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.

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 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 A guided approach for subtomogram averaging of challenging macromolecular assemblies

2020 ◽  
Author(s):  
Danielle Grotjahn ◽  
Saikat Chowdhury ◽  
Gabriel C. Lander
Keyword(s):  
Electron Tomography ◽  
A Priori ◽  
Three Dimensional ◽  
Structural Heterogeneity ◽  
Dimensional Structure ◽  
Diverse Range ◽  
Macromolecular Assemblies ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

AbstractCryo-electron tomography is a powerful biophysical technique enabling three-dimensional visualization of complex biological systems. Macromolecular targets of interest identified within cryo-tomograms can be computationally extracted, aligned, and averaged to produce a better-resolved structure through a process called subtomogram averaging (STA). However, accurate alignment of macromolecular machines that exhibit extreme structural heterogeneity and conformational flexibility remains a significant challenge with conventional STA approaches. To expand the applicability of STA to a broader range of pleomorphic complexes, we developed a user-guided, focused refinement approach that can be incorporated into the standard STA workflow to facilitate the robust alignment of particularly challenging samples. We demonstrate that it is possible to align visually recognizable portions of multi-subunit complexes by providing a priori information regarding their relative orientations within cryo-tomograms, and describe how this strategy was applied to successfully elucidate the first three-dimensional structure of the dynein-dynactin motor protein complex bound to microtubules. Our approach expands the application of STA for solving a more diverse range of heterogeneous biological structures, and establishes a conceptual framework for the development of automated strategies to deconvolve the complexity of crowded cellular environments and improve in situ structure determination technologies.

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 Density enhancements associated with equatorial spread <I>F</I>

2010 ◽  
Vol 28 (2) ◽  
pp. 327-337 ◽  
Author(s):  
J. Krall ◽  
J. D. Huba ◽  
G. Joyce ◽  
T. Yokoyama
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Simulation Code ◽  
Equatorial Spread F ◽  
Equatorial Ionization Anomaly ◽  
Spread F ◽  
Key Features ◽  
Field Lines ◽  
Dimensional Simulation

Abstract. Forces governing the three-dimensional structure of equatorial spread-F (ESF) plumes are examined using the NRL SAMI3/ESF three-dimensional simulation code. As is the case with the equatorial ionization anomaly (IA), density crests within the plume occur where gravitational and diffusive forces are in balance. Large E×B drifts within the ESF plume place these crests on field lines with apex heights higher than those of the background IA crests. Large poleward field-aligned ion velocities within the plume result in large ion-neutral diffusive forces that support these ionization crests at altitudes higher than background IA crest altitudes. We show examples in which density enhancements associated with ESF, also called "plasma blobs," can occur within an ESF plume on density-crest field lines, at or above the density crests. Simulated ESF density enhancements reproduce all key features of those that have been observed in situ.

scholarly journals Identification of CD34+/PGDFRα+ Valve Interstitial Cells (VICs) in Human Aortic Valves: Association of Their Abundance, Morphology and Spatial Organization with Early Calcific Remodeling

2020 ◽  
Vol 21 (17) ◽  
pp. 6330
Author(s):  
Grzegorz J. Lis ◽  
Andrzej Dubrowski ◽  
Maciej Lis ◽  
Bernard Solewski ◽  
Karolina Witkowska ◽  
...  
Keyword(s):  
Heart Valves ◽  
Spatial Organization ◽  
Three Dimensional ◽  
Interstitial Cells ◽  
Dimensional Structure ◽  
Aortic Valves ◽  
Valve Interstitial Cells ◽  
Valve Tissue ◽  
Early Signs ◽  
Younger Age

Aortic valve interstitial cells (VICs) constitute a heterogeneous population involved in the maintenance of unique valvular architecture, ensuring proper hemodynamic function but also engaged in valve degeneration. Recently, cells similar to telocytes/interstitial Cajal-like cells described in various organs were found in heart valves. The aim of this study was to examine the density, distribution, and spatial organization of a VIC subset co-expressing CD34 and PDGFRα in normal aortic valves and to investigate if these cells are associated with the occurrence of early signs of valve calcific remodeling. We examined 28 human aortic valves obtained upon autopsy. General valve morphology and the early signs of degeneration were assessed histochemically. The studied VICs were identified by immunofluorescence (CD34, PDGFRα, vimentin), and their number in standardized parts and layers of the valves was evaluated. In order to show the complex three-dimensional structure of CD34+/PDGFRα+ VICs, whole-mount specimens were imaged by confocal microscopy, and subsequently rendered using the Imaris (Bitplane AG, Zürich, Switzerland) software. CD34+/PDGFRα+ VICs were found in all examined valves, showing significant differences in the number, distribution within valve tissue, spatial organization, and morphology (spherical/oval without projections; numerous short projections; long, branching, occasionally moniliform projections). Such a complex morphology was associated with the younger age of the subjects, and these VICs were more frequent in the spongiosa layer of the valve. Both the number and percentage of CD34+/PDGFRα+ VICs were inversely correlated with the age of the subjects. Valves with histochemical signs of early calcification contained a lower number of CD34+/PDGFRα+ cells. They were less numerous in proximal parts of the cusps, i.e., areas prone to calcification. The results suggest that normal aortic valves contain a subpopulation of CD34+/PDGFRα+ VICs, which might be involved in the maintenance of local microenvironment resisting to pathologic remodeling. Their reduced number in older age could limit the self-regenerative properties of the valve stroma.

scholarly journals The centromere-kinetochore complex: a repeat subunit model.

