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1.
Chromatin dynamics in interphase cells revealed by tracking in a two-photon excitation microscope
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Increasing evidence points to a dynamical compartmentalization of the cell nucleus, yet the mechanisms by which interphase chromatin moves and is positioned within nuclei remain unclear. Here, we study the dynamics of chromatin in vivo applying a novel particle-tracking method in a two-photon microscope that provides approximately 10-fold higher spatial and temporal resolutions than previous measurements. We followed the motion of a chromatin sequence containing a lac-operator repeat in cells stably expressing lac repressor fused with enhanced green fluorescent protein, observing long periods of apparent constrained diffusion interrupted by relatively abrupt jumps of approximately 150 nm lasting 0.3-2 s. During these jumps, the particle moved an average of four times faster than in the periods between jumps and in paths more rectilinear than predicted for random diffusion motion. Additionally, the jumps were sensitive to the temperature and absent after ATP depletion. These experimental results point to an energy-dependent mechanism driving fast motion of chromatin in interphase cells. 相似文献
2.
Fereydoon Taheri Buse Isbilir Gabriele Müller Jan W. Krieger Giuseppe Chirico Jörg Langowski Katalin Tóth 《Biophysical journal》2018,114(10):2465-2472
Using fluorescence correlation spectroscopy in single-plane illumination microscopy, we investigated the dynamics of chromatin in interphase mouse adult fibroblast cell nuclei under the influence of the intermediate filament protein lamin A. We find that 1) lamin A-eGFP and histone H2A-mRFP show significant comobility, indicating that their motions are clearly interconnected in the nucleus, and 2) that the random motion of histones H2A within the chromatin network is subdiffusive, i.e., the effective diffusion coefficient decreases for slow timescales. Knocking out lamin A changes the diffusion back to normal. Thus, lamin A influences the dynamics of the entire chromatin network. Our conclusion is that lamin A plays a central role in determining the viscoelasticity of the chromatin network and helping to maintain local ordering of interphase chromosomes. 相似文献
3.
W.F A Straight J.F Marko J Swedlow A Dernburg A Belmont A.W Murray D.A Agard J.W Sedat 《Current biology : CB》1997,7(12)
Background: Structural studies of fixed cells have revealed that interphase chromosomes are highly organized into specific arrangements in the nucleus, and have led to a picture of the nucleus as a static structure with immobile chromosomes held in fixed positions, an impression apparently confirmed by recent photobleaching studies. Functional studies of chromosome behavior, however, suggest that many essential processes, such as recombination, require interphase chromosomes to move around within the nucleus.Results: To reconcile these contradictory views, we exploited methods for tagging specific chromosome sites in living cells of Saccharomyces cerevisiae with green fluorescent protein and in Drosophila melanogaster with fluorescently labeled topoisomerase ll. Combining these techniques with submicrometer single-particle tracking, we directly measured the motion of interphase chromatin, at high resolution and in three dimensions. We found that chromatin does indeed undergo significant diffusive motion within the nucleus, but this motion is constrained such that a given chromatin segment is free to move within only a limited subregion of the nucleus. Chromatin diffusion was found to be insensitive to metabolic inhibitors, suggesting that it results from classical Brownian motion rather than from active motility. Nocodazole greatly reduced chromatin confinement, suggesting a role for the cytoskeleton in the maintenance of nuclear architecture.Conclusions: We conclude that chromatin is free to undergo substantial Brownian motion, but that a given chromatin segment is confined to a subregion of the nucleus. This constrained diffusion is consistent with a highly defined nuclear architecture, but also allows enough motion for processes requiring chromosome motility to take place. These results lead to a model for the regulation of chromosome interactions by nuclear architecture. 相似文献
4.
