The spatial organization and extraction of the wall-forming bodies ofEimeria maxima

Parasitology ◽  
2013 ◽  
Vol 140 (7) ◽  
pp. 876-887 ◽  
Author(s):  
SONJA FRÖLICH ◽  
MICHAEL JOHNSON ◽  
MICHELLE ROBINSON ◽  
ROLF ENTZEROTH ◽  
MICHAEL WALLACH
Keyword(s):  
Spatial Organization ◽  
Fluorescent Microscopy ◽  
Three Dimensional ◽  
Wide Field ◽  
Oocyst Wall ◽  
Apicomplexan Parasites ◽  
Sds Page ◽  
Interest In Research

SUMMARYEimeria maximahas been used as a model apicomplexan parasite to study sexual stage development and oocyst wall formation. A complete understanding of the wall's biochemical and biophysical properties is of great interest in research on all apicomplexan parasites. Purified gametocytes, zygotes and oocysts were analysed by three-dimensional confocal microscopy, and wide-field fluorescent microscopy was used to investigate the appearance and spatial organization of the 2 types of wall-forming bodies (WFBs). In addition, a variety of staining procedures and immunoassays were used to assess the biosynthesis, metabolic activity, intactness and molecular composition of the WFBsin situ. WFBs were extracted from gametocytes/zygotes and their composition was assessed by microscopy and SDS-PAGE analysis. It was concluded that isolated gametocytes are intact and metabolically active. Additionally, it was observed that the Type 1 WFBs are aligned at the periphery of the parasite and fuse together producing neutral lipid rich patches that appear to be inserted into the space between 2 parasite-specific membranes. Finally, it was shown that the WFBs extracted from purified gametocytes had the same shape, size and staining properties as those observedin situ, and contained the major glycoprotein antigens known to be present in these organelles.

scholarly journals New Data on Organization and Spatial Localization of B-Chromosomes in Cell Nuclei of the Yellow-Necked Mouse Apodemus flavicollis

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1819
Author(s):  
Tatyana Karamysheva ◽  
Svetlana Romanenko ◽  
Alexey Makunin ◽  
Marija Rajičić ◽  
Alexey Bogdanov ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Sex Chromosomes ◽  
Interphase Nucleus ◽  
Spatial Organization ◽  
Three Dimensional ◽  
Dna Probes ◽  
B Chromosomes ◽  
Apodemus Flavicollis ◽  
Yellow Necked Mouse

The gene composition, function and evolution of B-chromosomes (Bs) have been actively discussed in recent years. However, the additional genomic elements are still enigmatic. One of Bs mysteries is their spatial organization in the interphase nucleus. It is known that heterochromatic compartments are not randomly localized in a nucleus. The purpose of this work was to study the organization and three-dimensional spatial arrangement of Bs in the interphase nucleus. Using microdissection of Bs and autosome centromeric heterochromatic regions of the yellow-necked mouse (Apodemus flavicollis) we obtained DNA probes for further two-dimensional (2D)- and three-dimensional (3D)- fluorescence in situ hybridization (FISH) studies. Simultaneous in situ hybridization of obtained here B-specific DNA probes and autosomal C-positive pericentromeric region-specific probes further corroborated the previously stated hypothesis about the pseudoautosomal origin of the additional chromosomes of this species. Analysis of the spatial organization of the Bs demonstrated the peripheral location of B-specific chromatin within the interphase nucleus and feasible contact with the nuclear envelope (similarly to pericentromeric regions of autosomes and sex chromosomes). It is assumed that such interaction is essential for the regulation of nuclear architecture. It also points out that Bs may follow the same mechanism as sex chromosomes to avoid a meiotic checkpoint.

scholarly journals Three-dimensional electron microscopy of ribosomal chromatin in two higher plants: a cytochemical, immunocytochemical, and in situ hybridization approach.

1991 ◽  
Vol 39 (11) ◽  
pp. 1495-1506 ◽  
Author(s):  
P M Motte ◽  
R Loppes ◽  
M Menager ◽  
R Deltour
Keyword(s):  
Electron Microscopy ◽  
In Situ Hybridization ◽  
Spatial Organization ◽  
Higher Plants ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Thin Sections ◽  
Cytoplasmic Dna ◽  
Immunocytochemical Techniques

