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1.
Elife ; 122023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37906089

RESUMO

Chromatin has been shown to undergo diffusional motion, which is affected during gene transcription by RNA polymerase activity. However, the relationship between chromatin mobility and other genomic processes remains unclear. Hence, we set out to label the DNA directly in a sequence unbiased manner and followed labeled chromatin dynamics in interphase human cells expressing GFP-tagged proliferating cell nuclear antigen (PCNA), a cell cycle marker and core component of the DNA replication machinery. We detected decreased chromatin mobility during the S-phase compared to G1 and G2 phases in tumor as well as normal diploid cells using automated particle tracking. To gain insight into the dynamical organization of the genome during DNA replication, we determined labeled chromatin domain sizes and analyzed their motion in replicating cells. By correlating chromatin mobility proximal to the active sites of DNA synthesis, we showed that chromatin motion was locally constrained at the sites of DNA replication. Furthermore, inhibiting DNA synthesis led to increased loading of DNA polymerases. This was accompanied by accumulation of the single-stranded DNA binding protein on the chromatin and activation of DNA helicases further restricting local chromatin motion. We, therefore, propose that it is the loading of replisomes but not their catalytic activity that reduces the dynamics of replicating chromatin segments in the S-phase as well as their accessibility and probability of interactions with other genomic regions.


Assuntos
Cromatina , Replicação do DNA , Humanos , Fase S , Ciclo Celular , DNA Helicases
2.
Med Image Anal ; 72: 102128, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34229189

RESUMO

Tracking of particles in temporal fluorescence microscopy image sequences is of fundamental importance to quantify dynamic processes of intracellular structures as well as virus structures. We introduce a probabilistic deep learning approach for fluorescent particle tracking, which is based on a recurrent neural network that mimics classical Bayesian filtering. Compared to previous deep learning methods for particle tracking, our approach takes into account uncertainty, both aleatoric and epistemic uncertainty. Thus, information about the reliability of the computed trajectories is determined. Manual tuning of tracking parameters is not necessary and prior knowledge about the noise statistics is not required. Short and long-term temporal dependencies of individual object dynamics are exploited for state prediction, and assigned detections are used to update the predicted states. For correspondence finding, we introduce a neural network which computes assignment probabilities jointly across multiple detections as well as determines the probabilities of missing detections. Training requires only simulated data and therefore tedious manual annotation of ground truth is not needed. We performed a quantitative performance evaluation based on synthetic and real 2D as well as 3D fluorescence microscopy images. We used image data of the Particle Tracking Challenge as well as real time-lapse fluorescence microscopy images displaying virus structures and chromatin structures. It turned out that our approach yields state-of-the-art results or improves the tracking results compared to previous methods.


Assuntos
Algoritmos , Redes Neurais de Computação , Teorema de Bayes , Humanos , Microscopia de Fluorescência , Reprodutibilidade dos Testes
3.
Nucleic Acids Res ; 48(22): 12751-12777, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33264404

RESUMO

To ensure error-free duplication of all (epi)genetic information once per cell cycle, DNA replication follows a cell type and developmental stage specific spatio-temporal program. Here, we analyze the spatio-temporal DNA replication progression in (un)differentiated mouse embryonic stem (mES) cells. Whereas telomeres replicate throughout S-phase, we observe mid S-phase replication of (peri)centromeric heterochromatin in mES cells, which switches to late S-phase replication upon differentiation. This replication timing reversal correlates with and depends on an increase in condensation and a decrease in acetylation of chromatin. We further find synchronous duplication of the Y chromosome, marking the end of S-phase, irrespectively of the pluripotency state. Using a combination of single-molecule and super-resolution microscopy, we measure molecular properties of the mES cell replicon, the number of replication foci active in parallel and their spatial clustering. We conclude that each replication nanofocus in mES cells corresponds to an individual replicon, with up to one quarter representing unidirectional forks. Furthermore, with molecular combing and genome-wide origin mapping analyses, we find that mES cells activate twice as many origins spaced at half the distance than somatic cells. Altogether, our results highlight fundamental developmental differences on progression of genome replication and origin activation in pluripotent cells.


Assuntos
Replicação do DNA/genética , Heterocromatina/genética , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular/genética , Centrômero/genética , Duplicação Cromossômica/genética , Cromossomos Humanos Y/genética , Genoma/genética , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Fase S/genética , Imagem Individual de Molécula
4.
Nucleus ; 10(1): 231-253, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31744372

RESUMO

Constitutive heterochromatin is considered as a functionally inert genome compartment, important for its architecture and stability. How such stable structure is maintained is not well understood. Here, we apply four different visualization schemes to label it and investigate its dynamics during DNA replication and repair. We show that replisomes assemble over the heterochromatin in a temporally ordered manner. Furthermore, heterochromatin undergoes transient decompaction locally at the active sites of DNA synthesis. Using selective laser microirradiation conditions that lead to damage repaired via processive DNA synthesis, we measured similarly local decompaction of heterochromatin. In both cases, we could not observe large-scale movement of heterochromatin to the domain surface. Instead, the processive DNA synthesis machinery assembled at the replication/repair sites. Altogether, our data are compatible with a progression of DNA replication/repair along the chromatin in a dynamic mode with localized and transient decompaction that does not globally remodels the whole heterochromatin compartment.


