DNA damage reduces heterogeneity and coherence of chromatin motions.

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.

The nuclear structural protein NuMA is a negative regulator of 53BP1 in DNA double-strand break repair.

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.

The nuclear mitotic apparatus protein NuMA controls rDNA transcription and mediates the nucleolar stress response in a p53-independent manner.

The nuclear mitotic apparatus protein, NuMA, is involved in major cellular events such as DNA damage response, apoptosis and p53-mediated growth-arrest, all of which are under the control of the nucleolus upon stress. Proteomic investigation has identified NuMA among hundreds of nucleolar proteins. Yet, the precise link between NuMA and nucleolar function remains undetermined. We confirm that NuMA is present in the nucleolus and reveal redistribution of NuMA upon actinomycin D or doxorubicin-induced nucleolar stress. NuMA coimmunoprecipitates with RNA polymerase I, with ribosomal proteins RPL26 and RPL24, and with components of B-WICH, an ATP-dependent chromatin remodeling complex associated with rDNA transcription. NuMA also binds to 18S and 28S rRNAs and localizes to rDNA promoter regions. Downregulation of NuMA expression triggers nucleolar stress, as shown by decreased nascent pre-rRNA synthesis, fibrillarin perinucleolar cap formation and upregulation of p27kip1, but not p53. Physiologically relevant nucleolar stress induction with reactive oxygen species reaffirms a p53-independent p27kip1 response pathway and leads to nascent pre-rRNA reduction. It also promotes the decrease in the amount of NuMA. This previously uncharacterized function of NuMA in rDNA transcription and p53-independent nucleolar stress response supports a central role for this nuclear structural protein in cellular homeostasis.

High-content image informatics of the structural nuclear protein NuMA parses trajectories for stem/progenitor cell lineages and oncogenic transformation.

Stem and progenitor cells that exhibit significant regenerative potential and critical roles in cancer initiation and progression remain difficult to characterize. Cell fates are determined by reciprocal signaling between the cell microenvironment and the nucleus; hence parameters derived from nuclear remodeling are ideal candidates for stem/progenitor cell characterization. Here we applied high-content, single cell analysis of nuclear shape and organization to examine stem and progenitor cells destined to distinct differentiation endpoints, yet undistinguishable by conventional methods. Nuclear descriptors defined through image informatics classified mesenchymal stem cells poised to either adipogenic or osteogenic differentiation, and oligodendrocyte precursors isolated from different regions of the brain and destined to distinct astrocyte subtypes. Nuclear descriptors also revealed early changes in stem cells after chemical oncogenesis, allowing the identification of a class of cancer-mitigating biomaterials. To capture the metrology of nuclear changes, we developed a simple and quantitative "imaging-derived" parsing index, which reflects the dynamic evolution of the high-dimensional space of nuclear organizational features. A comparative analysis of parsing outcomes via either nuclear shape or textural metrics of the nuclear structural protein NuMA indicates the nuclear shape alone is a weak phenotypic predictor. In contrast, variations in the NuMA organization parsed emergent cell phenotypes and discerned emergent stages of stem cell transformation, supporting a prognosticating role for this protein in the outcomes of nuclear functions.

Nanoscale histone localization in live cells reveals reduced chromatin mobility in response to DNA damage.

Nuclear functions including gene expression, DNA replication and genome maintenance intimately rely on dynamic changes in chromatin organization. The movements of chromatin fibers might play important roles in the regulation of these fundamental processes, yet the mechanisms controlling chromatin mobility are poorly understood owing to methodological limitations for the assessment of chromatin movements. Here, we present a facile and quantitative technique that relies on photoactivation of GFP-tagged histones and paired-particle tracking to measure chromatin mobility in live cells. We validate the method by comparing live cells to ATP-depleted cells and show that chromatin movements in mammalian cells are predominantly energy dependent. We also find that chromatin diffusion decreases in response to DNA breaks induced by a genotoxic drug or by the ISceI meganuclease. Timecourse analysis after cell exposure to ionizing radiation indicates that the decrease in chromatin mobility is transient and precedes subsequent increased mobility. Future applications of the method in the DNA repair field and beyond are discussed.

