scholarly journals Helical metaphase chromatid coiling is conserved

2021 ◽  
Author(s):  
Ivona Kubalova ◽  
Amanda Souza Camara ◽  
Petr Capal ◽  
Tomas Beseda ◽  
Jean-Marie Rouillard ◽  
...  
Keyword(s):  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Chromosome Organization ◽  
Published Data ◽  
Metaphase Chromosomes ◽  
Chromosome Conformation ◽  
Polymer Simulation ◽  
Base Analog ◽  
Chromatin Fibers ◽  
Chromatin Density

The higher-order metaphase chromosome organization has been under controversial discussion already for 140 years. Classical light and electron microscopy proposed chromatids to be composed of helically organized chromatin fibers, so-called chromonemata. More recently also non-helical models were suggested. We studied the chromosome organization in barley by interdisciplinary cutting-edge approaches, such as chromosome sorting, chromosome conformation capture, oligonucleotide-fluorescence in situ hybridization, base analog incorporation, super-resolution microscopy, and polymer simulation to elucidate the arrangement of chromatids of large mitotic metaphase chromosomes. Our data provide cumulative evidence for the presence of a helically arranged 400 nm chromatin fiber representing the chromonema within the chromatid arms. The number of turns is positively correlated with the arm length. Turn size and chromatin density decrease towards the telomeres. Due to the specialized functions of centromeres and nucleolus-organizing regions, the helical organization is interrupted at these regions, which display several thinners and straight chromatin fibers. Based on our findings and re-analyzing previously published data from other plant and non-plant species we conclude that the helical turning of metaphase chromatid arms is a conserved feature of large eukaryotic chromosomes.

scholarly journals Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

2021 ◽  
Vol 22 (4) ◽  
pp. 1903
Author(s):  
Ivona Kubalová ◽  
Alžběta Němečková ◽  
Klaus Weisshart ◽  
Eva Hřibová ◽  
Veit Schubert
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Chromatin Condensation ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Wide Field ◽  
Structured Illumination Microscopy ◽  
Metaphase Chromosomes ◽  
Localization Microscopy ◽  
Super Resolution Microscopy

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200–250 nm laterally, ~500–700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4′,6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

scholarly journals Learning the distribution of single-cell chromosome conformations in bacteria reveals emergent order across genomic scales

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joris J. B. Messelink ◽  
Muriel C. F. van Teeseling ◽  
Jacqueline Janssen ◽  
Martin Thanbichler ◽  
Chase P. Broedersz
Keyword(s):  
Single Cell ◽  
Caulobacter Crescentus ◽  
Model Organism ◽  
Super Resolution ◽  
Chromosome Organization ◽  
General Strategy ◽  
Cell Axis ◽  
Cellular Processes ◽  
Emergent Order ◽  
Genomic Clusters

AbstractThe order and variability of bacterial chromosome organization, contained within the distribution of chromosome conformations, are unclear. Here, we develop a fully data-driven maximum entropy approach to extract single-cell 3D chromosome conformations from Hi–C experiments on the model organism Caulobacter crescentus. The predictive power of our model is validated by independent experiments. We find that on large genomic scales, organizational features are predominantly present along the long cell axis: chromosomal loci exhibit striking long-ranged two-point axial correlations, indicating emergent order. This organization is associated with large genomic clusters we term Super Domains (SuDs), whose existence we support with super-resolution microscopy. On smaller genomic scales, our model reveals chromosome extensions that correlate with transcriptional and loop extrusion activity. Finally, we quantify the information contained in chromosome organization that may guide cellular processes. Our approach can be extended to other species, providing a general strategy to resolve variability in single-cell chromosomal organization.

scholarly journals An Easy Path for Correlative Electron and Super-Resolution Light Microscopy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Dorothea Pinotsi ◽  
Simona Rodighiero ◽  
Silvia Campioni ◽  
Gabor Csucs
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Light Microscopy ◽  
Amyloid Fibrils ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Wide Field ◽  
Set Up ◽  
Bio Compatibility ◽  
Scanning Electron

