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1.
Nucleic Acids Res ; 48(21): e122, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33053171

RESUMO

Protein-protein interactions are essential to ensure timely and precise recruitment of chromatin remodellers and repair factors to DNA damage sites. Conventional analyses of protein-protein interactions at a population level may mask the complexity of interaction dynamics, highlighting the need for a method that enables quantification of DNA damage-dependent interactions at a single-cell level. To this end, we integrated a pulsed UV laser on a confocal fluorescence lifetime imaging (FLIM) microscope to induce localized DNA damage. To quantify protein-protein interactions in live cells, we measured Förster resonance energy transfer (FRET) between mEGFP- and mCherry-tagged proteins, based on the fluorescence lifetime reduction of the mEGFP donor protein. The UV-FLIM-FRET system offers a unique combination of real-time and single-cell quantification of DNA damage-dependent interactions, and can distinguish between direct protein-protein interactions, as opposed to those mediated by chromatin proximity. Using the UV-FLIM-FRET system, we show the dynamic changes in the interaction between poly(ADP-ribose) polymerase 1, amplified in liver cancer 1, X-ray repair cross-complementing protein 1 and tripartite motif containing 33 after DNA damage. This new set-up complements the toolset for studying DNA damage response by providing single-cell quantitative and dynamic information about protein-protein interactions at DNA damage sites.


Assuntos
Osteoblastos/efeitos da radiação , Poli(ADP-Ribose) Polimerase-1/genética , Mapeamento de Interação de Proteínas/métodos , Fatores de Transcrição/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Cromatina/efeitos da radiação , Dano ao DNA , Transferência Ressonante de Energia de Fluorescência , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lasers , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Imagem Óptica , Osteoblastos/citologia , Osteoblastos/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ligação Proteica , Transdução de Sinais , Análise de Célula Única , Fatores de Transcrição/metabolismo , Raios Ultravioleta , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Proteína Vermelha Fluorescente
2.
BMC Biol ; 19(1): 136, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215263

RESUMO

BACKGROUND: Quantitative imaging of epithelial tissues requires bioimage analysis tools that are widely applicable and accurate. In the case of imaging 3D tissues, a common preprocessing step consists of projecting the acquired 3D volume on a 2D plane mapping the tissue surface. While segmenting the tissue cells is amenable on 2D projections, it is still very difficult and cumbersome in 3D. However, for many specimen and models used in developmental and cell biology, the complex content of the image volume surrounding the epithelium in a tissue often reduces the visibility of the biological object in the projection, compromising its subsequent analysis. In addition, the projection may distort the geometry of the tissue and can lead to strong artifacts in the morphology measurement. RESULTS: Here we introduce a user-friendly toolbox built to robustly project epithelia on their 2D surface from 3D volumes and to produce accurate morphology measurement corrected for the projection distortion, even for very curved tissues. Our toolbox is built upon two components. LocalZProjector is a configurable Fiji plugin that generates 2D projections and height-maps from potentially large 3D stacks (larger than 40 GB per time-point) by only incorporating signal of the planes with local highest variance/mean intensity, despite a possibly complex image content. DeProj is a MATLAB tool that generates correct morphology measurements by combining the height-map output (such as the one offered by LocalZProjector) and the results of a cell segmentation on the 2D projection, hence effectively deprojecting the 2D segmentation in 3D. In this paper, we demonstrate their effectiveness over a wide range of different biological samples. We then compare its performance and accuracy against similar existing tools. CONCLUSIONS: We find that LocalZProjector performs well even in situations where the volume to project also contains unwanted signal in other layers. We show that it can process large images without a pre-processing step. We study the impact of geometrical distortions on morphological measurements induced by the projection. We measured very large distortions which are then corrected by DeProj, providing accurate outputs.


Assuntos
Imageamento Tridimensional , Microscopia
3.
EMBO J ; 36(17): 2595-2608, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28694242

RESUMO

DNA double-strand breaks (DSBs) induce a cellular response that involves histone modifications and chromatin remodeling at the damaged site and increases chromosome dynamics both locally at the damaged site and globally in the nucleus. In parallel, it has become clear that the spatial organization and dynamics of chromosomes can be largely explained by the statistical properties of tethered, but randomly moving, polymer chains, characterized mainly by their rigidity and compaction. How these properties of chromatin are affected during DNA damage remains, however, unclear. Here, we use live cell microscopy to track chromatin loci and measure distances between loci on yeast chromosome IV in thousands of cells, in the presence or absence of genotoxic stress. We confirm that DSBs result in enhanced chromatin subdiffusion and show that intrachromosomal distances increase with DNA damage all along the chromosome. Our data can be explained by an increase in chromatin rigidity, but not by chromatin decondensation or centromeric untethering only. We provide evidence that chromatin stiffening is mediated in part by histone H2A phosphorylation. Our results support a genome-wide stiffening of the chromatin fiber as a consequence of DNA damage and as a novel mechanism underlying increased chromatin mobility.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Histonas/metabolismo , Saccharomycetales/genética , Bleomicina/farmacologia , DNA Fúngico/genética , Mutagênicos/farmacologia , Fosforilação , Saccharomycetales/efeitos dos fármacos , Saccharomycetales/metabolismo
4.
Nucleic Acids Res ; 45(16): 9741-9759, 2017 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-28934471