NuMA promotes homologous recombination repair by regulating the accumulation of the ISWI ATPase SNF2h at DNA breaks.

Chromatin remodeling factors play an active role in the DNA damage response by shaping chromatin to facilitate the repair process. The spatiotemporal regulation of these factors is key to their function, yet poorly understood. We report that the structural nuclear protein NuMA accumulates at sites of DNA damage in a poly[ADP-ribose]ylation-dependent manner and functionally interacts with the ISWI ATPase SNF2h/SMARCA5, a chromatin remodeler that facilitates DNA repair. NuMA coimmunoprecipitates with SNF2h, regulates its diffusion in the nucleoplasm and controls its accumulation at DNA breaks. Consistent with NuMA enabling SNF2h function, cells with silenced NuMA exhibit reduced chromatin decompaction after DNA cleavage, lesser focal recruitment of homologous recombination repair factors, impaired DNA double-strand break repair in chromosomal (but not in episomal) contexts and increased sensitivity to DNA cross-linking agents. These findings reveal a structural basis for the orchestration of chromatin remodeling whereby a scaffold protein promotes genome maintenance by directing a remodeler to DNA breaks.

Interconnected contribution of tissue morphogenesis and the nuclear protein NuMA to the DNA damage response.

Epithelial tissue morphogenesis is accompanied by the formation of a polarity axis--a feature of tissue architecture that is initiated by the binding of integrins to the basement membrane. Polarity plays a crucial role in tissue homeostasis, preserving differentiation, cell survival and resistance to chemotherapeutic drugs among others. An important aspect in the maintenance of tissue homeostasis is genome integrity. As normal tissues frequently experience DNA double-strand breaks (DSBs), we asked how tissue architecture might participate in the DNA damage response. Using 3D culture models that mimic mammary glandular morphogenesis and tumor formation, we show that DSB repair activity is higher in basally polarized tissues, regardless of the malignant status of cells, and is controlled by hemidesmosomal integrin signaling. In the absence of glandular morphogenesis, in 2D flat monolayer cultures, basal polarity does not affect DNA repair activity but enhances H2AX phosphorylation, an early chromatin response to DNA damage. The nuclear mitotic apparatus protein 1 (NuMA), which controls breast glandular morphogenesis by acting on the organization of chromatin, displays a polarity-dependent pattern and redistributes in the cell nucleus of basally polarized cells upon the induction of DSBs. This is shown using high-content analysis of nuclear morphometric descriptors. Furthermore, silencing NuMA impairs H2AX phosphorylation--thus, tissue polarity and NuMA cooperate to maintain genome integrity.

Plastid proteome assembly without Toc159: photosynthetic protein import and accumulation of N-acetylated plastid precursor proteins.

Import of nuclear-encoded precursor proteins from the cytosol is an essential step in chloroplast biogenesis that is mediated by protein translocon complexes at the inner and outer envelope membrane (TOC). Toc159 is thought to be the main receptor for photosynthetic proteins, but lacking a large-scale systems approach, this hypothesis has only been tested for a handful of photosynthetic and nonphotosynthetic proteins. To assess Toc159 precursor specificity, we quantitatively analyzed the accumulation of plastid proteins in two mutant lines deficient in this receptor. Parallel genome-wide transcript profiling allowed us to discern the consequences of impaired protein import from systemic transcriptional responses that contribute to the loss of photosynthetic capacity. On this basis, we defined putative Toc159-independent and Toc159-dependent precursor proteins. Many photosynthetic proteins accumulate in Toc159-deficient plastids, and, surprisingly, several distinct metabolic pathways are negatively affected by Toc159 depletion. Lack of Toc159 furthermore affects several proteins that accumulate as unprocessed N-acetylated precursor proteins outside of plastids. Together, our data show an unexpected client protein promiscuity of Toc159 that requires a far more differentiated view of Toc159 receptor function and regulation of plastid protein import, in which cytosolic Met removal followed by N-terminal acetylation of precursors emerges as an additional regulatory step.

Reversion of breast epithelial polarity alterations caused by obesity.

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.

Characterization and implementation of a miniature X-ray system for live cell microscopy.

