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.

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).

scholarly journals Characterizing Locus Specific Chromatin Structure and Dynamics with Correlative Conventional and Super Resolution imaging in living cells

2021 ◽  
Author(s):  
Dushyant Mehra ◽  
Santosh Adhikari ◽  
Chiranjib Banerjee ◽  
Elias M. Puchner
Keyword(s):  
Single Molecule ◽  
Chromatin Structure ◽  
Spatial Organization ◽  
Super Resolution ◽  
Living Cells ◽  
Diffraction Limit ◽  
Optical Diffraction ◽  
Acquisition Time ◽  
Structure And Dynamics ◽  
Chromatin Structure And Dynamics

The dynamic rearrangement of chromatin is critical for gene regulation, but mapping both the spatial organization of chromatin and its dynamics remains a challenge. Many structural conformations are too small to be resolved via conventional fluorescence microscopy and the long acquisition time of super-resolution PALM imaging precludes the structural characterization of chromatin below the optical diffraction limit in living cells due to chromatin motion. Here we develop a correlative conventional fluorescence and PALM imaging approach to quantitatively map time-averaged chromatin structure and dynamics below the optical diffraction limit in living cells. By assigning localizations to a locus as it moves, we reliably discriminate between bound and searching dCas9 molecules, whose mobility overlap. Our approach accounts for changes in DNA mobility and relates local chromatin motion to larger scale domain movement. In our experimental system, we show that compacted telomeres have a higher density of bound dCas9 molecules, but the relative motion of those molecules is more restricted than in less compacted telomeres. Correlative conventional and PALM imaging therefore improves the ability to analyze the mobility and time-averaged nanoscopic structural features of locus specific chromatin with single molecule precision and yields unprecedented insights across length and time scales.

scholarly journals Mutant D96V calmodulin induces unexpected remodeling of cardiac nanostructure and physiology

2021 ◽  
Vol 154 (9) ◽  
Author(s):  
Heather L. Struckman ◽  
Mikhail Tarasov ◽  
Yusuf Olgar ◽  
Alec Miller ◽  
Jonathan P. Davis ◽  
...  
Keyword(s):  
Single Molecule ◽  
Spatial Organization ◽  
Sodium Current ◽  
Close Association ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Super Resolution ◽  
Sodium Calcium Exchanger ◽  
Patch Clamp Recording ◽  
Specific Expression

Calmodulin (CaM) prevents proarrhythmic late sodium current (INa) by facilitating normal inactivation of sodium channels (NaV). Since dysfunction of NaV1.6 has been implicated in late INa-mediated arrhythmias, we investigated its role in arrhythmias promoted by CaM mutant D96V. Super-resolution STED microscopy revealed enlarged NaV1.6 clusters in NaV1.6-expressing Chinese hamster ovary cells transfected with D96V-CaM relative to those transfected with WT-CaM. Therefore, we examined NaV1.6 clustering in transgenic mice with cardiac-specific expression of D96V-CaM (cD96V) with a C-terminal FLAG tag. Confocal microscopy confirmed expression of NaV1.6 and FLAG-tagged D96V-CaM in a striated pattern along with RYR2 in cD96V hearts, consistent with T-tubular localization. In both WT and cD96V hearts, STORM single molecule localization microscopy revealed that ∼50% of NaV1.6 clusters localized <100 nm from RYR2. However, NaV1.6 density within these regions was 67% greater in cD96V relative to WT. Consistent with this result, SICM-guided “smart” patch clamp recording of NaV activity from T-tubule openings revealed more frequent late-burst openings involving larger NaV clusters in cD96V myocytes relative to WT. Previous work identifies the sodium-calcium exchanger (NCX) as a key link between aberrant late NaV1.6 activity and proarrhythmic Ca2+ mishandling. Therefore, we explored the spatial organization of NaV1.6 and NCX using STORM. Consistent with their close association, 89% of NaV1.6 clusters localized <100 nm from NCX in cD96V hearts, compared with 77% in WT. Notably, density of both NaV1.6 and NCX was increased at these sites by 48% and 31%, respectively, in cD96V relative to WT. Consistent with these data, cD96V myocytes displayed larger, more frequent Ca2+ sparks relative to WT. These proarrhythmic functional effects were abrogated by cardiac-specific knockout of NaV1.6. To our knowledge, this is the first demonstration of proarrhythmic cardiac structural remodeling secondary to a defect in calmodulin, offering novel mechanistic insight into calmodulinopathy.