Vesa Aho Sami Salminen Salla Mattola Alka Gupta Felix Flomm Beate Sodeik Jens B. Bosse Maija Vihinen-Ranta 《PLoS pathogens》2021,17(12)
Herpes simplex virus capsids are assembled and packaged in the nucleus and move by diffusion through the nucleoplasm to the nuclear envelope for egress. Analyzing their motion provides conclusions not only on capsid transport but also on the properties of the nuclear environment during infection. We utilized live-cell imaging and single-particle tracking to characterize capsid motion relative to the host chromatin. The data indicate that as the chromatin was marginalized toward the nuclear envelope it presented a restrictive barrier to the capsids. However, later in infection this barrier became more permissive and the probability of capsids to enter the chromatin increased. Thus, although chromatin marginalization initially restricted capsid transport to the nuclear envelope, a structural reorganization of the chromatin counteracted that to promote capsid transport later. Analyses of capsid motion revealed that it was subdiffusive, and that the diffusion coefficients were lower in the chromatin than in regions lacking chromatin. In addition, the diffusion coefficient in both regions increased during infection. Throughout the infection, the capsids were never enriched at the nuclear envelope, which suggests that instead of nuclear export the transport through the chromatin is the rate-limiting step for the nuclear egress of capsids. This provides motivation for further studies by validating the importance of intranuclear transport to the life cycle of HSV-1. 相似文献
5.
Misteli T 《Histochemistry and cell biology》2008,129(1):5-11
Trafficking of proteins and RNAs is essential for cellular function and homeostasis. While it has long been appreciated that
proteins and RNAs move within cells, only recently has it become possible to visualize trafficking events in vivo. Analysis
of protein and RNA motion within the cell nucleus have been particularly intriguing as they have revealed an unanticipated
degree of dynamics within the organelle. These methods have revealed that the intranuclear trafficking occurs largely by energy-independent
mechanisms and is driven by diffusion. RNA molecules and non-DNA binding proteins undergo constrained diffusion, largely limited
by the spatial constraint imposed by chromatin, and chromatin binding proteins move by a stop-and-go mechanism where their
free diffusion is interrupted by random association with the chromatin fiber. The ability and mode of motion of proteins and
RNAs has implications for how they find nuclear targets on chromatin and in nuclear subcompartments and how macromolecular
complexes are assembled in vivo. Most importantly, the dynamic nature of proteins and RNAs is emerging as a means to control
physiological cellular responses and pathways. 相似文献
6.
Chromatin dynamics play a major role in regulating genetic processes. Now, accumulating data suggest that chromatin structure may also affect the mechanical properties of the nucleus and cell migration. Global chromatin organization appears to modulate the shape, the size and the stiffness of the nucleus. Directed-cell migration, which often requires nuclear reshaping to allow passage of cells through narrow openings, is dependent not only on changes in cytoskeletal elements but also on global chromatin condensation. Conceivably, during cell migration a physical link between the chromatin and the cytoskeleton facilitates coordinated structural changes in these two components. Thus, in addition to regulating genetic processes, we suggest that alterations in chromatin structure could facilitate cellular reorganizations necessary for efficient migration. 相似文献
7.
《Biophysical journal》2022,121(14):2684-2692
8.
Marshall WF 《Current biology : CB》2002,12(5):R185-R192
Fluorescence in situ hybridization combined with three-dimensional microscopy has shown that chromosomes are not randomly strewn throughout the nucleus but are in fact fairly well organized, with different loci reproducibly found in different regions of the nucleus. At the same time, increasingly sophisticated methods to track and analyze the movements of specific chromosomal loci in vivo using four-dimensional microscopy have revealed that chromatin undergoes extensive Brownian motion. However, the diffusion of interphase chromatin is constrained, implying that chromosomes are physically anchored within the nucleus. This constraint on diffusion is the result of interactions between chromatin and structural elements within the nucleus, such as nuclear pores or the nuclear lamina. The combination of defined positioning with constrained diffusion has a strong impact on interactions between chromosomal loci, and appears to explain the tendency of certain chromosome rearrangements to occur during the development of cancer. 相似文献
9.
