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

Direkte Schätzung der Ausbreitungsgeschwindigkeit für akustische Anwendungen, die auf Zeitverzögerungen beruhen

Spatially distributed acoustic sensors increasingly find many new applications in speech- based human-machine interfaces. One well researched topic is the localization of sound sources from Time Of Arrival (TOA) and Time Difference Of Arrivals (TDOA) measurements. Typically, the propagation speed of sound waves is considered as a known constant, although in a real scenario its value is known only up to some uncertainty due to air temperature variations. In general, when the conditions of the propagation medium are unknown, the propagation speed has to be considered as an additional unknown of the localization problem. Typical examples are underwater communications or seismology. In these cases the propagation speed of the waves emitted by the source has to be estimated jointly with the source position. Surprisingly, the direct estimation of the propagation speed from the same delay measurements used for localizing the target source is considered only in very few works. This thesis focuses on joint localization and propagation speed estimation by means of efficient algorithms that benefit from closed form solutions. After a review of some basic knowledge on wave propagation and time delay estimation, the state of the art techniques are described. Thereafter, a novel approach for the direct estimation of the propagation speed from delay measurements is presented. Such a new method is computationally efficient and benefits from a general formulation that holds for both TOAs and TDOAs. Its mathematical derivation is followed by several simulations where the proposed method shows significant performance improvements over the standard methods. As a benchmark for the estimation accuracy the Cramer-Rao Bound (CRB) was derived. Furthermore, experimental results using both simulated and real data demonstrate the feasibility of the proposed method. In particular, the proposed method was tested in conjunction with typical delay-based audio applications that normally assume a fixed value for the speed of sound. The so obtained results highlight the improvements led by the direct estimation of the speed of sound from both TOAs and TDOAs even for small temperature variations. Against a small increase in the computational cost, the accuracy of localization tasks turns out to be dramatically improved and virtually independent of temperature variations. Moreover, we show also an innovative application of direct speed estimation as a mean to assist a challenging task such as the disambiguation of TDOAs in multi-source reverberant environments.Räumlich verteilte akustische Sensoren werden immer häufiger für neue Anwendungen eingesetzt, z.B., Mensch-Maschine-Schnittstellen, die auf Sprachsteuerung beruhen. Besondere Aufmerksamkeit gilt der akustischen Quellenlokalisierung durch an den Sensoren gemessene Laufzeiten (engl. Times-Of-Arrival, TOAs) oder Laufzeitunterschieden (engl. Time-Differences-Of-Arrivals, TDOAs). In der Regel wird die Schallgeschwindigkeit als bekannte Konstante betrachtet, obwohl diese Annahme nur unter festen Bedingungen, bzw. in Testlabors, realistisch ist, da die Schallgeschwindigkeit von der Temperatur abhängt. Daher führen unbekannte Eigenschaften des Ausbreitungsmediums zu einem Lokalisierungsproblem, bei dem die Ausbreitungsgeschwindigkeit als zusätzlicher unbekannter Parameter auftritt. Typische Beispiele sind akustische Unterwasserkommunikation oder Seismologie. In allen diesen Fällen muss die Ausbreitungsgeschwindigkeit zusammen mit der Quellenposition geschätzt werden. Allerdings finden sich in der Literatur nur wenige Arbeiten, die sich mit der direkten Schätzung der Ausbreitungsgeschwindigkeit anhand der für die Lokalisierung benötigten Zeitverzögerungen befassen. Diese Dissertation beschreibt ein Verfahren für eine effiziente gemeinsame Schätzung der Quellenposition und der Ausbreitungsgeschwindigkeit. Nach einer Einführung in die Grundlagen der Wellenausbreitung und der Laufzeitschätzung wird eine einheitliche und verständliche Darstellung über den aktuellen Stand der Technik gegeben. Danach wird ein neuen Ansatz zur Schätzung der Ausbreitungsgeschwindigkeit anhand der gemessenen Zeitverzögerungen vorgestellt. Diese neue Methode bietet eine einheitliche Darstellung, die unabhängig von der Art der Messungen (TOA oder TDOA) ist und sich durch effiziente Algorithmen implementieren lässt. Die mathematische Herleitung wird von zahlreichen numerischen Simulationen begleitet, wobei die Schätzergebnisse der neu eingeführten Methode untersucht und verglichen werden. Dazu wird eine untere Schranke für die Varianz der Schätzer, die sogenannte Cramer-Rao Grenze, berechnet und angewendet. Insbesondere wird die Methode für unterschiedliche Lokalisierungsaufgaben im Audio-Bereich eingesetzt, bei denen in der Regel ein bekannter Wert für die Schallgeschwindigkeit voraussetzt wird. Auch bei geringen Abweichungen der Lufttemperatur ermöglicht die direkte Schätzung der Schallgeschwindigkeit deutlich bessere Lokalisierungsergebnisse. Bei einer geringen Erhöhung des Rechenaufwands führt die vorgestellte Methode zu einer Temperatur-unabhängigen Lokalisierung. Abschließend wird auch ein besonderes Experiment gezeigt, in dem die direkte Schallgeschwindigkeitsschätzung für die Zuordnung der Laufzeitdifferenzen in halligen Umgebungen mit mehrere Sprechern erfolgreich angewendet wird

Investigating the Impact of Single Molecule Fluorescence Dynamics on Photo Activated Localization Microscopy Experiments

When fluorophores are densely packed in a biological sample, localizing them one at a time is one of the paths to optical super-resolution. Photo Activated Localization Microscopy (PALM) is one of the methods of this burgeoning field that presents a large promise for addressing physical and biological questions requiring non-invasive, high-resolution optical imaging at the nanoscale. Since this work began when PALM was only two years old, the thesis contains a significant portion of research on the technique itself, and has therefore a marked methodological cut. The principal, and recurring, theme is the investigation of the single molecule fluorescence dynamics of the fluorophores used, that in the end determine to a large extent the ensemble imaging performance as well as the interpretation of the data. A few assumptions reported in the literature are questioned, and, beginning with a discussion of the properties of a bright fluorescent protein, mEos2, we propose an original approach based on the use of the temporal information besides the spatial one when treating PALM data. This allows both a more accurate quantification of the number of fluorophores activated in the sample as well as the correct identification of relevant biological structures, such as clusters, on the plasma membrane of cells. The second theme is the application of PALM to probe the functional arrangement of an important class of cell membrane proteins, through the study of the prototypical G protein-coupled receptor [beta]2-Adrenergic Receptor. First, the well studied biological properties of this family of signaling proteins are used to validate the potential of this approach. Then, PALM is used to gain new insight on the basal arrangement of [beta]2-AR in a relevant biological context using tools from spatial point pattern analysis. Provided that appropriate control experiments are performed, PALM is shown to have the potential to be included in the palette of the available techniques to study membrane protein organization. An important finding is the quantification of a cell-type specific clustering of this receptor in cardiomyocite-like cells. Finally, some technical issues relevant to dual color imaging, in particular concerning the axial stability of the microscope, are addressed as a third, perspective theme, together with a detailed first-time characterization of three representative fluorescent protein pairs currently available for dual color PALM imaging

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