RESUMO

Poly(ADP-ribose) glycohydrolase (PARG) regulates cellular poly(ADP-ribose) (PAR) levels by rapidly cleaving glycosidic bonds between ADP-ribose units. PARG interacts with proliferating cell nuclear antigen (PCNA) and is strongly recruited to DNA damage sites in a PAR- and PCNA-dependent fashion. Here we identified PARG acetylation site K409 that is essential for its interaction with PCNA, its localization within replication foci and its recruitment to DNA damage sites. We found K409 to be part of a non-canonical PIP-box within the PARG disordered regulatory region. The previously identified putative N-terminal PIP-box does not bind PCNA directly but contributes to PARG localization within replication foci. X-ray structure and MD simulations reveal that the PARG non-canonical PIP-box binds PCNA in a manner similar to other canonical PIP-boxes and may represent a new type of PIP-box. While the binding of previously described PIP-boxes is based on hydrophobic interactions, PARG PIP-box binds PCNA via both stabilizing hydrophobic and fine-tuning electrostatic interactions. Our data explain the mechanism of PARG-PCNA interaction through a new PARG PIP-box that exhibits non-canonical sequence properties but a canonical mode of PCNA binding.


Assuntos
Glicosídeo Hidrolases/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Acetilação , Calorimetria/métodos , Cromatina/metabolismo , Cristalografia por Raios X , Dano ao DNA , Transferência Ressonante de Energia de Fluorescência , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Células HeLa , Humanos , Imunoprecipitação , Lasers , Lisina/genética , Lisina/metabolismo , Simulação de Dinâmica Molecular , Antígeno Nuclear de Célula em Proliferação/química , Conformação Proteica , Fase S/genética , Eletricidade Estática
5.
J Cell Sci ; 129(4): 681-92, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26763908

RESUMO

Eukaryotic chromosomes undergo movements that are involved in the regulation of functional processes such as DNA repair. To better understand the origin of these movements, we used fluorescence microscopy, image analysis and chromosome conformation capture to quantify the actin contribution to chromosome movements and interactions in budding yeast. We show that both the cytoskeletal and nuclear actin drive local chromosome movements, independently of Csm4, a putative LINC protein. Inhibition of actin polymerization reduces subtelomere dynamics, resulting in more confined territories and enrichment in subtelomeric contacts. Artificial tethering of actin to nuclear pores increased both nuclear pore complex (NPC) and subtelomere motion. Chromosome loci that were positioned away from telomeres exhibited reduced motion in the presence of an actin polymerization inhibitor but were unaffected by the lack of Csm4. We further show that actin was required for locus mobility that was induced by targeting the chromatin-remodeling protein Ino80. Correlated with this, DNA repair by homologous recombination was less efficient. Overall, interphase chromosome dynamics are modulated by the additive effects of cytoskeletal actin through forces mediated by the nuclear envelope and nuclear actin, probably through the function of actin in chromatin-remodeling complexes.


Assuntos
Actinas/fisiologia , Cromossomos Fúngicos/fisiologia , Saccharomyces cerevisiae/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas de Membrana/fisiologia , Poro Nuclear/metabolismo , Multimerização Proteica , Transporte Proteico , Reparo de DNA por Recombinação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Telômero/genética , Telômero/metabolismo
6.
Sci Adv ; 9(35): eadg7519, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37656795

RESUMO

The maintenance of neural stem cells (NSCs) in the adult brain depends on their activation frequency and division mode. Using long-term intravital imaging of NSCs in the zebrafish adult telencephalon, we reveal that apical surface area and expression of the Notch ligand DeltaA predict these NSC decisions. deltaA-negative NSCs constitute a bona fide self-renewing NSC pool and systematically engage in asymmetric divisions generating a self-renewing deltaAneg daughter, which regains the size and behavior of its mother, and a neurogenic deltaApos daughter, eventually engaged in neuronal production following further quiescence-division phases. Pharmacological and genetic manipulations of Notch, DeltaA, and apical size further show that the prediction of activation frequency by apical size and the asymmetric divisions of deltaAneg NSCs are functionally independent of Notch. These results provide dynamic qualitative and quantitative readouts of NSC lineage progression in vivo and support a hierarchical organization of NSCs in differently fated subpopulations.