Protein clustering and spatial organization in T-cells

2015 ◽  
Vol 43 (3) ◽  
pp. 315-321 ◽  
Author(s):  
Michael J. Shannon ◽  
Garth Burn ◽  
Andrew Cope ◽  
Georgina Cornish ◽  
Dylan M. Owen
Keyword(s):  
T Cell ◽  
Single Molecule ◽  
Spatial Organization ◽  
Super Resolution ◽  
Cell Protein ◽  
Single Molecule Level ◽  
Composition And Structure ◽  
Resolution Analysis ◽  
And Function ◽  
Super Resolution Microscopy

T-cell protein microclusters have until recently been investigable only as microscale entities with their composition and structure being discerned by biochemistry or diffraction-limited light microscopy. With the advent of super resolution microscopy comes the ability to interrogate the structure and function of these clusters at the single molecule level by producing highly accurate pointillist maps of single molecule locations at ~20nm resolution. Analysis tools have also been developed to provide rich descriptors of the pointillist data, allowing us to pose questions about the nanoscale organization which governs the local and cell wide responses required of a migratory T-cell.

scholarly journals A Pairwise Distance Distribution Correction (DDC) algorithm to eliminate blinking-caused artifacts in super-resolution microscopy

2019 ◽  
Author(s):  
Christopher H. Bohrer ◽  
Xinxing Yang ◽  
Xiaoli Weng ◽  
Brian Tenner ◽  
Shreyasi Thakur ◽  
...  
Keyword(s):  
Single Molecule ◽  
Spatial Organization ◽  
Super Resolution ◽  
Distance Distribution ◽  
Pairwise Distance ◽  
Imaging Sequence ◽  
Wide Range ◽  
Quantitative Analyses ◽  
Super Resolution Microscopy ◽  
Accurate Reconstruction

AbstractIn single-molecule localization based super-resolution microscopy (SMLM), a fluorophore stochastically switches between fluorescent- and dark-states, leading to intermittent emission of fluorescence, a phenomenon known as blinking. Intermittent emissions create multiple localizations belonging to the same molecule, resulting in blinking-artifacts within SMLM images. These artifacts are often interpreted as true biological assemblies, confounding quantitative analyses and interpretations. Multiple methods have been developed to eliminate these artifacts, but they either require additional experiments, arbitrary thresholds, or specific photo-kinetic models. Here we present a method, termed Distance Distribution Correction (DDC), to eliminate blinking-caused repeat localizations without any additional calibrations. The approach relies on the finding that the true pairwise distance distribution of different fluorophores in an SMLM image can be naturally obtained from the imaging sequence by using distances between localizations separated by a time much longer than the average fluorescence survival time. We show that using the true pairwise distribution we can define and then maximize the likelihood of obtaining a particular set of localizations void of blinking-artifacts, generating an accurate reconstruction of the underlying cellular structure. Using both simulated and experimental data, we show that DDC surpasses all previous existing blinking-artifact correction methodologies, resulting in drastic improvements in obtaining the closest estimate of the true spatial organization and number of fluorescent emitters in a wide range of applications. The simplicity and robustness of DDC will allow it to become the field standard in SMLM imaging, enabling the most accurate reconstruction and quantification of SMLM images to date.

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 Nanoscale characterization of drug-induced microtubule filament dysfunction using super-resolution microscopy

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Ashley M. Rozario ◽  
Sam Duwé ◽  
Cade Elliott ◽  
Riley B. Hargreaves ◽  
Gregory W. Moseley ◽  
...  
Keyword(s):  
Single Molecule ◽  
Anticancer Agents ◽  
Super Resolution ◽  
Short Exposure ◽  
Cellular Functions ◽  
Drug Induced ◽  
Nanoscale Characterization ◽  
Drug Regimens ◽  
Filament Networks ◽  
20 Nm