Assuntos
Reparo do DNA , Replicação do DNA , DNA/biossíntese , Heterocromatina/metabolismo , Animais , Células Cultivadas , DNA/química , Células HeLa , Heterocromatina/química , Humanos , Camundongos
5.
AIMS Genet ; 5(2): 113-123, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-31435516

RESUMO

Chromosome positioning in sperm nucleus may have a functional significance by influencing the sequence of post-fertilization events. In this study we present data on preferential locations of chromosomes 1, 29 and X in Bos taurus spermatozoa. Here we demonstrate that the position of X chromosome in the sperm nucleus is more restricted as compared to the position of chromosome 1, which is about of the same size. Our data support the concept of the functional significance of genome architecture in male germline cells.

6.
BMC Mol Biol ; 16: 18, 2015 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-26458748

RESUMO

BACKGROUND: Studies of DNA damage response are critical for the comprehensive understanding of age-related changes in cells, tissues and organisms. Syrian hamster cells halt proliferation and become presenescent after several passages in standard conditions of cultivation due to what is known as "culture stress". Using proliferating young and non-dividing presenescent cells in primary cultures of Syrian hamster fibroblasts, we defined their response to the action of radiomimetic drug bleomycin (BL) that induces DNA double-strand breaks (DSBs). RESULTS: The effect of the drug was estimated by immunoblotting and immunofluorescence microscopy using the antibody to phosphorylated histone H2AX (gH2AX), which is generally accepted as a DSB marker. At all stages of the cell cycle, both presenescent and young cells demonstrated variability of the number of gH2AX foci per nucleus. gH2AX focus induction was found to be independent from BL-hydrolase expression. Some differences in DSB repair process between BL-treated young and presenescent Syrian hamster cells were observed: (1) the kinetics of gH2AX focus loss in G0 fibroblasts of young culture was faster than in cells that prematurely stopped dividing; (2) presenescent cells were characterized by a slower recruitment of DSB repair proteins 53BP1, phospho-DNA-PK and phospho-ATM to gH2AX focal sites, while the rate of phosphorylated ATM/ATR substrate accumulation was the same as that in young cells. CONCLUSIONS: Our results demonstrate an impairment of DSB repair in prematurely aged Syrian hamster fibroblasts in comparison with young fibroblasts, suggesting age-related differences in response to BL therapy.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Bleomicina/farmacologia , Senescência Celular/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/genética , Histonas/metabolismo , Senilidade Prematura/genética , Animais , Anticorpos/imunologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Cricetinae , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Fase G1/genética , Histonas/genética , Histonas/imunologia , Mesocricetus/genética , Fosforilação , Ligação Proteica/fisiologia , Fase de Repouso do Ciclo Celular/genética , Proteína Supressora de Tumor p53/metabolismo , beta-Galactosidase/metabolismo
7.
Methods Mol Biol ; 1300: 43-65, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25916704

RESUMO

Genomic DNA of a eukaryotic cell is replicated once during the S-phase of the cell cycle to precisely maintain the complete genetic information. In the course of S-phase, semiconservative DNA synthesis is sequentially initiated and performed at thousands of discrete patches of the DNA helix termed replicons. At any given moment of S-phase, multiple replicons are active in parallel in different parts of the genome. In the last decades, tools and methods to visualize DNA synthesis inside cells have been developed. Pulse labeling with nucleotides as well as detecting components of the replication machinery yielded an overall picture of multiple discrete sites of active DNA synthesis termed replication foci (RFi) and forming spatiotemporal patterns within the cell nucleus. Recent advances in fluorescence microscopy and digital imaging in combination with computational image analysis allow a comprehensive quantitative analysis of RFi and provide valuable insights into the organization of the genomic DNA replication process and also of the genome itself. In this chapter, we describe in detail protocols for the visualization and quantification of RFi at different levels of optical and physical resolution.


