histamine, UTP or EGF, were studied. Photorelease of DAG in cyto immediately increased the amplitude and duration of relocation events, regardless of the agonist used. Together, the results demonstrate the usefulness of caged DAG for dissecting PKC signaling and suggest that DAG levels are limiting during signaling.

cell biology↗