6 research outputs found
Phase-contrast x-ray imaging and tomography of the nematode Caenorhabditis elegans
No full textWe have analyzed the model organism Caenorhabditis elegans with the help of phase-contrast x-ray tomography. This work combines techniques from x-ray imaging studies of single biological cells by in-line holography with three-dimensional reconstruction and furthermore extends these studies to the multicellular level. To preserve the sub-cellular ultrastructure of the nematodes, we used the near-native sample preparation of high-pressure freezing as commonly used in the field of electron microscopy. For the presented samples, a standard, non-magnifying parallel-beam setting, as well as a magnifying, divergent-beam setting using nanofocusing optics is evaluated based on their tomographic reconstruction potential. In this paper, we address difficulties in sample preparation and issues of image processing. By experimental refinement and through optimized reconstruction procedures, we were able to perform x-ray imaging studies on a living specimen
X-Ray Nano-Diffraction on Cytoskeletal Networks
No full textThe nano-scale structure of cytoskeletal biopolymers as well as sophisticated superstructures determine the versatile cellular shapes and specific mechanical properties. One example is keratin intermediate filaments in epithelial cells, which form thick bundles that can further organize in a cross-linked network. To study the native structure of keratin bundles in whole cells, high-resolution techniques are required, which do at the same time achieve high penetration depths. We employ scanning x-ray diffraction using a nano-focused x-ray beam to study the structure of keratin in freeze-dried eukaryotic cells. By scanning the sample through the beam we obtain x-ray dark-field images with a resolution of the order of the beam size, which clearly show the keratin network. Each individual diffraction pattern is further analyzed to yield insight into the local sample structure, which allows us to determine the local structure orientation. Due to the small beam size we access the structure in a small sample volume without performing the ensemble average over one complete cell.Open-Access-Publikationsfonds 201
UNC-108/RAB-2 and its effector RIC-19 are involved in dense core vesicle maturation in C. elegans
No full textSmall GTPases of the Rab family regulate intracellular vesicular trafficking. In C. elegans, two dominant alleles of unc-108/rab-2 have been isolated based on their movement defects. Using a combination of pharmacological assays and electron microscopy, we show that the locomotion defects of rab-2 mutant animals are cause by a reduced facilitation of synaptic vesicle (SV) release from pre-synaptic sites due to lower levels of diacylglycerol (DAG). DAG levels at release sites can be regulated by neuro-peptides secreted from dense core vesicles (DCV). We show that while their number and distribution is not affected, DCVs in dominant rab-2 mutants are enlarged and contain less neuropeptides. This implicates RAB-2 in the biogenesis of DCVs at the Golgi complex. In rab-2 mutants DCV cargo inappropriately enters late endosomal compartments. Finally we show that RIC-19, the C. elegans ortholog of the human diabetes autoantigen ICA69, is recruited to Golgi membranes by RAB-2 for proper DCV biogenesis.sponsorship: We thank Francis Barr for the hRab2 cDNA, Shweta Aggarwal, Reinhard Jahn, and Gottfried Mieskes for support, Marc Pilon for RIC-19 antibodies, Ken Miller and Josh Kaplan for strains, and Ken Miller for sharing unpublished results and discussions. We specially thank Virginie Lecaudey, Nikhil Sasidharan, and Reinhard Jahn for their comments on the manuscript. Some strains were provided by the Caenorhabditis Genetics Center, which is supported by the National Institutes of Health National Center for Research Resources. We gratefully acknowledge funding by the European Union (EU) Sixth Framework Programme coordination action program European Network of Neuroscience Institutes, Neuroscience Early Stage Research Training fellowships of the EU to L. Luo and M. Sumakovic, and a postdoctoral fellowship from the Research Foundation Flanders (FWO) to S.J. Husson. L. Schoofs is supported by the FWO, and S. Eimer is supported by the German Research Foundation Research Center for Molecular Physiology of the Brain. (National Institutes of Health National Center for Research Resources, European Union (EU), Research Foundation Flanders (FWO), German Research Foundation Research Center for Molecular Physiology of the Brain)status: Publishe
Low-dose three-dimensional hard x-ray imaging of bacterial cells
No full textWe have imaged the three-dimensional density distribution of unstained and unsliced, freeze-dried cells of the gram-positive bacterium Deinococcus radiodurans by tomographic x-ray propagation microscopy, i.e. projection tomography with phase contrast formation by free space propagation. The work extends previous x-ray imaging of biological cells in the simple in-line holography geometry to full three-dimensional reconstruction, based on a fast iterative phase reconstruction algorithm which circumvents the usual twin-image problem. The sample is illuminated by the highly curved wave fronts emitted from a virtual quasi-point source with 10 nm cross section, realized by two crossed x-ray waveguides. The experimental scheme allows for a particularly dose efficient determination of the 3D density distribution in the cellular structure
Eine mikroskopische Übersicht intrazellulärer Transportprozesse
