scholarly journals CENP-A nucleosome clusters form rosette-like structures around HJURP during G1

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Leonid Andronov ◽  
Khalid Ouararhni ◽  
Isabelle Stoll ◽  
Bruno P. Klaholz ◽  
Ali Hamiche
Keyword(s):  
Centromeric Region ◽  
Histone H3 ◽  
Super Resolution ◽  
Structural Description ◽  
Compact Structure ◽  
Segmentation Analysis ◽  
Histone H3 Variant ◽  
Localization Analysis ◽  
2D And 3D ◽  
Super Resolution Microscopy

Abstract CENP-A is an essential histone H3 variant that epigenetically marks the centromeric region of chromosomes. Here we show that CENP-A nucleosomes form characteristic clusters during the G1 phase of the cell cycle. 2D and 3D super-resolution microscopy and segmentation analysis reveal that these clusters encompass a globular rosette-like structure, which evolves into a more compact structure in late G1. The rosette-like clusters contain numerous CENP-A molecules and form a large cellular structure of ∼250–300 nm diameter with remarkably similar shapes for each centromere. Co-localization analysis shows that HJURP, the CENP-A chaperone, is located in the center of the rosette and serves as a nucleation point. The discovery of an HJURP-mediated CENP-A nucleation in human cells and its structural description provide important insights into the mechanism of CENP-A deposition and the organization of CENP-A chromatin in the centromeric region.

scholarly journals Only the Rye Derived Part of the 1BL/1RS Hybrid Centromere Incorporates CENH3 of Wheat

2021 ◽  
Vol 12 ◽  
Author(s):  
Raheleh Karimi-Ashtiyani ◽  
Veit Schubert ◽  
Andreas Houben
Keyword(s):  
Histone H3 ◽  
Super Resolution ◽  
Translocation Chromosome ◽  
Chromosome Stability ◽  
Histone H3 Variant ◽  
Super Resolution Microscopy

The precise assembly of the kinetochore complex at the centromere is epigenetically determined by substituting histone H3 with the centromere-specific histone H3 variant CENH3 in centromeric nucleosomes. The wheat-rye 1BL/1RS translocation chromosome in the background of wheat resulted from a centric misdivision followed by the fusion of the broken arms of chromosomes 1B and 1R from wheat and rye, respectively. The resulting hybrid (dicentric)centromere is composed of both wheat and rye centromeric repeats. As CENH3 is a marker for centromere activity, we applied Immuno-FISH followed by ultrastructural super-resolution microscopy to address whether both or only parts of the hybrid centromere are active. Our study demonstrates that only the rye-derived centromere part incorporates CENH3 of wheat in the 1BL/1RS hybrid centromere. This finding supports the notion that one centromere part of a translocated chromosome undergoes inactivation, creating functional monocentric chromosomes to maintain chromosome stability.

scholarly journals Quantitative Super-Resolution Microscopy of the Mammalian Glycocalyx

2018 ◽  
Author(s):  
Leonhard Möckl ◽  
Kayvon Pedram ◽  
Anish R. Roy ◽  
Venkatesh Krishnan ◽  
Anna-Karin Gustavsson ◽  
...  
Keyword(s):  
Epithelial To Mesenchymal Transition ◽  
Human Tumor ◽  
Super Resolution ◽  
Metabolic Labeling ◽  
Human Tumor Cells ◽  
Mesenchymal Transition ◽  
Health And Disease ◽  
20 Nm ◽  
2D And 3D ◽  
Super Resolution Microscopy

<div> <div> <div> <p>The mammalian glycocalyx is a heavily glycosylated extramembrane compartment found on nearly every cell. Despite its relevance in both health and disease, studies of the glycocalyx remain hampered by a paucity of methods to spatially classify its components. We combine metabolic labeling, bioorthogonal chemistry, and super-resolution localization microscopy to image two constituents of cell-surface glycans, N-acetylgalactosamine (GalNAc) and sialic acid, with 10-20 nm precision in 2D and 3D. This approach enables two measurements: glycocalyx height and the distribution of individual sugars distal from the membrane. These measurements show that the glycocalyx exhibits nanoscale architecture, on both cell lines and primary human tumor cells. Additionally, we observe enhanced glycocalyx height in response to epithelial-to- mesenchymal transition and to oncogenic KRAS activation. In the latter case, we trace increased height to an effector gene, GALNT7. These data highlight the power of advanced imaging methods to provide molecular and functional insights into glycocalyx biology. </p> </div> </div> </div>

scholarly journals Super-resolution microscopy reveals coupling between mammalian centriole subdistal appendages and distal appendages