The diffusive motion of DNA-containing chromatin in live cells and isolated nuclei is investigated using a two-photon standing wave fluorescence photobleaching experiment with 100 nm spatial resolution. The chromatin is labeled using the minor groove binding dye Hoechst 33342. In live cells, the mean diffusion rate is 5 × 10−4 μm2/s, with considerable cell-to-cell variation. This diffusion is highly constrained and cannot be observed in a standard, single beam fluorescence recovery after photobleaching experiment. To determine the chemical origin of the diffusion, we study motion in isolated nuclei and vary the strength of the histone-DNA interactions by changing the ionic strength and using chemical and photocross-linking experiments. At higher NaCl concentrations, we see increased chromatin diffusion as the histone-DNA interaction is weakened due to ionic screening, whereas photocross-linking the core histones to the DNA results in a complete absence of diffusive motion. These trends are consistent with the 100 nm scale motion being correlated with the interactions of histone proteins with the DNA. If chromatin diffusion is connected to the nucleosomal dynamics on much smaller length scales, this may provide a way to assay biochemical activity in vivo based on larger scale macromolecular dynamics observed via fluorescence microscopy. 相似文献
10.
The effect of chromatin organization on EGFP-tagged histone protein dynamics within the cell nucleus has been probed using fluorescence correlation and recovery measurements on single living HeLa cells. Our studies reveal that free fraction of core-particle histones exist as multimers within the cell nucleus whereas the linker histones exist in monomeric forms. The multimeric state of core histones is found to be invariant across mammalian and polytene chromosomes and this is ATP dependent. In contrast, the dynamics of the linker histones exhibits two distinct diffusion timescales corresponding to its transient binding and unbinding to chromatin governed by the tail domain residues. Under conditions of chromatin condensation induced by apoptosis, the free multimeric fraction of core histones is found to become immobile, while the monomeric linker histone mobility is partially reduced. In addition, we observe differences in nuclear colocalization of linker and core particle histones. These results are validated through Brownian dynamics simulation of core and linker histone mobility. Our findings provide a framework to understand the coupling between the state of chromatin assembly and histone protein dynamics that is central to accessing regulatory sites on the genome. 相似文献
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Dushyant Mehra Santosh Adhikari Chiranjib Banerjee Elias M Puchner 《Nucleic acids research》2022,50(13):e78
The dynamic rearrangement of chromatin is critical for gene regulation, but mapping both the spatial organization of chromatin and its dynamics remains a challenge. Many structural conformations are too small to be resolved via conventional fluorescence microscopy and the long acquisition time of super-resolution photoactivated localization microscopy (PALM) precludes the structural characterization of chromatin below the optical diffraction limit in living cells due to chromatin motion. Here we develop a correlative conventional fluorescence and PALM imaging approach to quantitatively map time-averaged chromatin structure and dynamics below the optical diffraction limit in living cells. By assigning localizations to a locus as it moves, we reliably discriminate between bound and unbound dCas9 molecules, whose mobilities overlap. Our approach accounts for changes in DNA mobility and relates local chromatin motion to larger scale domain movement. In our experimental system, we show that compacted telomeres move faster and have a higher density of bound dCas9 molecules, but the relative motion of those molecules is more restricted than in less compacted telomeres. Correlative conventional and PALM imaging therefore improves the ability to analyze the mobility and time-averaged nanoscopic structural features of locus specific chromatin with single molecule sensitivity and yields unprecedented insights across length and time scales. 相似文献
13.
Anna-Karin Gustavsson Rajarshi P. Ghosh Petar N. Petrov Jan T. Liphardt W. E. Moerner 《Molecular biology of the cell》2022,33(6)
Chromatin organization and dynamics are critical for gene regulation. In this work we present a methodology for fast and parallel three-dimensional (3D) tracking of multiple chromosomal loci of choice over many thousands of frames on various timescales. We achieved this by developing and combining fluorogenic and replenishable nanobody arrays, engineered point spread functions, and light sheet illumination. The result is gentle live-cell 3D tracking with excellent spatiotemporal resolution throughout the mammalian cell nucleus. Correction for both sample drift and nuclear translation facilitated accurate long-term tracking of the chromatin dynamics. We demonstrate tracking both of fast dynamics (50 Hz) and over timescales extending to several hours, and we find both large heterogeneity between cells and apparent anisotropy in the dynamics in the axial direction. We further quantify the effect of inhibiting actin polymerization on the dynamics and find an overall increase in both the apparent diffusion coefficient D* and anomalous diffusion exponent α and a transition to more-isotropic dynamics in 3D after such treatment. We think that in the future our methodology will allow researchers to obtain a better fundamental understanding of chromatin dynamics and how it is altered during disease progression and after perturbations of cellular function. 相似文献
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15.
