Consistent Sampling and Signal Recovery

An attractive formulation of the sampling problem is based on the principle of a consistent signal reconstruction. The requirement is that the reconstructed signal is indistinguishable from the input in the sense that it yields the exact same measurements. Such a system can be interpreted as an oblique projection onto a given reconstruction space. The standard formulation requires a one-to-one relationship between the input measurements and the reconstructed model. Unfortunately, this condition fails when the cross-correlation matrix between the analysis and reconstruction basis functions is not invertible; in particular, when there are less measurements than the number of reconstruction functions. In this paper, we propose an extension of consistent sampling that is applicable to those singular cases as well, and that yields a unique and well-defined solution. This solution also makes use of projection operators and has a geometric interpretation. The key idea is to exclude the null space of the sampling operator from the reconstruction space and to enforce consistency on its complement. We specify a class of consistent reconstruction algorithms corresponding to different choices of complementary reconstruction spaces. The formulation includes the Moore-Penrose generalized inverse, as well as other potentially more interesting reconstructions that preserve certain preferential signals. In particular, we display solutions that preserve polynomials or sinusoids, and therefore perform well in practical applications

High-resolution insights into macromolecular assembly: a yeast’s survival strategy

Cells grow in environments that can change suddenly. To cope with unpredictable perturbations, they have evolved mechanisms to adjust their metabolism according to the various types of environmental stress. Cells experiencing starvation, for example, have low energy levels and are forced to lower their metabolism and enter a protective quiescent state to survive until nutrients become available again. Recently, it has been shown that starved yeast cells experience a marked acidification of the cytoplasm, due to a passive influx of protons. This pH drop causes multiple rearrangements in the cytoplasm: increased crowding, reduced mobility of intracellular components and formation of stress-induced non-membrane bound compartments of specific metabolic enzymes. Cytoplasm rearrangements are required for cell survival and can be reversed upon replenishment of energy. However, there is little understanding of how cytoplasmic components reorganize in stressed quiescent cells. Using high-pressure freezing, correlative light and electron microscopy (CLEM) and electron tomography, coupled to high-resolution 3D-reconstruction techniques, I investigate the structural modi cations that happen in situ in yeast cells undergoing quiescence. I observe that the cytoplasm becomes increasingly crowded, due to a massive rearrangement of membranous structures, including accumulation of intracellular vesicles and pronounced invaginations in the plasma membrane. This is proved by quantification of the difference in ribosome densities between stressed and not stressed cells. The increased crowding, coupled to cytoplasm acidification, leads to the formation of non-membrane bound enzyme compartments, that appear as foci and elongated structures of fluorescently tagged enzymes. I prove that the fluorescent structures correspond to bundles of filaments. Among many essential enzymes, known to form mesoscale structure in stressed yeast, I demonstrate that the eukaryotic translation initiation factor 2B (eIF2B) forms bundles of filaments in situ, and the evolutionary conserved glutamine synthetase (Gln1) self-assembles into filaments in vitro. The present study on the energy depleted cytoplasm and the structural analysis of filament-forming enzymes provides insights into an unexplored survival strategy that is used by yeast, as well as other organisms, to cope with extreme environmental conditions and stress.:1 Introduction 1 Stress, survival and quiescence 2 1.1 Cytoplasm and cellular compartments 2 1.2 Membraneless compartmentalization in the cell 3 1.3 Stress-induced non-membrane bound assemblies 4 The quiescent sleeping yeast 6 1.4 The yeast S.cerevisiae as model organism 7 1.5 Growth and metabolism of yeast 9 1.5.1 Yeast eukaryotic translation initiation factor 2B: eIF2B 11 1.5.2 Yeast glutamine synthetase: Gln1 12 3D electron microscopy 14 Aims of the Thesis 18 2 Materials and methods 21 Room temperature electron microscopy (EM) 21 2.1 Yeast strains, media and energy depletion 21 2.2 High-pressure freezing of yeast cells 22 2.2.1 EM sample preparation for untagged eIF2B yeast strains 22 2.2.2 EM sample preparation for GFP-tagged eIF2B yeast strains 23 2.3 Electron tomography 23 2.4 Subtomogram averaging 24 2.5 Fiji script for automated ribosome counting 25 2.6 Immunofluorescence of eIF2B in yeast 26 2.7 Western-blot on yeast ribosomes 27 Single particle procedures 30 2.8 Protein purification protocols 30 2.8.1 Baculovirus-insect cell expression and purification of eIF2B 30 2.8.2 Gradient of fixation for fragile complexes 31 2.8.3 Yeast expression and purification of Gln1 33 2.9 Negative staining 34 2.9.1 Image acquisition and analysis—eIF2B 35 2.9.2 Image acquisition and analysis—Gln1 36 Cryo-electronmicroscopy(cryo-EM) 37 2.10 Plunge freezing 37 2.11 Image acquisition and 3D reconstruction 37 3 Results Visualizing yeast’s cytoplasmic reorganization 39 3.1 Quiescence is accompanied by reorganization of the cytoplasm 40 3.2 Ribosome density proves cytoplasmic crowding in starved cells 42 3.3 eIF2B organizes in bundles of filaments in energy-depleted cells 45 3.4 eIF2B filaments are polymers of the eIF2B complex 47 3.5 Filaments are found in wild-type energy-depleted cells 49 Structural analysis of filament forming enzymes 51 3.6 Purification of eIF2B complexes 51 3.7 Single particle analysis of eIF2B 53 3.8 Purification of Gln1 complexes 55 3.9 Single particle analysis of Gln1 56 3.10 Gln1 forms filaments in vitro 58 4 Discussion and Outlook 59 4.1 Yeast cytoplasm reorganizes in response of stress 59 4.2 Ribosomes density is a measure of increased macromolecular crowding 60 4.3 eIF2B forms filaments as a survival strategy 62 4.4 Molecular analysis of filament forming enzymes 64 4.5 Outlook 65 Appendix 67 Bibliography 8