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Weng Man Chong ◽  
Won-Jing Wang ◽  
Chien-Hui Lo ◽  
Tzu-Yuan Chiu ◽  
Ting-Jui Chang ◽  
...  
Keyword(s):  
Super Resolution ◽  
The Other ◽  
Functional Understanding ◽  
Localization Analysis ◽  
Architectural Framework ◽  
Distal Border ◽  
Super Resolution Microscopy

Subdistal appendages (sDAPs) are centriolar elements that are observed proximal to the distal appendages (DAPs) in vertebrates. Despite the obvious presence of sDAPs, structural and functional understanding of them remains elusive. Here, by combining super-resolved localization analysis and CRISPR-Cas9 genetic perturbation, we find that although DAPs and sDAPs are primarily responsible for distinct functions in ciliogenesis and microtubule anchoring, respectively, the presence of one element actually affects the positioning of the other. Specifically, we find dual layers of both ODF2 and CEP89, where their localizations are differentially regulated by DAP and sDAP integrity. DAP depletion relaxes longitudinal occupancy of sDAP protein ninein to cover the DAP region, implying a role of DAPs in sDAP positioning. Removing sDAPs alter the distal border of centrosomal γ-tubulins, illustrating a new role of sDAPs. Together, our results provide an architectural framework for sDAPs that sheds light on functional understanding, surprisingly revealing coupling between DAPs and sDAPs.

scholarly journals Prospects and limitations of expansion microscopy in chromatin ultrastructure determination

2020 ◽  
Author(s):  
Ivona Kubalová ◽  
Markéta Schmidt Černohorská ◽  
Martina Huranová ◽  
Klaus Weisshart ◽  
Andreas Houben ◽  
...  
Keyword(s):  
Super Resolution ◽  
Structural Features ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Tissue Slices ◽  
Nucleus Shape ◽  
Chromatin Ultrastructure ◽  
Histone H3 Variant ◽  
Super Resolution Microscopy ◽  
Physical Expansion

AbstractExpansion Microscopy (ExM) is a method to magnify physically a specimen with preserved ultrastructure. It has the potential to explore structural features beyond the diffraction limit of light. The procedure has been successfully used for different animal species, from isolated macromolecular complexes through cells to tissue slices. Expansion of plant-derived samples is still at the beginning, and little is known whether the chromatin ultrastructure becomes altered by physical expansion.In this study, we expanded isolated barley nuclei and compared whether ExM can provide a structural view of chromatin comparable with super-resolution microscopy. Different fixation and denaturation/digestion conditions were tested to maintain the chromatin ultrastructure. We achieved up to ∼4.2-times physically expanded nuclei corresponding to a maximal resolution of ∼50-60 nm when imaged by wild-field (WF) microscopy. By applying structured illumination microscopy (SIM, super-resolution) doubling the WF resolution the chromatin structures were observed at a resolution of ∼25-35 nm.WF microscopy showed a preserved nucleus shape and nucleoli. Moreover, we were able to detect chromatin domains, invisible in unexpanded nuclei. However, by applying SIM we observed that the preservation of the chromatin ultrastructure after expansion was not complete and that the majority of the tested conditions failed to keep the ultrastructure.Nevertheless, using expanded nuclei we detected successfully centromere repeats by fluorescence in situ hybridization (FISH) and the centromere-specific histone H3 variant CENH3 by indirect immunostaining. However, although these repeats and proteins were localized at the correct position within the nuclei (indicating a Rabl orientation) their ultrastructural arrangement was impaired.

scholarly journals Quantitative Super-Resolution Microscopy of the Mammalian Glycocalyx

2018 ◽  
Author(s):  
Leonhard Möckl ◽  
Kayvon Pedram ◽  
Anish R. Roy ◽  
Venkatesh Krishnan ◽  
Anna-Karin Gustavsson ◽  
...  
Keyword(s):  
Epithelial To Mesenchymal Transition ◽  
Human Tumor ◽  
Super Resolution ◽  
Metabolic Labeling ◽  
Human Tumor Cells ◽  
Mesenchymal Transition ◽  
Health And Disease ◽  
20 Nm ◽  
2D And 3D ◽  
Super Resolution Microscopy