Abstract A number of new Correlative Light and Electron Microscopy approaches have been developed over the past years, offering the opportunity to combine the specificity and bio-compatibility of light microscopy with the high resolution achieved in electron microscopy. More recently, these approaches have taken one step further and also super-resolution light microscopy was combined with transmission or scanning electron microscopy. This combination usually requires moving the specimen between different imaging systems, an expensive set-up and relatively complicated imaging workflows. Here we present a way to overcome these difficulties by exploiting a commercially available wide-field fluorescence microscope integrated in the specimen chamber of a Scanning Electron Microscope (SEM) to perform correlative LM/EM studies. Super-resolution light microscopy was achieved by using a recently developed algorithm - the Super-Resolution Radial Fluctuations (SRRF) - to improve the resolution of diffraction limited fluorescent images. With this combination of hardware/software it is possible to obtain correlative super-resolution light and scanning electron microscopy images in an easy and fast way. The imaging workflow is described and demonstrated on fluorescently labelled amyloid fibrils, fibrillar protein aggregates linked to the onset of multiple neurodegenerative diseases, revealing information about their polymorphism.

Correlative STED super-resolution light and electron microscopy on resin sections

2019 ◽  
Vol 52 (37) ◽  
pp. 374003
Author(s):  
Christian A Wurm ◽  
Heinz Schwarz ◽  
Daniel C Jans ◽  
Dietmar Riedel ◽  
Bruno M Humbel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Super Resolution

scholarly journals Direct Visualization of Actin Filaments and Actin-Binding Proteins in Neuronal Cells

2020 ◽  
Vol 8 ◽  
Author(s):  
Minkyo Jung ◽  
Doory Kim ◽  
Ji Young Mun
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Actin Binding ◽  
Direct Visualization ◽  
Actin Binding Proteins ◽  
Synapse Plasticity

Actin networks and actin-binding proteins (ABPs) are most abundant in the cytoskeleton of neurons. The function of ABPs in neurons is nucleation of actin polymerization, polymerization or depolymerization regulation, bundling of actin through crosslinking or stabilization, cargo movement along actin filaments, and anchoring of actin to other cellular components. In axons, ABP–actin interaction forms a dynamic, deep actin network, which regulates axon extension, guidance, axon branches, and synaptic structures. In dendrites, actin and ABPs are related to filopodia attenuation, spine formation, and synapse plasticity. ABP phosphorylation or mutation changes ABP–actin binding, which regulates axon or dendritic plasticity. In addition, hyperactive ABPs might also be expressed as aggregates of abnormal proteins in neurodegeneration. Those changes cause many neurological disorders. Here, we will review direct visualization of ABP and actin using various electron microscopy (EM) techniques, super resolution microscopy (SRM), and correlative light and electron microscopy (CLEM) with discussion of important ABPs in neuron.

scholarly journals Topographic contrast of ultrathin cryo-sections for correlative super-resolution light and electron microscopy

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
José María Mateos ◽  
Bruno Guhl ◽  
Jana Doehner ◽  
Gery Barmettler ◽  
Andres Kaech ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Super Resolution

scholarly journals Large-scale chromatin structure of inducible genes: transcription on a condensed, linear template

2009 ◽  
Vol 185 (1) ◽  
pp. 87-100 ◽  
Author(s):  
Yan Hu ◽  
Igor Kireev ◽  
Matt Plutz ◽  
Nazanin Ashourian ◽  
Andrew S. Belmont
Keyword(s):  
Chromatin Structure ◽  
Transcriptional Activation ◽  
Large Scale ◽  
Nuclear Speckles ◽  
Interphase Chromosome ◽  
Chromosome Conformation ◽  
Dynamic Events ◽  
Classical Models ◽  
Chromatin Fibers