No full textDas Ziel dieser Arbeit ist die Entwicklung eines verbesserten Verständnisses von intrazellulären Transportprozessen in multizellulären Organismen. Für diese Zwecke wurde in diesen Studien die Vorzüge des Modelorgansimus Caenorhabditis elegans benutzt um dessen Familie der Rab-GTPasen zu untersuchen, da diese kleinen GTPasesn sämtliche Transportprozesse organisieren. Diese Arbeit umfasst eine morphologische und phenotypische Beschreibung der verfügbaren Mutanten dieser Rab-Genfamilie auf zellulärer und subzellulärer Ebene in 2D und 3D, mit Hilfe von elektronen- und röntgen-mikroskopischen Methoden. Zusätzlich wurde zur Bewahrung der zellulären Ultrastruktur die Methode des Hoch-Druck-Gefrierens angewendet, da diese Methode eine instantane, native Konservierung des gesamten Wurmes ermöglicht. Eine Einführung in die Thematik des intrazellulären Transport ist gegeben in Kapitel I währen alle benutzen Methoden in Kapitel II zu finden sind. Die Ergebnisse der morphologischen Studie mit Hilfe von elektronenmikroskopischen Methoden sowie die Verhaltensstudien von den Rab-Mutanten sind in einem Rab-Atlas zusammengefasst und werden in einem frei zugänglichen Rab-Wiki der Forschungsgemeinschaft zur Verfügung gestellt. Die Ergebnisse aus allen vom Author, für diese Studie, durchgeführten und analysierten Experimenten sind in Kapitel III aufgeführt und diskutiert. Weitere Fokus wurde auf die ultra-schnellen Transportprozesse in Nervenzellen gerichtet. Für die Auswertung der Vesikel Verteilungen in Neuronen wurde hierfür das semi-automatisches Programm XtraCOunt entwickelt. Die Ergebnisse dieser Studien sind in Kapitel IV zusammengefasst und die erste Anwendung dieses vielseitigen Programmes in weiterführenden Studien wird in Kapitel V präsentiert. Zuletzt wurden erste röntgen-mikroskopische Untersuchungen für die Untersuchung von Hoch-Druck-Gefrorenen Proben ausprobiert. Diese Tests umfassen die Untersuchung in der Phasenkontrast-Bildgebung, Holotomographie und Röntgenspektroskopie. In Kapitel VI werden die zugrundeliegenden mathematisch-physikalischen Hintergründe erörtert und die vielversprechenden Ergebnisse dieser Untersuchungen präsentiert. Im abschließenden Kapitel VII werden alle die Zusammenhänge der Untersuchungen dieser Studie diskutiert und deren Beitrag zum weiteren Verständnis im Dienste der Forschungsgemeinschaft erörtert.The general aim of this thesis work is to gain an encompassing insight into the intracellular trafficking machinery of multicellular organisms. For this purpose, the previously well-described model organism Caenorhabdits elegans is analyzed in our studies, with a focus on the family of small Rab-GTPases. These Rab-proteins (RABs) are the central players that orchestrate all intracellular trafficking events. An introduction to the current understandings in this field of science is given in the introductory chapter I. This thesis work began with a morphological description and a behavioral characterization of mutants of all members of rab-gene (rab) family. The major technique used for this anatomic investigation on the cellular- and subcellular level was two- as well as three-dimensional electron microscopy of high-pressure frozen nematode with a near-native sample-preservation. An atlas comprising all morphological data together with the results of additional behavioral studies, is the ultimate outcome of this thesis work. Among the descriptions of several tissues and organelles, the findings that the Golgi shapes seems to adapt to the activity in a tissue-specific manner is of central importance as it suggests that intracellular transport processes must also be investigated in a tissue-specific fashion. The generated tissue- and Rab-Atlas, along with all related results, is presented in chapter III, while a detailed description of used materials and methods is given in chapter II. During the entire investigation a special focus was directed towards the ultra-fast trafficking events that ensure the transmission at synapses. To aid these studies, the image analysis tool XtraCOunt was generated during this thesis work in order to semi-automates the analysis of these trafficking studies. Chapter IV describes this tool along with all its features. In the atlas studies the rab-10 mutant was identified as potentially neuronally impaired. In chapter IV, this mutant is examined for its synaptic features and it has been found to resemble the rab-2 mutant alterations in dense-core vesicle biogenesis. XtraCOunt was conceived for the analysis of electron micrographs of nematode synapses but was shown to be applicable in a vast range of other trafficking studies. Current collaborating studies, using this tool, led to the understanding of the phenotype of Sydney Brenner's first described mutant dpy-1 (dumpy), whose cuticular components are not able to maintain their network integrity. The fundamental findings of this study are presented in chapter V. To overcome imaging-constrains presented by the mostly electron microscopic nature of my study, I have furthermore tested the possibility of studying high-pressure frozen specimen with x-rays. High resolution phase contrast imaging, holo-tomography as well as micro-fluorescence investigations were tested for their applicability, feasibility and limitations. Their underlying mathematical theory and the obtained experimental results are presented and discussed in chapter VI. The major results of these investigations is a full-worm-tomogram with subcellular resolution and a map of tissue-specific elemental distributions within worm thin-slices. The concluding chapter VII briefly summarizes the results of all performed investigations in the context with one another and their possible meaning for the intracellular trafficking community
RAB-5 and RAB-10 cooperate to regulate neuropeptide release in Caenorhabditis elegans
No full textNeurons secrete neuropeptides from dense core vesicles (DCVs) to modulate neuronal activity. Little is known about how neurons manage to differentially regulate the release of synaptic vesicles (SVs) and DCVs. To analyze this, we screened all Caenorhabditis elegans Rab GTPases and Tre2/Bub2/Cdc16 (TBC) domain containing GTPase-activating proteins (GAPs) for defects in DCV release from C. elegans motoneurons. rab-5 and rab-10 mutants show severe defects in DCV secretion, whereas SV exocytosis is unaffected. We identified TBC-2 and TBC-4 as putative GAPs for RAB-5 and RAB-10, respectively. Multiple Rabs and RabGAPs are typically organized in cascades that confer directionality to membrane-trafficking processes. We show here that the formation of release-competent DCVs requires a reciprocal exclusion cascade coupling RAB-5 and RAB-10, in which each of the two Rabs recruits the other's GAP molecule. This contributes to a separation of RAB-5 and RAB-10 domains at the Golgi-endosomal interface, which is lost when either of the two GAPs is inactivated. Taken together, our data suggest that RAB-5 and RAB-10 cooperate to locally exclude each other at an essential stage during DCV sorting