Assuntos
Replicação do DNA , Mamíferos/metabolismo , Microscopia de Fluorescência/métodos , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , DNA/metabolismo , Replicação do DNA/efeitos dos fármacos , Humanos , Soluções Hipotônicas/farmacologia , Camundongos , Nucleotídeos/metabolismo , Coloração e Rotulagem
8.
Cold Spring Harb Perspect Biol ; 2(4): a000737, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20452942

RESUMO

The discovery of the DNA double helix structure half a century ago immediately suggested a mechanism for its duplication by semi-conservative copying of the nucleotide sequence into two DNA daughter strands. Shortly after, a second fundamental step toward the elucidation of the mechanism of DNA replication was taken with the isolation of the first enzyme able to polymerize DNA from a template. In the subsequent years, the basic mechanism of DNA replication and its enzymatic machinery components were elucidated, mostly through genetic approaches and in vitro biochemistry. Most recently, the spatial and temporal organization of the DNA replication process in vivo within the context of chromatin and inside the intact cell are finally beginning to be elucidated. On the one hand, recent advances in genome-wide high throughput techniques are providing a new wave of information on the progression of genome replication at high spatial resolution. On the other hand, novel super-resolution microscopy techniques are just starting to give us the first glimpses of how DNA replication is organized within the context of single intact cells with high spatial resolution. The integration of these data with time lapse microscopy analysis will give us the ability to film and dissect the replication of the genome in situ and in real time.


Assuntos
Replicação do DNA/fisiologia , Genoma , Animais , Ciclo Celular/genética , Epigênese Genética , Humanos , Microscopia Confocal
9.
Artigo em Inglês | MEDLINE | ID: mdl-19964529

RESUMO

Fluorescently tagged proteins such as GFP-PCNA produce rich dynamically varying textural patterns of foci distributed in the nucleus. This enables the behavioral study of sub-cellular structures during different phases of the cell cycle. The varying punctuate patterns of fluorescence, drastic changes in SNR, shape and position during mitosis and abundance of touching cells, however, require more sophisticated algorithms for reliable automatic cell segmentation and lineage analysis. Since the cell nuclei are non-uniform in appearance, a distribution-based modeling of foreground classes is essential. The recently proposed graph partitioning active contours (GPAC) algorithm supports region descriptors and flexible distance metrics. We extend GPAC for fluorescence-based cell segmentation using regional density functions and dramatically improve its efficiency for segmentation from O(N(4)) to O(N(2)), for an image with N(2) pixels, making it practical and scalable for high throughput microscopy imaging studies.


Assuntos
Microscopia de Fluorescência/métodos , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares/metabolismo , Algoritmos , Ciclo Celular , Núcleo Celular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Microscopia de Fluorescência/estatística & dados numéricos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores de Tempo
10.
Med Image Comput Comput Assist Interv ; 12(Pt 2): 617-24, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-20426163

RESUMO

Current chemical biology methods for studying spatiotemporal correlation between biochemical networks and cell cycle phase progression in live-cells typically use fluorescence-based imaging of fusion proteins. Stable cell lines expressing fluorescently tagged protein GFP-PCNA produce rich, dynamically varying sub-cellular foci patterns characterizing the cell cycle phases, including the progress during the S-phase. Variable fluorescence patterns, drastic changes in SNR, shape and position changes and abundance of touching cells require sophisticated algorithms for reliable automatic segmentation and cell cycle classification. We extend the recently proposed graph partitioning active contours (GPAC) for fluorescence-based nucleus segmentation using regional density functions and dramatically improve its efficiency, making it scalable for high content microscopy imaging. We utilize surface shape properties of GFP-PCNA intensity field to obtain descriptors of foci patterns and perform automated cell cycle phase classification, and give quantitative performance by comparing our results to manually labeled data.


Assuntos
Algoritmos , Ciclo Celular , Núcleo Celular/ultraestrutura , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Reconhecimento Automatizado de Padrão/métodos , Células HeLa , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
11.
Chromosome Res ; 15(6): 787-97, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17874213

RESUMO

Double-strand DNA breaks (DSBs) induced by ionizing radiation can be visualized in human cells using antibodies against Ser-139 phosphorylated histone H2AX (gamma-H2AX). Large gamma-H2AX foci are seen in the nucleus fixed 1 hour after irradiation and their number corresponds to the number of DSBs, allowing analysis of these genome lesions after low doses. We estimated whether transcription is affected in chromatin domains containing gamma-H2AX by following in vivo incorporation of 5-bromouridine triphosphate (BrUTP) loaded by cell scratching (run-on assay). We found that BrUTP incorporation is strongly suppressed at gamma-H2AX foci, suggesting that H2AX phosphorylation inhibits transcription. This is not caused by preferential association of gamma-H2AX foci with constitutive or facultative heterochromatin, which was visualized in irradiated cells using antibodies against histone H3 trimethylated at lysine-9 (H3-K9m3) or histone H3 trimethylated at lysine-27 (H3-K27m3). Apparently, formation of gamma-H2AX induces changes of chromatin that inhibit assembly of transcription complexes without heterochromatin formation. Inhibition of transcription by phosphorylation of histone H2AX can decrease chromatin movement at DSBs and frequency of misjoining of DNA ends.


Assuntos
Núcleo Celular/metabolismo , Histonas/metabolismo , Transcrição Gênica , Bromodesoxiuridina/farmacologia , Linhagem Celular Tumoral , Metilação de DNA , Nucleotídeos de Desoxiuracil/metabolismo , Fibroblastos/metabolismo , Histonas/química , Humanos , Microscopia Confocal , Fosforilação
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