Single cell visualization of transcription kinetics variance of highly mobile identical genes using 3D nanoimaging

Both multi-cell biochemical assays and single cell fluorescence measurements have revealed that the elongation rate of Polymerase II (PolII) in eukaryotes varies largely across different cell types and genes. However, there is not yet a consensus whether intrinsic factors such as the position, local mobility or the engagement by an active molecular mechanism of a genetic locus could be the determinants of the observed heterogeneity. Employing high-speed 3D fluorescence nanoimaging we resolve here at the single cell level multiple, distinct regions of mRNA synthesis within a labeled transgene array. By employing phasor analysis, a fluorescence fluctuation spectroscopy technique, we demonstrate that these regions are active transcription sites that release mRNA molecules in the nucleoplasm, and we extract the local PolII elongation rate. While we detect a range of 10-100 bp/s for PolII elongation from cell to cell, we are now also able to measure up to a four-fold variation in the average elongation between identical copies of the same gene measured simultaneously within the same cell. Furthermore, we are able to visualize changes of PolII elongation as a function of time. We observe a correlation between the average elongation rate measured in a locus and its local mobility. Finally, by cross-correlating the transcriptional activity with the nm-sized movements of the active loci, we provide evidence of an active molecular mechanism determining displacements of the transcription sites concomitant to increases in transcriptional activity. Together these observations demonstrate that local factors, such as chromatin local mobility and the microenvironment of the transcription site, are an important source of transcription kinetics variability.Comment: 56 pages, 5 main figures and 10 supplementary figure

Linescan microscopy data to extract diffusion coefficient of a fluorescent species using a commercial confocal microscope

We are grateful to the Max Delbrück Center for Molecular Medicine in the Helmholtz Association for core support and funding. P.A. and M.J.L. would like to acknowledge funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project-ID 421152132-SFB1423 subproject C03.We report here on the measurement of the diffusion coefficient of fluorescent species using a commercial microscope possessing a resonant scanner. Sequential linescans with a rate of up to 12 kHz yield a temporal resolution of 83 μs, making the setup amenable to measure diffusion rates over a range covering at least three orders of magnitude, from 100 μm2/s down to 0.1 μm2/s. We share representative data sets covering (i) the diffusion of a dye molecule, observed in media of different viscosities and (ii) the diffusion of a prototypical membrane receptor.  The data can be valuable for researchers interested in the rapid diffusion properties of nuclear, cytosolic or membrane bound proteins fused to fluorescent tags.Publisher PDFPeer reviewe

Differential Signaling Profiles of MC4R Mutations with Three Different Ligands

The melanocortin 4 receptor (MC4R) is a key player in hypothalamic weight regulation and energy expenditure as part of the leptin-melanocortin pathway. Mutations in this G protein coupled receptor (GPCR) are the most common cause for monogenetic obesity, which appears to be mediated by changes in the anorectic action of MC4R via GS-dependent cyclic adenosine-monophosphate (cAMP) signaling as well as other signaling pathways. To study potential bias in the effects of MC4R mutations between the different signaling pathways, we investigated three major MC4R mutations: a GS loss-of-function (S127L) and a GS gain-of-function mutant (H158R), as well as the most common European single nucleotide polymorphism (V103I). We tested signaling of all four major G protein families plus extracellular regulated kinase (ERK) phosphorylation and β-arrestin2 recruitment, using the two endogenous agonists, α- and β-melanocyte stimulating hormone (MSH), along with a synthetic peptide agonist (NDP-α-MSH). The S127L mutation led to a full loss-of-function in all investigated pathways, whereas V103I and H158R were clearly biased towards the Gq/11 pathway when challenged with the endogenous ligands. These results show that MC4R mutations can cause vastly different changes in the various MC4R signaling pathways and highlight the importance of a comprehensive characterization of receptor mutations

Automated Test Case Generation as a Many-Objective Optimisation Problem with Dynamic Selection of the Targets