The structure of interphase chromosomes, and in particular the changes in large-scale chromatin structure accompanying transcriptional activation, remain poorly characterized. Here we use light microscopy and in vivo immunogold labeling to directly visualize the interphase chromosome conformation of 1–2 Mbp chromatin domains formed by multi-copy BAC transgenes containing 130–220 kb of genomic DNA surrounding the DHFR, Hsp70, or MT gene loci. We demonstrate near-endogenous transcription levels in the context of large-scale chromatin fibers compacted nonuniformly well above the 30-nm chromatin fiber. An approximately 1.5–3-fold extension of these large-scale chromatin fibers accompanies transcriptional induction and active genes remain mobile. Heat shock–induced Hsp70 transgenes associate with the exterior of nuclear speckles, with Hsp70 transcripts accumulating within the speckle. Live-cell imaging reveals distinct dynamic events, with Hsp70 transgenes associating with adjacent speckles, nucleating new speckles, or moving to preexisting speckles. Our results call for reexamination of classical models of interphase chromosome organization.

scholarly journals Somatic junctions connect microglia and developing neurons

2021 ◽  
Author(s):  
Csaba Cserép ◽  
Anett D. Schwarcz ◽  
Balázs Pósfai ◽  
Zsófia I. László ◽  
Anna Kellermayer ◽  
...  
Keyword(s):  
Adult Neurogenesis ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Adult Brain ◽  
Neuronal Progenitors ◽  
Immature Neurons ◽  
The Status ◽  
Underlying Mechanisms ◽  
Developing Neurons ◽  
Early Developmental

SummaryMicroglia are the resident immune cells of the brain with multiple homeostatic and regulatory roles. Emerging evidence also highlights the fundamental transformative role of microglia in brain development. While tightly controlled, bi-directional communication between microglia and neuronal progenitors or immature neurons has been postulated, the main sites of interaction and the underlying mechanisms remain elusive. By using correlated light and electron microscopy together with super-resolution imaging, here we provide evidence that microglial processes form specialized nanoscale contacts with the cell bodies of developing and immature neurons throughout embryonic, early postnatal and adult neurogenesis. These early developmental contacts are highly reminiscent to somatic purinergic junctions that are instrumental for microglia-neuron communication in the adult brain. We propose that early developmental formation of somatic purinergic junctions represents an ideal interface for microglia to monitor the status of developing neurons and to direct prenatal, early postnatal and adult neurogenesis.

scholarly journals RIM-Binding Protein 2 organizes Ca2+ channel topography and regulates release probability and vesicle replenishment at a fast central synapse

2021 ◽  
Author(s):  
Tanvi Butola ◽  
Theocharis Alvanos ◽  
Anika Hintze ◽  
Peter Koppensteiner ◽  
David Kleindienst ◽  
...  
Keyword(s):  
Binding Protein ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Auditory Pathway ◽  
Nerve Fibers ◽  
Release Probability ◽  
High Release ◽  
Train Stimulation ◽  
Bushy Cells

RIM-Binding Protein 2 (RIM-BP2) is a multi-domain protein of the presynaptic active zone (AZ). By binding to Rab-interacting protein (RIM), bassoon and voltage-gated Ca2+ channels (CaV), it is considered to be a central organizer of the topography of CaV and release sites of synaptic vesicles (SVs) at the AZ. Here, we investigated the role of RIM-BP2 at the endbulb of Held synapse of auditory nerve fibers with bushy cells of the cochlear nucleus, a fast relay of the auditory pathway with high release probability. Disruption of RIM-BP2 lowered release probability altering short-term plasticity and reduced evoked excitatory postsynaptic currents (EPSCs). Analysis of SV pool dynamics during high frequency train stimulation indicated a reduction of SVs with high release probability but an overall normal size of the readily releasable SV pool (RRP). The Ca2+-dependent fast component of SV replenishment after RRP depletion was slowed. Augmenting Ca2+ influx by adding extracellular Ca2+ restored release probability but not EPSC amplitude, and uncovered an impairment of SV replenishment during train stimulation. Ultrastructural analysis by super-resolution light and electron microscopy revealed an impaired topography of presynaptic CaV and a reduction of docked and membrane-proximal SVs at the AZ. We conclude that RIM-BP2 organizes the topography of CaV, and promotes SV tethering and docking. This way RIM-BP2 is critical for establishing a high initial release probability as required to reliably signal sound onset information that we found to be degraded in bushy cells of RIM-BP2-deficient mice in vivo.

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