scholarly journals Single-molecule localization microscopy reveals molecular transactions during RAD51 filament assembly at cellular DNA damage sites

2018 ◽  
Vol 46 (5) ◽  
pp. 2398-2416 ◽  
Author(s):  
Kalina T Haas ◽  
MiYoung Lee ◽  
Alessandro Esposito ◽  
Ashok R Venkitaraman
Keyword(s):  
Dna Damage ◽  
Single Molecule ◽  
Localization Microscopy ◽  
Filament Assembly ◽  
Cellular Dna ◽  
Single Molecule Localization Microscopy

scholarly journals Elucidation of the Clustered Nano-Architecture of Radiation-Induced DNA Damage Sites and Surrounding Chromatin in Cancer Cells: A Single Molecule Localization Microscopy Approach

2021 ◽  
Vol 22 (7) ◽  
pp. 3636
Author(s):  
Michael Hausmann ◽  
Martin Falk ◽  
Charlotte Neitzel ◽  
Andreas Hofmann ◽  
Abin Biswas ◽  
...  
Keyword(s):  
Dna Damage ◽  
Single Molecule ◽  
Radiation Treatment ◽  
Critical Role ◽  
Chromatin Domain ◽  
Dsb Repair ◽  
Localization Microscopy ◽  
Radiation Induced ◽  
Structural Aspects ◽  
Single Molecule Localization Microscopy

In cancer therapy, the application of (fractionated) harsh radiation treatment is state of the art for many types of tumors. However, ionizing radiation is a “double-edged sword”—it can kill the tumor but can also promote the selection of radioresistant tumor cell clones or even initiate carcinogenesis in the normal irradiated tissue. Individualized radiotherapy would reduce these risks and boost the treatment, but its development requires a deep understanding of DNA damage and repair processes and the corresponding control mechanisms. DNA double strand breaks (DSBs) and their repair play a critical role in the cellular response to radiation. In previous years, it has become apparent that, beyond genetic and epigenetic determinants, the structural aspects of damaged chromatin (i.e., not only of DSBs themselves but also of the whole damage-surrounding chromatin domains) form another layer of complex DSB regulation. In the present article, we summarize the application of super-resolution single molecule localization microscopy (SMLM) for investigations of these structural aspects with emphasis on the relationship between the nano-architecture of radiation-induced repair foci (IRIFs), represented here by γH2AX foci, and their chromatin environment. Using irradiated HeLa cell cultures as an example, we show repair-dependent rearrangements of damaged chromatin and analyze the architecture of γH2AX repair clusters according to topological similarities. Although HeLa cells are known to have highly aberrant genomes, the topological similarity of γH2AX was high, indicating a functional, presumptively genome type-independent relevance of structural aspects in DSB repair. Remarkably, nano-scaled chromatin rearrangements during repair depended both on the chromatin domain type and the treatment. Based on these results, we demonstrate how the nano-architecture and topology of IRIFs and chromatin can be determined, point to the methodological relevance of SMLM, and discuss the consequences of the observed phenomena for the DSB repair network regulation or, for instance, radiation treatment outcomes.

scholarly journals Establishing Live-Cell Single-Molecule Localization Microscopy Imaging and Single-Particle Tracking in the Archaeon Haloferax volcanii

2020 ◽  
Vol 11 ◽  
Author(s):  
Bartosz Turkowyd ◽  
Sandra Schreiber ◽  
Julia Wörtz ◽  
Ella Shtifman Segal ◽  
Moshe Mevarech ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Proteins ◽  
Single Particle Tracking ◽  
Haloferax Volcanii ◽  
Microscopy Imaging ◽  
Localization Microscopy ◽  
Cellular Dna ◽  
Eukaryotic Organisms ◽  
Single Molecule Localization Microscopy

In recent years, fluorescence microscopy techniques for the localization and tracking of single molecules in living cells have become well-established and are indispensable tools for the investigation of cellular biology and in vivo biochemistry of many bacterial and eukaryotic organisms. Nevertheless, these techniques are still not established for imaging archaea. Their establishment as a standard tool for the study of archaea will be a decisive milestone for the exploration of this branch of life and its unique biology. Here, we have developed a reliable protocol for the study of the archaeon Haloferax volcanii. We have generated an autofluorescence-free H. volcanii strain, evaluated several fluorescent proteins for their suitability to serve as single-molecule fluorescence markers and codon-optimized them to work under optimal H. volcanii cultivation conditions. We found that two of them, Dendra2Hfx and PAmCherry1Hfx, provide state-of-the-art single-molecule imaging. Our strategy is quantitative and allows dual-color imaging of two targets in the same field of view (FOV) as well as DNA co-staining. We present the first single-molecule localization microscopy (SMLM) images of the subcellular organization and dynamics of two crucial intracellular proteins in living H. volcanii cells, FtsZ1, which shows complex structures in the cell division ring, and RNA polymerase, which localizes around the periphery of the cellular DNA.This work should provide incentive to develop SMLM strategies for other archaeal organisms in the near future.

scholarly journals Single Molecule Localization Microscopy of DNA Damage Response Pathways in Cancer.