SONAR Images Denoising

International audienc

X-ray computed tomography

X-ray computed tomography (CT) can reveal the internal details of objects in three dimensions non-destructively. In this Primer, we outline the basic principles of CT and describe the ways in which a CT scan can be acquired using X-ray tubes and synchrotron sources, including the different possible contrast modes that can be exploited. We explain the process of computationally reconstructing three-dimensional (3D) images from 2D radiographs and how to segment the 3D images for subsequent visualization and quantification. Whereas CT is widely used in medical and heavy industrial contexts at relatively low resolutions, here we focus on the application of higher resolution X-ray CT across science and engineering. We consider the application of X-ray CT to study subjects across the materials, metrology and manufacturing, engineering, food, biological, geological and palaeontological sciences. We examine how CT can be used to follow the structural evolution of materials in three dimensions in real time or in a time-lapse manner, for example to follow materials manufacturing or the in-service behaviour and degradation of manufactured components. Finally, we consider the potential for radiation damage and common sources of imaging artefacts, discuss reproducibility issues and consider future advances and opportunities

Studien zur Proteintranslokation in Escherichia coli : Untersuchung der Membranproteine SecYEG und YidC unter Verwendung biochemischer und kristallographischer Methoden

Transport of proteins into or across cellular membranes is mediated by the conserved and ubiquitous Sec-machinery. The Sec-homologue in the inner membrane of Escherichia coli is SecYEG. Sec-mediated insertion of numerous membrane proteins is aided by YidC, another protein integral to the inner membrane of Escherichia coli. YidC fulfils in addition the integration of a variety of membrane proteins Sec-independently. It belongs to a conserved but structurally uncharacterised family of proteins important for membrane protein biogenesis and comprises homologues in mitochondria and chloroplasts. By modification of a former crystallisation protocol two-dimensional crystals of SecYEG were grown in presence of the signal sequence peptide of LamB. Recording of structural data by electron cryo-microscopy and calculation of a difference structure comparing a former SecYEG projection structure with the one of SecYEG crystallised in presence of the substrate revealed several new and vacant densities. These hint to signal peptide binding close to the translocation pore and to significant rearrangements in proximity to the lateral exit site for transmembrane domains in SecYEG. The difference structure suggests that dimeric SecYEG is an asymmetric molecule consisting of one active and one inactive SecYEG monomer. Detergent removal from a mixture of purified YidC and lipids produced two-dimensional crystals that were highly dependent on the ionic strength and lipid composition for their growth. Electron cryo-microscopy on the frozen-hydrated crystals and image processing visualised structural details at about 10 Å resolution. Averaging two alternative projection structures in p2 and p121_a symmetry, respectively, yielded essentially the same features. Four YidC monomers form one unit cell (dimensions 82 x 71 Å, included angle 85 ° and 90 °, respectively) and seem to be arranged as two sets of dimers integrated in an anti-parallel fashion into the membrane. An area of low density in the centre of each YidC monomer resembles possibly a constriction of the membrane, which could have particular relevance for the integration of substrate proteins into the lipid bilayer.Der Transport von Proteinen in zelluläre Membranen hinein oder durch diese hindurch wird durch die konservierte und überall anzutreffende Sec-Maschinerie vermittelt. Das Sec-Homolog in der inneren Membran von Escherichia coli ist SecYEG. Der Sec-vermittelte Einbau von vielen Membranproteinen wird unterstützt von YidC, einem weiteren Protein in der inneren Membran von Escherichia coli. YidC führt zusätzlich den Einbau einiger Proteine Sec-unabhängig durch. YidC gehört zu einer konservierten aber