We report the 3-D arrangement of DNA within the nucleolar subcomponents from two evolutionary distant higher plants, Zea mays and Sinapis alba. These species are particularly convenient to study the spatial organization of plant intranucleolar DNA, since their nucleoli have been previously reconstructed in 3-D from serial ultra-thin sections. We used the osmium ammine-B complex (a specific DNA stain) on thick sections of Lowicryl-embedded root fragments. Immunocytochemical techniques using anti-DNA antibodies and rDNA/rDNA in situ hybridization were also applied on ultra-thin sections. We showed on tilted images that the OA-B stains DNA throughout the whole thickness of the section. In addition, very low quantities of cytoplasmic DNA were stained by this complex, which is now the best DNA stain used in electron microscopy. Within the nucleoli the DNA was localized in the fibrillar centers, where large clumps of dense chromatin were also visible. In the two plant species intranucleolar chromatin forms a complex network with strands partially linked to chromosomal nucleolar-organizing regions identified by in situ hybridization. This study describes for the first time the spatial arrangement of the intranucleolar chromatin in nucleoli of higher plants using high-resolution techniques.

scholarly journals Structure of hibernating ribosomes studied by cryoelectron tomography in vitro and in situ

2010 ◽  
Vol 190 (4) ◽  
pp. 613-621 ◽  
Author(s):  
Julio O. Ortiz ◽  
Florian Brandt ◽  
Valério R.F. Matias ◽  
Lau Sennels ◽  
Juri Rappsilber ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Spatial Organization ◽  
Three Dimensional ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Three Dimensional Configuration

Ribosomes arranged in pairs (100S) have been related with nutritional stress response and are believed to represent a “hibernation state.” Several proteins have been identified that are associated with 100S ribosomes but their spatial organization has hitherto not been characterized. We have used cryoelectron tomography to reveal the three-dimensional configuration of 100S ribosomes isolated from starved Escherichia coli cells and we have described their mode of interaction. In situ studies with intact E. coli cells allowed us to demonstrate that 100S ribosomes do exist in vivo and represent an easily reversible state of quiescence; they readily vanish when the growth medium is replenished.

scholarly journals Integration of Multiple Resolution Data in 3D Chromatin Reconstruction Using ChromStruct

Biology ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 338
Author(s):  
Claudia Caudai ◽  
Monica Zoppè ◽  
Anna Tonazzini ◽  
Ivan Merelli ◽  
Emanuele Salerno
Keyword(s):  
Spatial Organization ◽  
Local Level ◽  
3D Structure ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Rna Seq ◽  
Chromosome Conformation ◽  
Experimental Protocols ◽  
Easy Integration

The three-dimensional structure of chromatin in the cellular nucleus carries important information that is connected to physiological and pathological correlates and dysfunctional cell behaviour. As direct observation is not feasible at present, on one side, several experimental techniques have been developed to provide information on the spatial organization of the DNA in the cell; on the other side, several computational methods have been developed to elaborate experimental data and infer 3D chromatin conformations. The most relevant experimental methods are Chromosome Conformation Capture and its derivatives, chromatin immunoprecipitation and sequencing techniques (CHIP-seq), RNA-seq, fluorescence in situ hybridization (FISH) and other genetic and biochemical techniques. All of them provide important and complementary information that relate to the three-dimensional organization of chromatin. However, these techniques employ very different experimental protocols and provide information that is not easily integrated, due to different contexts and different resolutions. Here, we present an open-source tool, which is an expansion of the previously reported code ChromStruct, for inferring the 3D structure of chromatin that, by exploiting a multilevel approach, allows an easy integration of information derived from different experimental protocols and referred to different resolution levels of the structure, from a few kilobases up to Megabases. Our results show that the introduction of chromatin modelling features related to CTCF CHIA-PET data, histone modification CHIP-seq, and RNA-seq data produce appreciable improvements in ChromStruct’s 3D reconstructions, compared to the use of HI-C data alone, at a local level and at a very high resolution.

scholarly journals Major Reorganization of Chromosome Conformation During Muscle Development in Pig

2021 ◽  
Vol 12 ◽  
Author(s):  
Maria Marti-Marimon ◽  
Nathalie Vialaneix ◽  
Yvette Lahbib-Mansais ◽  
Matthias Zytnicki ◽  
Sylvie Camut ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Muscle Development ◽  
Three Dimensional ◽  
Eukaryotic Cell ◽  
Chromosome Conformation ◽  
Cell Functions ◽  
A Genome ◽  
Structural Regulation ◽  
Complex Landscape

