scholarly journals Spatial Organization of Chromatin: Emergence of Chromatin Structure During Development

2021 ◽  
Vol 37 (1) ◽  
Author(s):  
Rajarshi P. Ghosh ◽  
Barbara J. Meyer
Keyword(s):  
Single Molecule ◽  
Genome Organization ◽  
Spatial Organization ◽  
Super Resolution ◽  
Annual Review ◽  
Publication Date ◽  
Cellular Processes ◽  
Regulatory Processes ◽  
Genome Wide ◽  
Resolution Imaging

Nuclei are central hubs for information processing in eukaryotic cells. The need to fit large genomes into small nuclei imposes severe restrictions on genome organization and the mechanisms that drive genome-wide regulatory processes. How a disordered polymer such as chromatin, which has vast heterogeneity in its DNA and histone modification profiles, folds into discernibly consistent patterns is a fundamental question in biology. Outstanding questions include how genomes are spatially and temporally organized to regulate cellular processes with high precision and whether genome organization is causally linked to transcription regulation. The advent of next-generation sequencing, super-resolution imaging, multiplexed fluorescent in situ hybridization, and single-molecule imaging in individual living cells has caused a resurgence in efforts to understand the spatiotemporal organization of the genome. In this review, we discuss structural and mechanistic properties of genome organization at different length scales and examine changes in higher-order chromatin organization during important developmental transitions. Expected final online publication date for the Annual Review of Cell and Developmental Biology, Volume 37 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals Cryogenic Super-Resolution Fluorescence and Electron Microscopy Correlated at the Nanoscale

2021 ◽  
Vol 72 (1) ◽  
Author(s):  
Peter D. Dahlberg ◽  
W.E. Moerner
Keyword(s):  
Electron Microscopy ◽  
Single Molecule ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Nanometer Scale ◽  
Annual Review ◽  
Publication Date ◽  
Fluorescence And Electron Microscopy ◽  
Cellular Context

We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and cryogenic electron tomography (CET) are both powerful techniques for observing subcellular organization, but each approach has unique limitations. The combination of the two brings the single-molecule sensitivity and specificity of SR to the detailed cellular context and molecular scale resolution of CET. The resulting correlative data is more informative than the sum of its parts. The correlative images can be used to pinpoint the positions of fluorescently labeled proteins in the high-resolution context of CET with nanometer-scale precision and/or to identify proteins in electron-dense structures. The execution of srCryoCLEM is challenging and the approach is best described as a method that is still in its infancy with numerous technical challenges. In this review, we describe state-of-the-art srCryoCLEM experiments, discuss the most pressing challenges, and give a brief outlook on future applications. Expected final online publication date for the Annual Review of Physical Chemistry, Volume 72 is April 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair

2013 ◽  
Vol 202 (3) ◽  
pp. 579-595 ◽  
Author(s):  
Sébastien Britton ◽  
Julia Coates ◽  
Stephen P. Jackson
Keyword(s):  
High Resolution ◽  
Single Molecule ◽  
Anticancer Agents ◽  
Spatial Organization ◽  
System Development ◽  
Super Resolution ◽  
Strand Break ◽  
Resistance Mechanisms ◽  
Original Method ◽  
Immune System Development

DNA double-strand breaks (DSBs) are the most toxic of all genomic insults, and pathways dealing with their signaling and repair are crucial to prevent cancer and for immune system development. Despite intense investigations, our knowledge of these pathways has been technically limited by our inability to detect the main repair factors at DSBs in cells. In this paper, we present an original method that involves a combination of ribonuclease- and detergent-based preextraction with high-resolution microscopy. This method allows direct visualization of previously hidden repair complexes, including the main DSB sensor Ku, at virtually any type of DSB, including those induced by anticancer agents. We demonstrate its broad range of applications by coupling it to laser microirradiation, super-resolution microscopy, and single-molecule counting to investigate the spatial organization and composition of repair factories. Furthermore, we use our method to monitor DNA repair and identify mechanisms of repair pathway choice, and we show its utility in defining cellular sensitivities and resistance mechanisms to anticancer agents.

