scholarly journals Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

10.3791/4251 ◽  
2012 ◽  
Author(s):  
Jason M. Beckta ◽  
Scott C. Henderson ◽  
Kristoffer Valerie
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Three Dimensional ◽  
Live Cell ◽  
Damage Response

scholarly journals Dynamics of the DNA damage response: insights from live-cell imaging

2013 ◽  
Vol 12 (2) ◽  
pp. 109-117 ◽  
Author(s):  
K. Karanam ◽  
A. Loewer ◽  
G. Lahav
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Damage Response

Cost-Effective High-Speed, Three-Dimensional Live-Cell Imaging of HIV-1 Transfer at the T Cell Virological Synapse

2021 ◽  
Author(s):  
Alice Sandmeyer ◽  
Lili Wang ◽  
Wolfgang Hübner ◽  
Marcel Müller ◽  
Benjamin Chen ◽  
...  
Keyword(s):  
T Cell ◽  
Live Cell Imaging ◽  
High Speed ◽  
Cell Imaging ◽  
Three Dimensional ◽  
Cost Effective ◽  
Live Cell ◽  
Virological Synapse ◽  
Hiv 1

scholarly journals A computational and structural analysis of germline and somatic variants affecting the DDR mechanism, and their impact on human diseases

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lorena Magraner-Pardo ◽  
Roman A. Laskowski ◽  
Tirso Pons ◽  
Janet M. Thornton
Keyword(s):  
Prostate Cancer ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Protein Interaction ◽  
Three Dimensional ◽  
Protein Protein Interaction ◽  
Variants Of Unknown Significance ◽  
Damage Response ◽  
Dna Variants ◽  
Unknown Significance

AbstractDNA-Damage Response (DDR) proteins are crucial for maintaining the integrity of the genome by identifying and repairing errors in DNA. Variants affecting their function can have severe consequences since failure to repair damaged DNA can result in cells turning cancerous. Here, we compare germline and somatic variants in DDR genes, specifically looking at their locations in the corresponding three-dimensional (3D) structures, Pfam domains, and protein–protein interaction interfaces. We show that somatic variants in metastatic cases are more likely to be found in Pfam domains and protein interaction interfaces than are pathogenic germline variants or variants of unknown significance (VUS). We also show that there are hotspots in the structures of ATM and BRCA2 proteins where pathogenic germline, and recurrent somatic variants from primary and metastatic tumours, cluster together in 3D. Moreover, in the ATM, BRCA1 and BRCA2 genes from prostate cancer patients, the distributions of germline benign, pathogenic, VUS, and recurrent somatic variants differ across Pfam domains. Together, these results provide a better characterisation of the most recurrent affected regions in DDRs and could help in the understanding of individual susceptibility to tumour development.

scholarly journals CRISPR-mediated Multiplexed Live Cell Imaging of Nonrepetitive Genomic Loci

2020 ◽  
Author(s):  
Patricia A. Clow ◽  
Nathaniel Jillette ◽  
Jacqueline J. Zhu ◽  
Albert W. Cheng
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Repetitive Sequences ◽  
Binary Sequence ◽  
Three Dimensional ◽  
Live Cell ◽  
Live Cells ◽  
Imaging Method ◽  
Genomic Locus ◽  
Time Dynamics

AbstractThree-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and low signal-to-noise ratios (SNRs), and are thus mostly applicable to repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method, with an enhanced SNR, that allows for one nonrepetitive genomic locus to be labeled using a single sgRNA. We constructed Casilio dual-color probes to visualize the dynamic interactions of cohesin-bound elements in single live cells. By forming a binary sequence of multiple Casilio probes (PISCES) across a continuous stretch of DNA, we track the dynamic 3D folding of a 74kb genomic region over time. This method offers unprecedented resolution and scalability for delineating the dynamic 4D nucleome.One Sentence SummaryCasilio enables multiplexed live cell imaging of nonrepetitive DNA loci for illuminating the real-time dynamics of genome structures.

Three-Dimensional Unstained Live-Cell Imaging Using Stimulated Parametric Emission Microscopy

2009 ◽  
Vol 48 (9) ◽  
pp. 097003 ◽  
Author(s):  
Hieu M. Dang ◽  
Takehito Kawasumi ◽  
Gen Omura ◽  
Toshiyuki Umano ◽  
Shin'ichiro Kajiyama ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Three Dimensional ◽  
Live Cell ◽  
Emission Microscopy

scholarly journals Persistence and Dynamics of DNA Damage Signal Amplification Determined by Microcolony Formation and Live-cell Imaging

2011 ◽  
Vol 52 (6) ◽  
pp. 766-774 ◽  
Author(s):  
Yasuyoshi OKA ◽  
Motohiro YAMAUCHI ◽  
Masatoshi SUZUKI ◽  
Shunichi YAMASHITA ◽  
Keiji SUZUKI
Keyword(s):  
Dna Damage ◽  
Signal Amplification ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell

CBIO-17. A NOVEL ROLE FOR ATR KINASE DETERMINES GLIOBLASTOMA TUMOUR CELL INVASION

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi30-vi30
Author(s):  
ross carruthers ◽  
Sarah Derby ◽  
Karen Strathdee ◽  
Anthony Chalmers ◽  
Jim Norman ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Invasion ◽  
Tumour Cell ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Tumour Cell Invasion ◽  
Atr Kinase

Abstract BACKGROUND: Widespread contamination of the brain with malignant cells is a predominant feature of glioblastoma (GBM) and fatal brainstem infiltration is frequently observed at autopsy. Whilst radiotherapy improves survival, irradiation increases GBM cell invasion, resulting in sublethal dose to cells migrating outside the irradiated volume. Tumour cell invasion should be a therapeutic priority if survival is to be improved. The responsible molecular mechanisms are key to improving outcomes but remain enigmatic. Ataxia telangiectasia and rad3-related (ATR) is a DNA damage response (DDR) kinase involved in DNA replication stress (RS) response and is an established therapeutic target for GBM. In this study we demonstrate a novel role for ATR kinase in facilitating malignant cell invasion. METHODS AND RESULTS: Invading margins of human GBM samples demonstrated increased pATR expression relative to core. Live cell imaging demonstrated a reduction in cell velocity following ATR inhibition (ATRi; VE822) or ATR siRNA, and a retraction defect was evident in vitro. Extensive cytoplasmic vacuolation occurred following ATRi or siRNA which were single walled structures on electron microscopy which could engulf high molecular weight dextran, suggesting blockade of macropinosome processing. Live cell imaging with GFP-integrin α5 and integrin recycling assays showed integrin sequestration within macropinosomes and reduced integrin internalisation respectively. Interrogation of a published ‘ATR interactome’ revealed ATR targets with functions in endocytic vesicle trafficking. Intravital in vivo imaging of murine xenograft tumours confirmed vacuolation and dextran uptake following ATRi, whilst a further study demonstrated reduced invading tumour cells following ATRi in intracranial xenografts. CONCLUSION: We demonstrate a novel role for ATR in facilitating macropinocytic vesicle trafficking and integrin internalisation. ATRi results in a profound motility defect in vitro and in vivo. ATR inhibitors are entering early phase trials as radiation sensitisers and we propose that therapeutic benefit will extend beyond DNA damage potentiation.

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