The study of radiation effects on biological tissues is a diverse field of research with direct applications to improve human health, in particular in the contexts of radiation therapy and space exploration. Understanding the DNA damage response following radiation exposure, which is a key determinant for mutagenesis, requires reproducible methods for delivering known doses of ionizing radiation (IR) in a controlled environment. Multiple IR sources, including research X-ray and gamma-ray irradiators are routinely used in basic and translational research with cell and animal models. These systems are however not ideal when a high temporal resolution is needed, for example to study early DNA damage responses with live cell microscopy. Here, we characterize the dose rate and beam properties of a commercial, miniature, affordable, and versatile X-ray source (Mini-X). We describe how to use Mini-X on the stage of a fluorescence microscope to deliver high IR dose rates (up to 29 Gy/min) or lower dose rates (≤ 0.1 Gy/min) in live cell imaging experiments. This article provides a blueprint for radiation biology applications with high temporal resolution, with a step-by-step guide to implement a miniature X-ray system on an imaging platform, and the information needed to characterize the system.

Three-dimensional tracking using a single-spot rotating point spread function created by a multiring spiral phase plate.

Significance: Three-dimensional (3D) imaging and object tracking is critical for medical and biological research and can be achieved by multifocal imaging with diffractive optical elements (DOEs) converting depth ( z ) information into a modification of the two-dimensional image. Physical insight into DOE designs will spur this expanding field. Aim: To precisely track microscopic fluorescent objects in biological systems in 3D with a simple low-cost DOE system. Approach: We designed a multiring spiral phase plate (SPP) generating a single-spot rotating point spread function (SS-RPSF) in a microscope. Our simple, analytically transparent design process uses Bessel beams to avoid rotational ambiguities and achieve a significant depth range. The SPP was inserted into the Nomarski prism slider of a standard microscope. Performance was evaluated using fluorescent beads and in live cells expressing a fluorescent chromatin marker. Results: Bead localization precision was < 25 nm in the transverse dimensions and ≤ 70 nm along the axial dimension over an axial range of 6 µ m . Higher axial precision ( ≤ 50 nm ) was achieved over a shallower focal depth of 2.9 µ m . 3D diffusion constants of chromatin matched expected values. Conclusions: Precise 3D localization and tracking can be achieved with a SS-RPSF SPP in a standard microscope with minor modifications.

Performance of deep learning restoration methods for the extraction of particle dynamics in noisy microscopy image sequences.

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.

Diet Alters Entero-Mammary Signaling to Regulate the Breast Microbiome and Tumorigenesis.

Obesity and poor diet often go hand-in-hand, altering metabolic signaling and thereby impacting breast cancer risk and outcomes. We have recently demonstrated that dietary patterns modulate mammary microbiota populations. An important and largely open question is whether the microbiome of the gut and mammary gland mediates the dietary effects on breast cancer. To address this, we performed fecal transplants between mice on control or high-fat diets (HFD) and recorded mammary tumor outcomes in a chemical carcinogenesis model. HFD induced protumorigenic effects, which could be mimicked in animals fed a control diet by transplanting HFD-derived microbiota. Fecal transplants altered both the gut and mammary tumor microbiota populations, suggesting a link between the gut and breast microbiomes. HFD increased serum levels of bacterial lipopolysaccharide (LPS), and control diet-derived fecal transplant reduced LPS bioavailability in HFD-fed animals. In vitro models of the normal breast epithelium showed that LPS disrupts tight junctions (TJ) and compromises epithelial permeability. In mice, HFD or fecal transplant from animals on HFD reduced expression of TJ-associated genes in the gut and mammary gland. Furthermore, infecting breast cancer cells with an HFD-derived microbiome increased proliferation, implicating tumor-associated bacteria in cancer signaling. In a double-blind placebo-controlled clinical trial of patients with breast cancer administered fish oil supplements before primary tumor resection, dietary intervention modulated the microbiota in tumors and normal breast tissue. This study demonstrates a link between the gut and breast that mediates the effect of diet on cancer. SIGNIFICANCE: This study demonstrates that diet shifts the microbiome in the gut and the breast tumor microenvironment to affect tumorigenesis, and oral dietary interventions can modulate the tumor microbiota in patients with breast cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/14/3890/F1.large.jpg.