Measuring Absolute Membrane Potential Across Space and Time

2021 ◽  
Vol 50 (1) ◽  
Author(s):  
Julia R. Lazzari-Dean ◽  
Anneliese M.M. Gest ◽  
Evan W. Miller
Keyword(s):  
Membrane Potential ◽  
Neurotransmitter Release ◽  
Cell Cycle Control ◽  
The Other ◽  
Annual Review ◽  
Publication Date ◽  
Short Term ◽  
Cellular Processes ◽  
Sensing Platform ◽  
The One

Membrane potential (Vmem) is a fundamental biophysical signal present in all cells. Vmem signals range in time from milliseconds to days, and they span lengths from microns to centimeters. Vmem affects many cellular processes, ranging from neurotransmitter release to cell cycle control to tissue patterning. However, existing tools are not suitable for Vmem quantification in many of these areas. In this review, we outline the diverse biology of Vmem, drafting a wish list of features for a Vmem sensing platform. We then use these guidelines to discuss electrode-based and optical platforms for interrogating Vmem. On the one hand, electrode-based strategies exhibit excellent quantification but are most effective in short-term, cellular recordings. On the other hand, optical strategies provide easier access to diverse samples but generally only detect relative changes in Vmem. By combining the respective strengths of these technologies, recent advances in optical quantification of absolute Vmem enable new inquiries into Vmem biology. Expected final online publication date for the Annual Review of Biophysics, Volume 50 is May 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals Comparing lifeact and phalloidin for super-resolution imaging of actin in fixed cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0246138
Author(s):  
Hanieh Mazloom-Farsibaf ◽  
Farzin Farzam ◽  
Mohamadreza Fazel ◽  
Michael J. Wester ◽  
Marjolein B. M. Meddens ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Cell Movement ◽  
Super Resolution ◽  
Actin Binding ◽  
Thin Filaments ◽  
Reversible Binding ◽  
Multiple Regions ◽  
Resolution Imaging ◽  
Division Cell

Visualizing actin filaments in fixed cells is of great interest for a variety of topics in cell biology such as cell division, cell movement, and cell signaling. We investigated the possibility of replacing phalloidin, the standard reagent for super-resolution imaging of F-actin in fixed cells, with the actin binding peptide ‘lifeact’. We compared the labels for use in single molecule based super-resolution microscopy, where AlexaFluor 647 labeled phalloidin was used in a dSTORM modality and Atto 655 labeled lifeact was used in a single molecule imaging, reversible binding modality. We found that imaging with lifeact had a comparable resolution in reconstructed images and provided several advantages over phalloidin including lower costs, the ability to image multiple regions of interest on a coverslip without degradation, simplified sequential super-resolution imaging, and more continuous labeling of thin filaments.

scholarly journals Single Molecule Localization Microscopy (SMLM) imaging of bacteria: A sample preparation guide

2020 ◽  
Author(s):  
Sachith D. Gunasinghe ◽  
Kirstin D. Elgass ◽  
Toby D. M. Bell ◽  
Trevor Lithgow
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Single Molecule ◽  
Spatial Organization ◽  
Outer Membrane Proteins ◽  
Fluorescent Microscopy ◽  
Super Resolution ◽  
Model Systems ◽  
Invaluable Tool ◽  
Optical Reconstruction

Abstract In recent years Super-resolution microscopy has become an invaluable tool to noninvasively interrogate the membrane architecture of bacteria to study the spatial organization of proteins associated with membranes, which in turn help us to understand how bacteria have evolved to exploit environmental niches. Model systems like Escherichia coli and Caulobacter cresentus have been used to study the spatiotemporal organization of membrane proteins. Like most gram-negative bacteria, the outer membrane of E.coli is populated with β-barrel proteins, which serve as selective channels where exchange of small molecules take place. Surface exposed domains in these channels provide means to fluorescently label and utilise them for fluorescent microscopy studies to investigate their spatial organization at the outer membrane. Here, we describe a methodology to fluorescently label outer membrane proteins in E.coli and study their spatial organization using direct stochastic optical reconstruction microscopy (dSTORM).