2016 ◽  
Vol 22 (S3) ◽  
pp. 1016-1017 ◽  
Author(s):  
Donna R. Whelan ◽  
Yandong Yin ◽  
Keria Bermudez-Hernandez ◽  
Sarah Keegan ◽  
David Fenyö ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Single Molecule ◽  
Localization Microscopy ◽  
Damage Response ◽  
Single Molecule Localization Microscopy

scholarly journals Establishing live-cell single-molecule localization microscopy imaging and single-particle tracking in the archaeon Haloferax volcanii

2020 ◽  
Author(s):  
Bartosz Turkowyd ◽  
Sandra Schreiber ◽  
Julia Wörtz ◽  
Ella Shtifman Segal ◽  
Moshe Mevarech ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Proteins ◽  
Single Particle Tracking ◽  
Haloferax Volcanii ◽  
Microscopy Imaging ◽  
Localization Microscopy ◽  
Cellular Dna ◽  
Eukaryotic Organisms ◽  
Single Molecule Localization Microscopy

AbstractIn recent years, fluorescence microscopy techniques for the localization and tracking of single molecules in living cells have become well-established and indispensable tools for the investigation of cellular biology and in vivo biochemistry of many bacterial and eukaryotic organisms. Nevertheless, these techniques are still not established for imaging archaea. Their establishment as a standard tool for the study of archaea will be a decisive milestone for the exploration of this branch of life and its unique biology.Here we have developed a reliable protocol for the study of the archaeon Haloferax volcanii. We have generated an autofluorescence-free H. volcanii strain, evaluated several fluorescent proteins for their suitability to serve as single-molecule fluorescence markers and codon-optimized them to work under optimal H. volcanii cultivation conditions. We found that two of them, Dendra2Hfx and PAmCherry1Hfx, provide state-of-the-art single-molecule imaging. Our strategy is quantitative and allows dual-color imaging of two targets in the same field of view as well as DNA co-staining. We present the first single-molecule localization microscopy (SMLM) images of the subcellular organization and dynamics of two crucial intracellular proteins in living H. volcanii cells, FtsZ1, which shows complex structures in the cell division ring, and RNA polymerase, which localizes around the periphery of the cellular DNA. This work should provide incentive to develop SMLM strategies for other archaeal organisms in the near future.

Single-Molecule Imaging of Active Mitochondrial Nitroreductases using a Photo-Crosslinking Fluorescent Sensor

2019 ◽  
Author(s):  
Zacharias Thiel ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Subcellular Localization ◽  
Fluorescent Probe ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Fluorescent Sensor ◽  
Biological Functions ◽  
Localization Microscopy ◽  
Drug Activation ◽  
Nitroreductase Activity ◽  
Single Molecule Localization Microscopy

Many biomacromolecules are known to cluster in microdomains with specific subcellular localization. In the case of enzymes, this clustering greatly defines their biological functions. Nitroreductases are enzymes capable of reducing nitro groups to amines and play a role in detoxification and pro-drug activation. Although nitroreductase activity has been detected in mammalian cells, the subcellular localization of this activity remains incompletely characterized. Here, we report a fluorescent probe that enables super-resolved imaging of pools of nitroreductase activity within mitochondria. This probe is activated sequentially by nitroreductases and light to give a photo-crosslinked adduct of active enzymes. In combination with a general photoactivatable marker of mitochondria, we performed two-color, threedimensional, single-molecule localization microscopy. These experiments allowed us to image the sub-mitochondrial organization of microdomains of nitroreductase activity.<br>

Single-Molecule Imaging of Active Mitochondrial Nitroreductases using a Photo-Crosslinking Fluorescent Sensor

2019 ◽  
Author(s):  
Zacharias Thiel ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Subcellular Localization ◽  
Fluorescent Probe ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Fluorescent Sensor ◽  
Biological Functions ◽  
Localization Microscopy ◽  
Drug Activation ◽  
Nitroreductase Activity ◽  
Single Molecule Localization Microscopy

Many biomacromolecules are known to cluster in microdomains with specific subcellular localization. In the case of enzymes, this clustering greatly defines their biological functions. Nitroreductases are enzymes capable of reducing nitro groups to amines and play a role in detoxification and pro-drug activation. Although nitroreductase activity has been detected in mammalian cells, the subcellular localization of this activity remains incompletely characterized. Here, we report a fluorescent probe that enables super-resolved imaging of pools of nitroreductase activity within mitochondria. This probe is activated sequentially by nitroreductases and light to give a photo-crosslinked adduct of active enzymes. In combination with a general photoactivatable marker of mitochondria, we performed two-color, threedimensional, single-molecule localization microscopy. These experiments allowed us to image the sub-mitochondrial organization of microdomains of nitroreductase activity.<br>

scholarly journals Single-molecule localization microscopy

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Mickaël Lelek ◽  
Melina T. Gyparaki ◽  
Gerti Beliu ◽  
Florian Schueder ◽  
Juliette Griffié ◽  
...  
Keyword(s):  
Single Molecule ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

scholarly journals Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Alan M. Szalai ◽  
Bruno Siarry ◽  
Jerónimo Lukin ◽  
David J. Williamson ◽  
Nicolás Unsain ◽  
...  
Keyword(s):  
Single Molecule ◽  
Total Internal Reflection ◽  
Single Molecules ◽  
Fluorescence Microscope ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Axial Position ◽  
Localization Microscopy ◽  
Localization Precision ◽  
Single Molecule Localization Microscopy

AbstractSingle-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule’s image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

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