Browsing by Author "Vidi, Pierre-Alexandre"

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    DNA damage reduces heterogeneity and coherence of chromatin motions
    (National Academy of Science, 2022) Locatelli, Maëlle; Lawrimore, Josh; Lin, Hua; Sanaullah, Sarvath; Seitz, Clayton; Segall, Dave; Kefer, Paul; Moreno, Naike Salvador; Lietz, Benton; Anderson, Rebecca; Holmes, Julia; Yuan, Chongli; Holzwarth, George; Bloom, Kerry S.; Liu, Jing; Bonin, Keith; Vidi, Pierre-Alexandre; Physics, School of Science
    Chromatin motions depend on and may regulate genome functions, in particular the DNA damage response. In yeast, DNA double-strand breaks (DSBs) globally increase chromatin diffusion, whereas in higher eukaryotes the impact of DSBs on chromatin dynamics is more nuanced. We mapped the motions of chromatin microdomains in mammalian cells using diffractive optics and photoactivatable chromatin probes and found a high level of spatial heterogeneity. DNA damage reduces heterogeneity and imposes spatially defined shifts in motions: Distal to DNA breaks, chromatin motions are globally reduced, whereas chromatin retains higher mobility at break sites. These effects are driven by context-dependent changes in chromatin compaction. Photoactivated lattices of chromatin microdomains are ideal to quantify microscale coupling of chromatin motion. We measured correlation distances up to 2 µm in the cell nucleus, spanning chromosome territories, and speculate that this correlation distance between chromatin microdomains corresponds to the physical separation of A and B compartments identified in chromosome conformation capture experiments. After DNA damage, chromatin motions become less correlated, a phenomenon driven by phase separation at DSBs. Our data indicate tight spatial control of chromatin motions after genomic insults, which may facilitate repair at the break sites and prevent deleterious contacts of DSBs, thereby reducing the risk of genomic rearrangements.
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    The nuclear structural protein NuMA is a negative regulator of 53BP1 in DNA double-strand break repair
    (Oxford University Press, 2019-04-08) Salvador Moreno, Naike; Liu, Jing; Haas, Karen M.; Parker, Laurie L.; Chakraborty, Chaitali; Kron, Stephen J.; Hodges, Kurt; Miller, Lance D.; Langefeld, Carl; Robinson, Paul J.; Lelièvre, Sophie A.; Vidi, Pierre-Alexandre; Physics, School of Science
    P53-binding protein 1 (53BP1) mediates DNA repair pathway choice and promotes checkpoint activation. Chromatin marks induced by DNA double-strand breaks and recognized by 53BP1 enable focal accumulation of this multifunctional repair factor at damaged chromatin. Here, we unveil an additional level of regulation of 53BP1 outside repair foci. 53BP1 movements are constrained throughout the nucleoplasm and increase in response to DNA damage. 53BP1 interacts with the structural protein NuMA, which controls 53BP1 diffusion. This interaction, and colocalization between the two proteins in vitro and in breast tissues, is reduced after DNA damage. In cell lines and breast carcinoma NuMA prevents 53BP1 accumulation at DNA breaks, and high NuMA expression predicts better patient outcomes. Manipulating NuMA expression alters PARP inhibitor sensitivity of BRCA1-null cells, end-joining activity, and immunoglobulin class switching that rely on 53BP1. We propose a mechanism involving the sequestration of 53BP1 by NuMA in the absence of DNA damage. Such a mechanism may have evolved to disable repair functions and may be a decisive factor for tumor responses to genotoxic treatments.
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    Performance of deep learning restoration methods for the extraction of particle dynamics in noisy microscopy image sequences
    (American Society for Cell Biology, 2021-04-19) Kefer, Paul; Iqbal, Fadil; Locatelli, Maelle; Lawrimore, Josh; Zhang, Mengdi; Bloom, Kerry; Bonin, Keith; Vidi, Pierre-Alexandre; Liu, Jing; Physics, School of Science
    Particle tracking in living systems requires low light exposure and short exposure times to avoid phototoxicity and photobleaching and to fully capture particle motion with high-speed imaging. Low-excitation light comes at the expense of tracking accuracy. Image restoration methods based on deep learning dramatically improve the signal-to-noise ratio in low-exposure data sets, qualitatively improving the images. However, it is not clear whether images generated by these methods yield accurate quantitative measurements such as diffusion parameters in (single) particle tracking experiments. Here, we evaluate the performance of two popular deep learning denoising software packages for particle tracking, using synthetic data sets and movies of diffusing chromatin as biological examples. With synthetic data, both supervised and unsupervised deep learning restored particle motions with high accuracy in two-dimensional data sets, whereas artifacts were introduced by the denoisers in three-dimensional data sets. Experimentally, we found that, while both supervised and unsupervised approaches improved tracking results compared with the original noisy images, supervised learning generally outperformed the unsupervised approach. We find that nicer-looking image sequences are not synonymous with more precise tracking results and highlight that deep learning algorithms can produce deceiving artifacts with extremely noisy images. Finally, we address the challenge of selecting parameters to train convolutional neural networks by implementing a frugal Bayesian optimizer that rapidly explores multidimensional parameter spaces, identifying networks yielding optimal particle tracking accuracy. Our study provides quantitative outcome measures of image restoration using deep learning. We anticipate broad application of this approach to critically evaluate artificial intelligence solutions for quantitative microscopy.
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    Reversion of breast epithelial polarity alterations caused by obesity
    (Springer Nature, 2023-05-09) Holmes, Julia; Gaber, Mohamed; Jenks, Mónica Z.; Wilson, Adam; Loy, Tucker; Lepetit, Cassandra; Vitolins, Mara Z.; Herbert, Brittney-Shea; Cook, Katherine L.; Vidi, Pierre-Alexandre; Medical and Molecular Genetics, School of Medicine
    Molecular links between breast cancer risk factors and pro-oncogenic tissue alterations are poorly understood. The goal of this study was to characterize the impact of overweight and obesity on tissue markers of risk, using normal breast biopsies, a mouse model of diet-induced obesity, and cultured breast acini. Proliferation and alteration of epithelial polarity, both necessary for tumor initiation, were quantified by immunostaining. High BMI (>30) and elevated leptin were associated with compromised epithelial polarity whereas overweight was associated with a modest increase in proliferation in human and mice mammary glands. Human serum with unfavorable adipokine levels altered epithelial polarization of cultured acini, recapitulating the effect of leptin. Weight loss in mice led to metabolic improvements and restored epithelial polarity. In acini cultures, alteration of epithelial polarity was prevented by antioxidants and could be reverted by normalizing culture conditions. This study shows that obesity and/or dietary factors modulate tissue markers of risk. It provides a framework to set target values for metabolic improvements and to assess the efficacy of interventional studies aimed at reducing breast cancer risk.