The Influence of the Western Diet on Microbiota and Gastrointestinal Immunity

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Vanessa Las Heras ◽  
Silvia Melgar ◽  
John MacSharry ◽  
Cormac G.M. Gahan
Keyword(s):  
Immune Function ◽  
Western Diet ◽  
Major Influence ◽  
Annual Review ◽  
Food Science ◽  
Publication Date ◽  
Gastrointestinal Microbiota ◽  
Human Diet ◽  
Regulatory Processes ◽  
Inflammatory Bowel

Diet exerts a major influence upon host immune function and the gastrointestinal microbiota. Although components of the human diet (including carbohydrates, fats, and proteins) are essential sources of nutrition for the host, they also influence immune function directly through interaction with innate and cell-mediated immune regulatory mechanisms. Regulation of the microbiota community structure also provides a mechanism by which food components influence host immune regulatory processes. Here, we consider the complex interplay between components of the modern (Western) diet, the microbiota, and host immunity in the context of obesity and metabolic disease, inflammatory bowel disease, and infection. Expected final online publication date for the Annual Review of Food Science and Technology, Volume 13 is March 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Single molecule Coordinate and Height super-resolution Imaging with Dithering and Orientation (CHIDO)

2020 ◽  
Author(s):  
Luis A. Alemán-Castañeda ◽  
Valentina Curcio ◽  
Thomas G. Brown ◽  
Sophie Brasselet ◽  
Miguel A. Alonso
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Resolution Imaging

scholarly journals Mapping 3D genome organization relative to nuclear compartments using TSA-Seq as a cytological ruler

2018 ◽  
Vol 217 (11) ◽  
pp. 4025-4048 ◽  
Author(s):  
Yu Chen ◽  
Yang Zhang ◽  
Yuchuan Wang ◽  
Liguo Zhang ◽  
Eva K. Brinkman ◽  
...  
Keyword(s):  
Genome Organization ◽  
Spatial Organization ◽  
Essential Feature ◽  
K562 Cells ◽  
Housekeeping Genes ◽  
Mapping Method ◽  
Spatial Gradient ◽  
Nuclear Speckles ◽  
Nuclear Compartments ◽  
Genome Wide

While nuclear compartmentalization is an essential feature of three-dimensional genome organization, no genomic method exists for measuring chromosome distances to defined nuclear structures. In this study, we describe TSA-Seq, a new mapping method capable of providing a “cytological ruler” for estimating mean chromosomal distances from nuclear speckles genome-wide and for predicting several Mbp chromosome trajectories between nuclear compartments without sophisticated computational modeling. Ensemble-averaged results in K562 cells reveal a clear nuclear lamina to speckle axis correlated with a striking spatial gradient in genome activity. This gradient represents a convolution of multiple spatially separated nuclear domains including two types of transcription “hot zones.” Transcription hot zones protruding furthest into the nuclear interior and positioning deterministically very close to nuclear speckles have higher numbers of total genes, the most highly expressed genes, housekeeping genes, genes with low transcriptional pausing, and super-enhancers. Our results demonstrate the capability of TSA-Seq for genome-wide mapping of nuclear structure and suggest a new model for spatial organization of transcription and gene expression.

scholarly journals Fast, Simultaneous Multiple Fluorophore Fitting in Single Molecule Super-Resolution Imaging

2011 ◽  
Vol 100 (3) ◽  
pp. 349a
Author(s):  
Fang Huang ◽  
Samantha L. Schwartz ◽  
Jason M. Byars ◽  
Keith A. Lidke
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Resolution Imaging ◽  
Fitting In
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