Abstract Background The integrity of microtubule filament networks is essential for the roles in diverse cellular functions, and disruption of its structure or dynamics has been explored as a therapeutic approach to tackle diseases such as cancer. Microtubule-interacting drugs, sometimes referred to as antimitotics, are used in cancer therapy to target and disrupt microtubules. However, due to associated side effects on healthy cells, there is a need to develop safer drug regimens that still retain clinical efficacy. Currently, many questions remain open regarding the extent of effects on cellular physiology of microtubule-interacting drugs at clinically relevant and low doses. Here, we use super-resolution microscopies (single-molecule localization and optical fluctuation based) to reveal the initial microtubule dysfunctions caused by nanomolar concentrations of colcemid. Results We identify previously undetected microtubule (MT) damage caused by clinically relevant doses of colcemid. Short exposure to 30–80 nM colcemid results in aberrant microtubule curvature, with a trend of increased curvature associated to increased doses, and curvatures greater than 2 rad/μm, a value associated with MT breakage. Microtubule fragmentation was detected upon treatment with ≥ 100 nM colcemid. Remarkably, lower doses (< 20 nM after 5 h) led to subtle but significant microtubule architecture remodelling characterized by increased curvature and suppression of microtubule dynamics. Conclusions Our results support the emerging hypothesis that microtubule-interacting drugs induce non-mitotic effects in cells, and establish a multi-modal imaging assay for detecting and measuring nanoscale microtubule dysfunction. The sub-diffraction visualization of these less severe precursor perturbations compared to the established antimitotic effects of microtubule-interacting drugs offers potential for improved understanding and design of anticancer agents.

Multi-colour DNA-qPAINT reveals how Csk nano-clusters regulate T-cell receptor signalling

2019 ◽  
Author(s):  
Sabrina Simoncelli ◽  
Juliette Griffié ◽  
David J. Williamson ◽  
Jack Bibby ◽  
Cara Bray ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Single Molecule ◽  
Spatial Organization ◽  
Regulatory Element ◽  
Tyrosine Phosphatase ◽  
Super Resolution ◽  
Cell Receptor ◽  
Knock Out ◽  
Spatio Temporal

AbstractPhosphorylation of the negative regulatory element of the tyrosine kinase Lck by Csk down-modulates T-cell receptor induced signalling. Being constitutively active, Csk spatial organization is responsible for regulating this signalling interaction. Here, we used stoichiometrically accurate, multiplexed, single-molecule super-resolution microscopy (DNA-qPAINT) to image the nanoscale spatial architecture of Csk and two binding partners implicated in its membrane association – PAG and TRAF3. Combined with a newly developed co-clustering analysis framework, we provide a powerful resource for dissecting signalling pathways regulated by spatio-temporal organisation. We found that Csk forms nanoscale clusters proximal to the plasma membrane that are lost post-stimulation and re-recruited at later time points. Unexpectedly, these clusters do not directly co-localise with PAG at the membrane, but instead provide a ready pool of monomers to down-regulate signalling. By generating CRISPR/Cas9 knock-out T-cells, our data also identify that protein tyrosine phosphatase non-receptor type 22 (PTPN22) is essential for Csk nanocluster re-recruitment and for maintenance of the synaptic PAG population.

scholarly journals How Single-Molecule Localization Microscopy Expanded Our Mechanistic Understanding of RNA Polymerase II Transcription

2021 ◽  
Vol 22 (13) ◽  
pp. 6694
Author(s):  
Peter Hoboth ◽  
Ondřej Šebesta ◽  
Pavel Hozák
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Spatial Organization ◽  
Super Resolution ◽  
Temporal Organization ◽  
Transcription Machinery ◽  
Mechanistic Understanding ◽  
Rnapii Transcription

Classical models of gene expression were built using genetics and biochemistry. Although these approaches are powerful, they have very limited consideration of the spatial and temporal organization of gene expression. Although the spatial organization and dynamics of RNA polymerase II (RNAPII) transcription machinery have fundamental functional consequences for gene expression, its detailed studies have been abrogated by the limits of classical light microscopy for a long time. The advent of super-resolution microscopy (SRM) techniques allowed for the visualization of the RNAPII transcription machinery with nanometer resolution and millisecond precision. In this review, we summarize the recent methodological advances in SRM, focus on its application for studies of the nanoscale organization in space and time of RNAPII transcription, and discuss its consequences for the mechanistic understanding of gene expression.

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