The spatial organization of the genome in the nucleus plays a crucial role in eukaryotic cell functions, yet little is known about chromatin structure variations during late fetal development in mammals. We performed in situ high-throughput chromosome conformation capture (Hi-C) sequencing of DNA from muscle samples of pig fetuses at two late stages of gestation. Comparative analysis of the resulting Hi-C interaction matrices between both groups showed widespread differences of different types. First, we discovered a complex landscape of stable and group-specific Topologically Associating Domains (TADs). Investigating the nuclear partition of the chromatin into transcriptionally active and inactive compartments, we observed a genome-wide fragmentation of these compartments between 90 and 110 days of gestation. Also, we identified and characterized the distribution of differential cis- and trans-pairwise interactions. In particular, trans-interactions at chromosome extremities revealed a mechanism of telomere clustering further confirmed by 3D Fluorescence in situ Hybridization (FISH). Altogether, we report major variations of the three-dimensional genome conformation during muscle development in pig, involving several levels of chromatin remodeling and structural regulation.

scholarly journals Three-dimensional mapping identifies distinct vascular niches for myelopoiesis

2020 ◽  
Author(s):  
Jizhou Zhang ◽  
Qingqing Wu ◽  
Courtney B. Johnson ◽  
Andre Olsson ◽  
Anastasiya Slaughter ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Dendritic Cell ◽  
Spatial Organization ◽  
Three Dimensional ◽  
The Body ◽  
Hematopoietic Stem ◽  
Dendritic Cell Differentiation ◽  
Hematopoietic Stem Cell Niche ◽  
Three Dimensional Mapping

SummaryIn contrast to virtually all other tissues in the body the anatomy of differentiation in the bone marrow remains unknown. This is due to the lack of strategies to examine blood cell production in situ, which are required to better understand differentiation, lineage commitment decisions, and to define how spatial organizing cues inform tissue function. Here we developed imaging approaches to map all myeloid cells in whole bones and generated 3D atlases of granulocyte and monocyte/dendritic cell differentiation during homeostasis. We found that myeloid progenitors leave the hematopoietic stem cell niche during differentiation. Granulocyte and monocyte dendritic cell progenitors (MDP) do not interact, instead they localize to different sinusoids where they give rise to clusters of immature cells. MDP cluster with Ly6Clo monocytes and conventional dendritic cells; these localize to a unique subset of colony stimulating factor 1 (CSF1, the major regulator of monopoiesis1) -expressing sinusoids. Csf1 deletion in the vasculature disrupted the MDP clusters and their interaction with sinusoids, leading to reduced MDP numbers and differentiation ability, with subsequent loss of peripheral Ly6Clo monocytes and dendritic cells. These data indicate that there is a specific spatial organization of definitive hematopoiesis and that local cues produced by distinct blood vessels are responsible for this organization. These maps provide a blueprint for in situ analyses of hematopoiesis in blood disorders.

scholarly journals Electron Tomography of Metaphase Nucleolar Organizer Regions: Evidence for a Twisted-Loop Organization

1997 ◽  
Vol 8 (11) ◽  
pp. 2199-2216 ◽  
Author(s):  
Laurent Heliot ◽  
Hervé Kaplan ◽  
Laurent Lucas ◽  
Christophe Klein ◽  
Adrien Beorchia ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Spatial Organization ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Nucleolar Organizer ◽  
Nucleolar Organizer Regions ◽  
Binding Factor ◽  
Upstream Binding Factor ◽  
Polymerase I

Metaphase nucleolar organizer regions (NORs), one of four types of chromosome bands, are located on human acrocentric chromosomes. They contain r-chromatin, i.e., ribosomal genes complexed with proteins such as upstream binding factor and RNA polymerase I, which are argyrophilic NOR proteins. Immunocytochemical and cytochemical labelings of these proteins were used to reveal r-chromatin in situ and to investigate its spatial organization within NORs by confocal microscopy and by electron tomography. For each labeling, confocal microscopy revealed small and large double-spotted NORs and crescent-shaped NORs. Their internal three-dimensional (3D) organization was studied by using electron tomography on specifically silver-stained NORs. The 3D reconstructions allow us to conclude that the argyrophilic NOR proteins are grouped as a fiber of 60–80 nm in diameter that constitutes either one part of a turn or two or three turns of a helix within small and large double-spotted NORs, respectively. Within crescent-shaped NORs, virtual slices reveal that the fiber constitutes several longitudinally twisted loops, grouped as two helical 250- to 300-nm coils, each centered on a nonargyrophilic axis of condensed chromatin. We propose a model of the 3D organization of r-chromatin within elongated NORs, in which loops are twisted and bent to constitute one basic chromatid coil.

scholarly journals A Single Cell Atlas of Lung Development

2021 ◽  
Author(s):  
Nicholas M. Negretti ◽  
Erin J. Plosa ◽  
John T. Benjamin ◽  
Bryce A. Schuler ◽  
A. Christian Habermann ◽  
...  
Keyword(s):  
Single Cell ◽  
Lung Development ◽  
Spatial Organization ◽  
Single Cell Rna Sequencing ◽  
And Function ◽  
Parenchymal Cells ◽  
Computational Analyses ◽  
Spatiotemporal Localization