A mutation in the Arabidopsis mTERF-related plastid protein SOLDAT10 activates retrograde signaling and suppresses (1)O(2)-induced cell death.

The conditional flu mutant of Arabidopsis thaliana generates singlet oxygen ((1)O(2)) in plastids during a dark-to-light shift. Seedlings of flu bleach and die, whereas mature plants stop growing and develop macroscopic necrotic lesions. Several suppressor mutants, dubbed singlet oxygen-linked death activator (soldat), were identified that abrogate (1)O(2)-mediated cell death of flu seedlings. One of the soldat mutations, soldat10, affects a gene encoding a plastid-localized protein related to the human mitochondrial transcription termination factor mTERF. As a consequence of this mutation, plastid-specific rRNA levels decrease and protein synthesis in plastids of soldat10 is attenuated. This disruption of chloroplast homeostasis in soldat10 seedlings affects communication between chloroplasts and the nucleus and leads to changes in the steady-state concentration of nuclear gene transcripts. The soldat10 seedlings suffer from mild photo-oxidative stress, as indicated by the constitutive up-regulation of stress-related genes. Even though soldat10/flu seedlings overaccumulate the photosensitizer protochlorophyllide in the dark and activate the expression of (1)O(2)-responsive genes after a dark-to-light shift they do not show a (1)O(2)-dependent cell death response. Disturbance of chloroplast homeostasis in emerging soldat10/flu seedlings seems to antagonize a subsequent (1)O(2)-mediated cell death response without suppressing (1)O(2)-dependent retrograde signaling. The results of this work reveal the unexpected complexity of what is commonly referred to as 'plastid signaling'.

Intersection of the tocopherol and plastoquinol metabolic pathways at the plastoglobule.

Plastoglobules, lipid-protein bodies in the stroma of plant chloroplasts, are enriched in non-polar lipids, in particular prenyl quinols. In the present study we show that, in addition to the thylakoids, plastoglobules also contain a considerable proportion of the plastidial PQ-9 (plastoquinol-9), the redox component of photosystem II, and of the cyclized product of PQ-9, PC-8 (plastochromanol-8), a tocochromanol with a structure similar to gamma-tocopherol and gamma-tocotrienol, but with a C-40 prenyl side chain. PC-8 formation was abolished in the Arabidopsis thaliana tocopherol cyclase mutant vte1, but accumulated in VTE1-overexpressing plants, in agreement with a role of tocopherol cyclase (VTE1) in PC-8 synthesis. VTE1 overexpression resulted in the proliferation of the number of plastoglobules which occurred in the form of clusters in the transgenic lines. Simultaneous overexpression of VTE1 and of the methyltransferase VTE4 resulted in the accumulation of a compound tentatively identified as 5-methyl-PC-8, the methylated form of PC-8. The results of the present study suggest that the existence of a plastoglobular pool of PQ-9, along with the partial conversion of PQ-9 into PC-8, might represent a mechanism for the regulation of the antioxidant content in thylakoids and of the PQ-9 pool that is available for photosynthesis.

Fluorescent and bioluminescent protein-fragment complementation assays in the study of G protein-coupled receptor oligomerization and signaling.

Most cellular functions, including signaling by G protein-coupled receptors (GPCRs), are mediated by protein-protein interactions, making the identification and localization of protein complexes key to the understanding of cellular processes. In complement to traditional biochemical techniques, noninvasive resonance energy transfer (RET) and protein-fragment complementation assays (PCAs) now allow protein interactions to be detected in the context of living cells. In this review, fluorescent and bioluminescent PCAs are discussed and their application illustrated with studies on GPCR signaling. Newly developed techniques combining PCA and RET assays for the detection of ternary and quaternary protein complexes are also presented.

Essential role of the G-domain in targeting of the protein import receptor atToc159 to the chloroplast outer membrane.