The test case generation is intrinsically a multi-objective problem, since the goal is covering multiple test targets (e.g., branches). Existing search-based approaches either consider one target at a time or aggregate all targets into a single fitness function (whole-suite approach). Multi and many-objective optimisation algorithms (MOAs) have never been applied to this problem, because existing algorithms do not scale to the number of coverage objectives that are typically found in real-world software. In addition, the final goal for MOAs is to find alternative trade-off solutions in the objective space, while in test generation the interesting solutions are only those test cases covering one or more uncovered targets. In this paper, we present DynaMOSA (Dynamic Many-Objective Sorting Algorithm), a novel many-objective solver specifically designed to address the test case generation problem in the context of coverage testing. DynaMOSA extends our previous many-objective technique MOSA (Many-Objective Sorting Algorithm) with dynamic selection of the coverage targets based on the control dependency hierarchy. Such extension makes the approach more effective and efficient in case of limited search budget. We carried out an empirical study on 346 Java classes using three coverage criteria (i.e., statement, branch, and strong mutation coverage) to assess the performance of DynaMOSA with respect to the whole-suite approach (WS), its archive-based variant (WSA) and MOSA. The results show that DynaMOSA outperforms WSA in 28% of the classes for branch coverage (+8% more coverage on average) and in 27% of the classes for mutation coverage (+11% more killed mutants on average). It outperforms WS in 51% of the classes for statement coverage, leading to +11% more coverage on average. Moreover, DynaMOSA outperforms its predecessor MOSA for all the three coverage criteria in 19% of the classes with +8% more code coverage on average

Fluorescence spectroscopy of low-level endogenous β-adrenergic receptor expression at the plasma membrane of differentiating human iPSC-derived cardiomyocytes

Funding: This project was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through Project 421152132 SFB1423 subproject C03 (PA) and SFB 1470 subproject A01 (PA).The potential of human-induced pluripotent stem cells (hiPSCs) to be differentiated into cardiomyocytes (CMs) mimicking adult CMs functional morphology, marker genes and signaling characteristics has been investigated since over a decade. The evolution of the membrane localization of CM-specific G protein-coupled receptors throughout differentiation has received, however, only limited attention to date. We employ here advanced fluorescent spectroscopy, namely linescan Fluorescence Correlation Spectroscopy (FCS), to observe how the plasma membrane abundance of the β1- and β2-adrenergic receptors (β1/2-ARs), labelled using a bright and photostable fluorescent antagonist, evolves during the long-term monolayer culture of hiPSC-derived CMs. We compare it to the kinetics of observed mRNA levels in wildtype (WT) hiPSCs and in two CRISPR/Cas9 knock-in clones. We conduct these observations against the backdrop of our recent report of cell-to-cell expression variability, as well as of the subcellular localization heterogeneity of β-ARs in adult CMs.Publisher PDFPeer reviewe

The impact of membrane protein diffusion on GPCR signaling

This research was carried out as part of the Math-+ excellence cluster (DFG EXC 2046, Project A01-11 [HHB, PA]) and was partially funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through the following grants: Project 421152132 SFB1423 subproject C03 (PA), SFB 1470 subproject A01 (PA) and SFB 1114/2 (SW).Spatiotemporal signal shaping in G protein-coupled receptor (GPCR) signaling is now a well-established and accepted notion to explain how signaling specificity can be achieved by a superfamily sharing only a handful of downstream second messengers. Dozens of Gs-coupled GPCR signals ultimately converge on the production of cAMP, a ubiquitous second messenger. This idea is almost always framed in terms of local concentrations, the differences in which are maintained by means of spatial separation. However, given the dynamic nature of the reaction-diffusion processes at hand, the dynamics, in particular the local diffusional properties of the receptors and their cognate G proteins, are also important. By combining some first principle considerations, simulated data, and experimental data of the receptors diffusing on the membranes of living cells, we offer a short perspective on the modulatory role of local membrane diffusion in regulating GPCR-mediated cell signaling. Our analysis points to a diffusion-limited regime where the effective production rate of activated G protein scales linearly with the receptor–G protein complex’s relative diffusion rate and to an interesting role played by the membrane geometry in modulating the efficiency of coupling.Publisher PDFPeer reviewe