Nucleosomes are the ubiquitous and fundamental packaging for eukaryotic genomes, and are the substrate for many processes in the nucleus. Nucleosomes are not static entities but can readily be moved by thermal energy and ATP-dependent chromatin remodeling complexes in a process known as sliding or shifting. We summarize from a mechanical perspective the twist defect and bulge diffusion mechanisms proposed as the most likely pathway for nucleosome mobilization. We then consider the elastic properties of DNA and how this affects the potential for each mechanism, concentrating on kinetic aspects of twist diffusion and possible planar bulge sizes and summarize the experimental evidence reflecting on each. Either, or both, mechanisms could occur, and careful experimentation focusing on their uniquely distinguishing features will be required to determine their relative contributions to chromatin dynamics. 相似文献
16.
We present a quantitative fluctuation-based assay to measure the degree of local chromatin compaction and investigate how chromatin density regulates the diffusive path adopted by an inert protein in dividing cells. The assay uses CHO-K1 cells coexpressing untagged enhanced green fluorescent protein (EGFP) and histone H2B tagged mCherry. We measure at the single-cell level the EGFP localization and molecular flow patterns characteristic of each stage of chromatin compaction from mitosis through interphase by means of pair-correlation analysis. We find that the naturally occurring changes in chromatin organization impart a regulation on the spatial distribution and temporal dynamics of EGFP within the nucleus. Combined with the analysis of Ca(2+) intracellular homeostasis during cell division, EGFP flow regulation can be interpreted as the result of controlled changes in chromatin compaction. For the first time, to our knowledge, we were able to probe chromatin compaction on the micrometer scale, where the regulation of molecular diffusion may become relevant for many cellular processes. 相似文献
17.
S. S. Bogachev E. V. Likhacheva I. V. Borisevich E. B. Kokoza 《Russian Journal of Developmental Biology》2000,31(4):197-203
In this article, we attempt to analyze the relationship between the processes which occur in the nucleus and the dynamics
of chromatin, as well as to classify the changes in the position of chromatin in the cell nucleus during the lifetime of the
cell. The proposed concept integrates possible types of chromatin movement within the nucleus. 相似文献
18.
In this article we attempt to analyze the relationship between the processes, which occur in the nucleus, and the dynamics of chromatin, as well as to classify the changes in the position of chromatin in the cell nucleus during the lifetime of the cell. The proposed concept integrates possible types of chromatin movement within the nucleus. 相似文献
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真核生物的DNA以染色质形式通过逐级折叠压缩形成高级结构存在于细胞核中。染色质高级结构直接参与了真核基因的转录调控和其它与DNA相关的生物学事件,因此研究染色质高级结构对了解表观遗传学分子机制有着至关重要的作用。近些年,研究者们针对30 nm染色质高级结构提出了两个模型:螺线管模型和Zig-Zag模型。2014年,我们利用体外染色质组装体系重建了30 nm染色质纤维,运用高精度冷冻电镜技术得到了分辨率为11?的30 nm染色质纤维的精细结构,提出了30 nm染色质高级结构的左手双螺旋Zig-Zag模型。本文综述了30 nm染色质纤维结构研究方面的相关进展,并对30 nm染色质高级结构的表观遗传调控机理以及单分子成像和操纵技术在研究30 nm染色质高级结构中潜在的应用作出讨论和展望。 相似文献