Assuntos
Células-Tronco Neurais , Peixe-Zebra , Animais , Neurônios/fisiologia , Divisão Celular , Neurogênese
7.
Microsc Microanal ; 18(6): 1419-29, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23113972

RESUMO

For over a decade fluorescence microscopy has demonstrated the capacity to achieve single-molecule localization accuracies of a few nanometers, well below the ≈ 200 nm lateral and ≈ 500 nm axial resolution limit of conventional microscopy. Yet, only the recent development of new fluorescence labeling modalities, the increase in sensitivity of imaging hardware, and the creation of novel image analysis tools allow for the emergence of single-molecule-based super-resolution imaging techniques. Novel methods such as photoactivated localization microscopy and stochastic optical reconstruction microscopy can typically reach a tenfold increase in resolution compared to standard microscopy methods. Their implementation is relatively easy only requiring minimal changes to a conventional wide-field or total internal reflection fluorescence microscope. The recent translation of these two methods into commercial imaging systems has made them further accessible to researchers in biology. However, these methods are still evolving rapidly toward imaging live samples with high temporal resolution and depth. In this review, we recall the roots of single-molecule localization microscopy, summarize major recent developments, and offer perspective on potential applications.

8.
Cell Stem Cell ; 28(8): 1457-1472.e12, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-33823144

RESUMO

Neural stem cell (NSC) populations persist in the adult vertebrate brain over a lifetime, and their homeostasis is controlled at the population level through unknown mechanisms. Here, we combine dynamic imaging of entire NSC populations in their in vivo niche over several weeks with pharmacological manipulations, mathematical modeling, and spatial statistics and demonstrate that NSCs use spatiotemporally resolved local feedback signals to coordinate their decision to divide in adult zebrafish brains. These involve Notch-mediated short-range inhibition from transient neural progenitors and a dispersion effect from the dividing NSCs themselves exerted with a delay of 9-12 days. Simulations from a stochastic NSC lattice model capturing these interactions demonstrate that these signals are linked by lineage progression and control the spatiotemporal distribution of output neurons. These results highlight how local and temporally delayed interactions occurring between brain germinal cells generate self-propagating dynamics that maintain NSC population homeostasis and coordinate specific spatiotemporal correlations.


Assuntos
Células-Tronco Neurais , Neurogênese , Animais , Encéfalo , Proliferação de Células , Retroalimentação , Peixe-Zebra
9.
Nucleus ; 9(1): 474-491, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30205747

RESUMO

Fluorescence microscopy in combination with the induction of localized DNA damage using focused light beams has played a major role in the study of protein recruitment kinetics to DNA damage sites in recent years. Currently published methods are dedicated to the study of single fluorophore/single protein kinetics. However, these methods may be limited when studying the relative recruitment dynamics between two or more proteins due to cell-to-cell variability in gene expression and recruitment kinetics, and are not suitable for comparative analysis of fast-recruiting proteins. To tackle these limitations, we have established a time-lapse fluorescence microscopy method based on simultaneous dual-channel acquisition following UV-A-induced local DNA damage coupled with a standardized image and recruitment analysis workflow. Simultaneous acquisition is achieved by spectrally splitting the emitted light into two light paths, which are simultaneously imaged on two halves of the same camera chip. To validate this method, we studied the recruitment of poly(ADP-ribose) polymerase 1 (PARP1), poly (ADP-ribose) glycohydrolase (PARG), proliferating cell nuclear antigen (PCNA) and the chromatin remodeler ALC1. In accordance with the published data based on single fluorophore imaging, simultaneous dual-channel imaging revealed that PARP1 regulates fast recruitment and dissociation of PARG and that in PARP1-depleted cells PARG and PCNA are recruited with comparable kinetics. This approach is particularly advantageous for analyzing the recruitment sequence of fast-recruiting proteins such as PARP1 and ALC1, and revealed that PARP1 is recruited faster than ALC1. Split-view imaging can be incorporated into any laser microirradiation-adapted microscopy setup together with a recruitment-dedicated image analysis package.