strukturell uncharakterisierten Familie von Proteinen, die wichtig für die Biogenese von Membranproteinen sind und hat Homologe in Mitochondrien und Chloroplasten. Durch Modifikation eines früheren Kristallisations-Protokolles wurde zweidimensionale Kristalle von SecYEG in Gegenwart des Signalsequenz-Peptides von LamB gezüchtet. Das Aufnehmen von Strukturdaten mittels Elektronen-Kryomikroskopie und die Berechnung einer Differenzstruktur, welche eine frühere SecYEG-Projektionsstruktur mit der von SecYEG kristallisiert in Gegenwart von Substrat vergleicht, demonstrierte eine Reihe neuer bzw. fehlender Dichten. Diese deuten auf das Binden von Signalpeptid in räumlicher Nähe zur Translokationspore sowie auf signifikante Umlagerungen in der Nähe des lateralen Austrittsortes für Transmembrandomänen in SecYEG hin. Die Differenzstruktur legt nahe, dass dimeres SecYEG ein asymmetrisches Molekül ist, bestehend aus einem aktiven und einem inaktiven SecYEG-Monomer. Detergenzentfernung aus einem Gemisch von gereinigtem YidC und Lipiden brachte zweidimensionale Kristalle hervor, welche bezüglich ihrer Bildung sehr abhängig von Ionenstärke und Lipidkomposition waren. Eine elektronen-kryomikroskopische Untersuchung der in hydratisiertem Zustand gefrorenen Kristalle und Bildverarbeitung machten strukturelle Details mit einer Auflösung von etwa 10 Å sichtbar. Mitteln von Einzelbildern zu zwei alternativen Projektionsstrukturen in p2 bzw. p121_a Symmetrie brachte grundsätzlich gleiche strukturelle Merkmale hervor. Vier YidC-Monomere bilden eine Einheitszelle (Dimensionen 82 x 71 Å, eingeschlossener Winkel 85 ° bzw. 90 °) und scheinen als zwei Dimere vorzuliegen, welche in gegensätzlicher Orientierung in die Membran eingebettet sind. Ein Bereich geringer Dichte im Zentrum eines jeden YidC-Monomers stellt möglicherweise eine Einstülpung der Membran dar, die besondere Bedeutung für den Einbau von Substratproteinen in die Lipid-Doppelschicht haben könnte

Fast wide-volume functional imaging of engineered in vitro brain tissues

The need for in vitro models that mimic the human brain to replace animal testing and allow high-throughput screening has driven scientists to develop new tools that reproduce tissue-like features on a chip. Three-dimensional (3D) in vitro cultures are emerging as an unmatched platform that preserves the complexity of cell-to-cell connections within a tissue, improves cell survival, and boosts neuronal differentiation. In this context, new and flexible imaging approaches are required to monitor the functional states of 3D networks. Herein, we propose an experimental model based on 3D neuronal networks in an alginate hydrogel, a tunable wide-volume imaging approach, and an efficient denoising algorithm to resolve, down to single cell resolution, the 3D activity of hundreds of neurons expressing the calcium sensor GCaMP6s. Furthermore, we implemented a 3D co-culture system mimicking the contiguous interfaces of distinct brain tissues such as the cortical-hippocampal interface. The analysis of the network activity of single and layered neuronal co-cultures revealed cell-type-specific activities and an organization of neuronal subpopulations that changed in the two culture configurations. Overall, our experimental platform represents a simple, powerful and cost-effective platform for developing and monitoring living 3D layered brain tissue on chip structures with high resolution and high throughput

Three-dimensional reconstruction of particle holograms: a fast and accurate multiscale approach.

10 pagesInternational audienceIn-line digital holography is an imaging technique that is being increasingly used for studying three-dimensional flows. It has been previously shown that very accurate reconstructions of objects could be achieved with the use of an inverse problem framework. Such approaches, however, suffer from higher computational times compared to less accurate conventional reconstructions based on hologram backpropagation. To overcome this computational issue, we propose a coarse-to-fine multiscale approach to strongly reduce the algorithm complexity. We illustrate that an accuracy comparable to that of state-of-the-art methods can be reached while accelerating parameter-space scanning