Two homologous GTP-binding proteins, atToc33 and atToc159, control access of cytosolic precursor proteins to the chloroplast. atToc33 is a constitutive outer chloroplast membrane protein, whereas the precursor receptor atToc159 also exists in a soluble, cytosolic form. This suggests that atToc159 may be able to switch between a soluble and an integral membrane form. By transient expression of GFP fusion proteins, mutant analysis, and biochemical experimentation, we demonstrate that the GTP-binding domain regulates the targeting of cytosolic atToc159 to the chloroplast and mediates the switch between cytosolic and integral membrane forms. Mutant atToc159, unable to bind GTP, does not reinstate a green phenotype in an albino mutant (ppi2) lacking endogenous atToc159, remaining trapped in the cytosol. Thus, the function of atToc159 in chloroplast biogenesis is dependent on an intrinsic GTP-regulated switch that controls localization of the receptor to the chloroplast envelope.

Radial Profile Analysis of Epithelial Polarity in Breast Acini: A Tool for Primary (Breast) Cancer Prevention.

Preventing cancer is vastly better than treating the disease in terms of a patient's quality of life and healthcare costs. Yet, to screen for chemopreventative drugs or evaluate interventions aimed at lowering cancer risk, quantitative readouts of risk are needed. In the breast and in other organs of epithelial origin, apical-basal polarity is key to homeostasis and is one of the first tissue characteristics lost during cancer initiation. Therefore, apical-basal polarity may be leveraged as an "architectural" determinant of cancer risk. A classic approach to quantify the localization of epithelial polarity markers is visual scoring at the microscope by trained investigators. This approach is time-intensive and limited to low throughput. To increase the speed, accuracy, and scoring volume, we developed an algorithm that essentially replaces the human eye to objectively quantify epithelial polarity in microscopy images of breast glandular units (acini). Acini in culture are identified based on a nuclear stain and the corresponding masks are divided into concentric terraces of equal width. This positional information is used to calculate radial intensity profiles (RP) of polarity markers. Profiles with a steep slope represent polarized structures, whereas more horizontal curves are indicative of non-polarized acini. To compare treatment effects, RP curves are integrated into summary values of polarity. We envision applications of this method for primary cancer prevention research with acini organoids, specifically (1) to screen for chemoprevention drugs, (2) for toxicological assessment of suspected carcinogens and pharmacological hit compounds, and (3) for personalized evaluation of cancer risk and risk-reducing interventions. The RadialProfiler algorithm developed for the MATLAB computing environment and for users without prior informatics knowledge is publicly available on the Open Science Framework (OSF).

Silver nanoparticles selectively treat triple-negative breast cancer cells without affecting non-malignant breast epithelial cells in vitro and in vivo.

Silver nanoparticles (AgNPs) show promise for treatment of aggressive cancers including triple-negative breast cancer (TNBC) in preclinical cancer models. For clinical development of AgNP-based therapeutics, it will be necessary to clearly define the specific physicochemical features of the nanoparticles that will be used, and to tie these properties to biological outcomes. To fill this knowledge gap, we performed thorough structure/function, mechanistic, safety, and efficacy studies to assess the potential for AgNPs to treat TNBC. We establish that AgNPs, regardless of size, shape, or stabilizing agent, are highly cytotoxic to TNBC cells at doses that are not cytotoxic to non-malignant breast epithelial cells. In contrast, TNBC cells and non-malignant breast epithelial cells are similarly sensitive to exposure to silver cation (Ag+), indicating that the nanoparticle formulation is essential for the TNBC-specific cytotoxicity. Mechanistically, AgNPs are internalized by both TNBC and non-malignant breast cells, but are rapidly degraded only in TNBC cells. Exposure to AgNPs depletes cellular antioxidants and causes endoplasmic reticulum stress in TNBC cells without causing similar damage in non-malignant breast epithelial cells. AgNPs also cause extensive DNA damage in 3D TNBC tumor nodules in vitro, but do not disrupt the normal architecture of breast acini in 3D cell culture, nor cause DNA damage or induce apoptosis in these structures. Lastly, we show that systemically administered AgNPs are effective at non-toxic doses for reducing the growth of TNBC tumor xenografts in mice. This work provides a rationale for development of AgNPs as a safe and specific TNBC treatment.