SummaryLung organogenesis requires precisely timed shifts in the spatial organization and function of parenchymal cells, especially during the later stages of lung development. To investigate the mechanisms governing lung parenchymal dynamics during development, we performed a single cell RNA sequencing (scRNA-seq) time-series yielding 92,238 epithelial, endothelial, and mesenchymal cells across 8 time points from embryonic day 12 (E12) to postnatal day 14 (P14) in mice. We combined new computational analyses with RNA in situ hybridization to explore transcriptional velocity, fate likelihood prediction, and spatiotemporal localization of cell populations during the transition between the saccular and alveolar stages. We interrogated this atlas to illustrate the complexity of type 1 pneumocyte function during the saccular and alveolar stages, and we demonstrate an integrated view of the cellular dynamics during lung development.

scholarly journals Distinct 3D Structural Patterns of Lamin A/C Expression in Hodgkin and Reed-Sternberg Cells

Cancers ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 286 ◽  
Author(s):  
Fabio Contu ◽  
Aline Rangel-Pozzo ◽  
Peter Trokajlo ◽  
Landon Wark ◽  
Ludger Klewes ◽  
...  
Keyword(s):  
Cell Lines ◽  
Spatial Organization ◽  
Expression Patterns ◽  
Fluorescent Microscopy ◽  
Three Dimensional ◽  
Lymphocyte Activation ◽  
B Cell Lymphoma ◽  
Peripheral Blood Lymphocytes ◽  
Lamin A ◽  
C Protein

Classical Hodgkin’s lymphoma (cHL) is a B-Cell lymphoma comprised of mononuclear Hodgkin cells (H) and bi- to multi-nucleated Reed-Sternberg (RS) cells. Previous studies revealed that H and RS cells express lamin A/C, a component of the lamina of the nuclear matrix. Since no information was available about the three-dimensional (3D) expression patterns of lamin A/C in H and RS cells, we analyzed the 3D spatial organization of lamin in such cells, using 3D fluorescent microscopy. H and RS cells from cHL derived cell lines stained positive for lamin A/C, in contrast to peripheral blood lymphocytes (PBLs), in which the lamin A/C protein was not detected or weak, although its presence could be transiently increased with lymphocyte activation by lipopolysaccharide (LPS). Most importantly, in H and RS cells, the regular homogeneous and spherically shaped lamin A/C pattern, identified in activated lymphocytes, was absent. Instead, in H and RS cells, lamin staining showed internal lamin A/C structures, subdividing the nuclei into two or more smaller compartments. Analysis of pre-treatment cHL patients’ samples replicated the lamin patterns identified in cHL cell lines. We conclude that the investigation of lamin A/C protein could be a useful tool for understanding nuclear remodeling in cHL.

scholarly journals Preservation of three-dimensional spatial structure in the gut microbiome

2017 ◽  
Author(s):  
Yuko Hasegawa ◽  
Jessica L. Mark Welch ◽  
Blair J. Rossetti ◽  
Gary G. Borisy
Keyword(s):  
Spatial Structure ◽  
Spatial Organization ◽  
Three Dimensional ◽  
Cutting Temperature ◽  
Dimensional Structure ◽  
Preparation Methods ◽  
Optimal Cutting Temperature ◽  
Food Particles ◽  
Intestinal Preparation

AbstractPreservation of three-dimensional structure in the gut is necessary in order to analyze the spatial organization of the gut microbiota and gut luminal contents. In this study, we evaluated preparation methods for mouse gut with the goal of preserving micron-scale spatial structure while performing fluorescence imaging assays. Our evaluation of embedding methods showed that commonly used media such as Tissue-Tek Optimal Cutting Temperature (OCT) compound, paraffin, and polyester waxes resulted in redistribution of luminal contents. By contrast, a hydrophilic methacrylate resin, Technovit H8100, preserved three-dimensional organization. Our mouse intestinal preparation protocol optimized using the Technovit H8100 embedding method was compatible with microbial fluorescencein situhybridization (FISH) and other labeling techniques, including immunostaining and staining with both wheat germ agglutinin (WGA) and 4’,6-diamidino-2-phenylindole (DAPI). Mucus labeling patterns of the samples fixed with paraformaldehyde (PFA) and Carnoy’s fixative were comparable. The protocol optimized in this study enabled simultaneous visualization of micron-scale spatial patterns formed by microbial cells in the mouse intestines along with biogeographical landmarks such as host-derived mucus and food particles.

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