Assuntos
Dano ao DNA , DNA Helicases/análise , Proteínas de Ligação a DNA/análise , Glicosídeo Hidrolases/análise , Lasers , Imagem Óptica , Poli(ADP-Ribose) Polimerase-1/análise , Antígeno Nuclear de Célula em Proliferação/análise , Raios Ultravioleta , Linhagem Celular Tumoral , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Glicosídeo Hidrolases/metabolismo , Humanos , Cinética , Microscopia de Fluorescência , Poli(ADP-Ribose) Polimerase-1/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo
10.
Elife ; 72018 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-30132756

RESUMO

Hematopoiesis leads to the formation of blood and immune cells. Hematopoietic stem cells emerge during development, from vascular components, via a process called the endothelial-to-hematopoietic transition (EHT). Here, we reveal essential biomechanical features of the EHT, using the zebrafish embryo imaged at unprecedented spatio-temporal resolution and an algorithm to unwrap the aorta into 2D-cartography. We show that the transition involves anisotropic contraction along the antero-posterior axis, with heterogenous organization of contractile circumferential actomyosin. The biomechanics of the contraction is oscillatory, with unusually long periods in comparison to other apical constriction mechanisms described so far in morphogenesis, and is supported by the anisotropic reinforcement of junctional contacts. Finally, we show that abrogation of blood flow impairs the actin cytoskeleton, the morphodynamics of EHT cells, and the orientation of the emergence. Overall, our results underline the peculiarities of the EHT biomechanics and the influence of the mechanical forces exerted by blood flow.


Assuntos
Actomiosina/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Peixe-Zebra/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Anisotropia , Fenômenos Biomecânicos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/citologia , Hemodinâmica , Junções Intercelulares/metabolismo , Modelos Biológicos , Mutação/genética , Cadeias Leves de Miosina/metabolismo , Fenótipo , Fosforilação , Fatores de Tempo
12.
Genome Biol ; 18(1): 81, 2017 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-28468672

RESUMO

BACKGROUND: The structure and mechanical properties of chromatin impact DNA functions and nuclear architecture but remain poorly understood. In budding yeast, a simple polymer model with minimal sequence-specific constraints and a small number of structural parameters can explain diverse experimental data on nuclear architecture. However, how assumed chromatin properties affect model predictions was not previously systematically investigated. RESULTS: We used hundreds of dynamic chromosome simulations and Bayesian inference to determine chromatin properties consistent with an extensive dataset that includes hundreds of measurements from imaging in fixed and live cells and two Hi-C studies. We place new constraints on average chromatin fiber properties, narrowing down the chromatin compaction to ~53-65 bp/nm and persistence length to ~52-85 nm. These constraints argue against a 20-30 nm fiber as the exclusive chromatin structure in the genome. Our best model provides a much better match to experimental measurements of nuclear architecture and also recapitulates chromatin dynamics measured on multiple loci over long timescales. CONCLUSION: This work substantially improves our understanding of yeast chromatin mechanics and chromosome architecture and provides a new analytic framework to infer chromosome properties in other organisms.


Assuntos
Núcleo Celular/genética , Montagem e Desmontagem da Cromatina , Cromatina/química , Simulação por Computador , Saccharomyces cerevisiae/genética , Teorema de Bayes , Cromatina/genética
13.
Curr Biol ; 22(20): 1881-90, 2012 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-22940469

RESUMO

BACKGROUND: Despite the absence of internal membranes, the nucleus of eukaryotic cells is spatially organized, with chromosomes and individual loci occupying dynamic, but nonrandom, spatial positions relative to nuclear landmarks and to each other. These positional preferences correlate with gene expression and DNA repair, recombination, and replication. Yet the principles that govern nuclear organization remain poorly understood and detailed predictive models are lacking. RESULTS: We present a computational model of dynamic chromosome configurations in the interphase yeast nucleus that is based on first principles and is able to statistically predict the positioning of any locus in nuclear space. Despite its simplicity, the model agrees with extensive previous and new measurements on locus positioning and with genome-wide DNA contact frequencies. Notably, our model recapitulates the position and morphology of the nucleolus, the observed variations in locus positions, and variations in contact frequencies within and across chromosomes, as well as subchromosomal contact features. The model is also able to correctly predict nuclear reorganization accompanying a reduction in ribosomal DNA transcription, and sites of chromosomal rearrangements tend to occur where the model predicted high contact frequencies. CONCLUSIONS: Our results suggest that large-scale yeast nuclear architecture can be largely understood as a consequence of generic properties of crowded polymers rather than of specific DNA-binding factors and that configurations of chromosomes and DNA contacts are dictated mainly by genomic location and chromosome lengths. Our model provides a quantitative framework to understand and predict large-scale spatial genome organization and its interplay with functional processes.


Assuntos
Núcleo Celular/fisiologia , Núcleo Celular/ultraestrutura , Cromossomos Fúngicos , Biologia Computacional/métodos , Interfase , Saccharomyces cerevisiae/fisiologia , Núcleo Celular/genética , Cromatina , Simulação por Computador , Replicação do DNA , Região Organizadora do Nucléolo/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Análise de Sequência de DNA , Transcrição Gênica
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