<div> <div> <div> <p>The mammalian glycocalyx is a heavily glycosylated extramembrane compartment found on nearly every cell. Despite its relevance in both health and disease, studies of the glycocalyx remain hampered by a paucity of methods to spatially classify its components. We combine metabolic labeling, bioorthogonal chemistry, and super-resolution localization microscopy to image two constituents of cell-surface glycans, N-acetylgalactosamine (GalNAc) and sialic acid, with 10-20 nm precision in 2D and 3D. This approach enables two measurements: glycocalyx height and the distribution of individual sugars distal from the membrane. These measurements show that the glycocalyx exhibits nanoscale architecture, on both cell lines and primary human tumor cells. Additionally, we observe enhanced glycocalyx height in response to epithelial-to- mesenchymal transition and to oncogenic KRAS activation. In the latter case, we trace increased height to an effector gene, GALNT7. These data highlight the power of advanced imaging methods to provide molecular and functional insights into glycocalyx biology. </p> </div> </div> </div>

scholarly journals Prospects and limitations of expansion microscopy in chromatin ultrastructure determination

2020 ◽  
Vol 28 (3-4) ◽  
pp. 355-368 ◽  
Author(s):  
Ivona Kubalová ◽  
Markéta Schmidt Černohorská ◽  
Martina Huranová ◽  
Klaus Weisshart ◽  
Andreas Houben ◽  
...  
Keyword(s):  
Super Resolution ◽  
Structural Features ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Tissue Slices ◽  
Nucleus Shape ◽  
Chromatin Ultrastructure ◽  
Histone H3 Variant ◽  
Super Resolution Microscopy ◽  
Physical Expansion

AbstractExpansion microscopy (ExM) is a method to magnify physically a specimen with preserved ultrastructure. It has the potential to explore structural features beyond the diffraction limit of light. The procedure has been successfully used for different animal species, from isolated macromolecular complexes through cells to tissue slices. Expansion of plant-derived samples is still at the beginning, and little is known, whether the chromatin ultrastructure becomes altered by physical expansion. In this study, we expanded isolated barley nuclei and compared whether ExM can provide a structural view of chromatin comparable with super-resolution microscopy. Different fixation and denaturation/digestion conditions were tested to maintain the chromatin ultrastructure. We achieved up to ~4.2-times physically expanded nuclei corresponding to a maximal resolution of ~50–60 nm when imaged by wild-field (WF) microscopy. By applying structured illumination microscopy (SIM, super-resolution) doubling the WF resolution, the chromatin structures were observed at a resolution of ~25–35 nm. WF microscopy showed a preserved nucleus shape and nucleoli. Moreover, we were able to detect chromatin domains, invisible in unexpanded nuclei. However, by applying SIM, we observed that the preservation of the chromatin ultrastructure after the expansion was not complete and that the majority of the tested conditions failed to keep the ultrastructure. Nevertheless, using expanded nuclei, we localized successfully centromere repeats by fluorescence in situ hybridization (FISH) and the centromere-specific histone H3 variant CENH3 by indirect immunolabelling. However, although these repeats and proteins were localized at the correct position within the nuclei (indicating a Rabl orientation), their ultrastructural arrangement was impaired.

scholarly journals Optimized localization analysis for single-molecule tracking and super-resolution microscopy

2010 ◽  
Vol 7 (5) ◽  
pp. 377-381 ◽  
Author(s):  
Kim I Mortensen ◽  
L Stirling Churchman ◽  
James A Spudich ◽  
Henrik Flyvbjerg
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Single Molecule Tracking ◽  
Localization Analysis ◽  
Super Resolution Microscopy

Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

Acta Naturae ◽  
2017 ◽  
Vol 9 (4) ◽  
pp. 42-51
Author(s):  
S. S. Ryabichko ◽  
◽  
A. N. Ibragimov ◽  
L. A. Lebedeva ◽  
E. N. Kozlov ◽  
...  
Keyword(s):  
Cell Nucleus ◽  
Super Resolution ◽  
Structure And Function ◽  
And Function ◽  
Super Resolution Microscopy

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

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