1991 ◽  
Vol 113 (5) ◽  
pp. 1091-1110 ◽  
Author(s):  
R P Zinkowski ◽  
J Meyne ◽  
B R Brinkley
Keyword(s):  
Three Dimensional ◽  
Protein Composition ◽  
Cytoplasmic Dynein ◽  
Dimensional Structure ◽  
Electron Microscopic ◽  
Microtubule Motor ◽  
A Subunit ◽  
Kinetochore Region ◽  
Subunit Organization

The three-dimensional structure of the kinetochore and the DNA/protein composition of the centromere-kinetochore region was investigated using two novel techniques, caffeine-induced detachment of unreplicated kinetochores and stretching of kinetochores by hypotonic and/or shear forces generated in a cytocentrifuge. Kinetochore detachment was confirmed by EM and immunostaining with CREST autoantibodies. Electron microscopic analyses of serial sections demonstrated that detached kinetochores represented fragments derived from whole kinetochores. This was especially evident for the seven large kinetochores in the male Indian muntjac that gave rise to 80-100 fragments upon detachment. The kinetochore fragments, all of which interacted with spindle microtubules and progressed through the entire repertoire of mitotic movements, provide evidence for a subunit organization within the kinetochore. Further support for a repeat subunit model was obtained by stretching or uncoiling the metaphase centromere-kinetochore complex by hypotonic treatments. When immunostained with CREST autoantibodies and subsequently processed for in situ hybridization using synthetic centromere probes, stretched kinetochores displayed a linear array of fluorescent subunits arranged in a repetitive pattern along a centromeric DNA fiber. In addition to CREST antigens, each repetitive subunit was found to bind tubulin and contain cytoplasmic dynein, a microtubule motor localized in the zone of the corona. Collectively, the data suggest that the kinetochore, a plate-like structure seen by EM on many eukaryotic chromosomes is formed by the folding of a linear DNA fiber consisting of tandemly repeated subunits interspersed by DNA linkers. This model, unlike any previously proposed, can account for the structural and evolutional diversity of the kinetochore and its relationship to the centromere of eukaryotic chromosomes of many species.

scholarly journals Molecular architecture of inner dynein arms in situ in Chlamydomonas reinhardtii flagella

2008 ◽  
Vol 183 (5) ◽  
pp. 923-932 ◽  
Author(s):  
Khanh Huy Bui ◽  
Hitoshi Sakakibara ◽  
Tandis Movassagh ◽  
Kazuhiro Oiwa ◽  
Takashi Ishikawa
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Molecular Architecture ◽  
Heavy Chains ◽  
Distal Side ◽  
Cryo Electron Tomography ◽  
Dynein Arms

The inner dynein arm regulates axonemal bending motion in eukaryotes. We used cryo-electron tomography to reconstruct the three-dimensional structure of inner dynein arms from Chlamydomonas reinhardtii. All the eight different heavy chains were identified in one 96-nm periodic repeat, as expected from previous biochemical studies. Based on mutants, we identified the positions of the AAA rings and the N-terminal tails of all the eight heavy chains. The dynein f dimer is located close to the surface of the A-microtubule, whereas the other six heavy chain rings are roughly colinear at a larger distance to form three dyads. Each dyad consists of two heavy chains and has a corresponding radial spoke or a similar feature. In each of the six heavy chains (dynein a, b, c, d, e, and g), the N-terminal tail extends from the distal side of the ring. To interact with the B-microtubule through stalks, the inner-arm dyneins must have either different handedness or, more probably, the opposite orientation of the AAA rings compared with the outer-arm dyneins.

scholarly journals The role of CTCF in regulating nuclear organization

2008 ◽  
Vol 205 (4) ◽  
pp. 747-750 ◽  
Author(s):  
Adam Williams ◽  
Richard A. Flavell
Keyword(s):  
Long Range ◽  
Dna Sequences ◽  
Spatial Organization ◽  
Zinc Finger Protein ◽  
Nuclear Organization ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Binding Factor ◽  
Long Range Interactions ◽  
Regulatory Functions

The spatial organization of the genome is thought to play an important part in the coordination of gene regulation. New techniques have been used to identify specific long-range interactions between distal DNA sequences, revealing an ever-increasing complexity to nuclear organization. CCCTC-binding factor (CTCF) is a versatile zinc finger protein with diverse regulatory functions. New data now help define how CTCF mediates both long-range intrachromosomal and interchromosomal interactions, and highlight CTCF as an important factor in determining the three-dimensional structure of the genome.

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