34 research outputs found
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RNA, Genome Output and Input
RNA, the transcriptional output of genomes, not only templates protein synthesis or directly engages in catalytic functions, but can feed back to the genome and serve as regulatory input for gene expression. Transcripts affecting the RNA abundance of other genes act by mechanisms similar to and in concert with protein factors that control transcription. Through recruitment or blocking of activating and silencing complexes to specific genomic loci, RNA and protein factors can favor transcription or lower the local gene expression potential. Most regulatory proteins enter nuclei from all directions to start the search for increased affinity to specific DNA sequences or to other proteins nearby genuine gene targets. In contrast, RNAs emerge from spatial point sources within nuclei, their encoding genes. A transcriptional burst can result in the local appearance of multiple nascent RNA copies at once, in turn increasing local nucleic acid density and RNA motif abundance before diffusion into the nuclear neighborhood. The confined initial localization of regulatory RNAs causing accumulation of protein co-factors raises the intriguing possibility that target specificity of non-coding, and probably coding, RNAs is achieved through gene/RNA positioning and spatial proximity to regulated genomic regions. Here we review examples of positional cis conservation of regulatory RNAs with respect to target genes, spatial proximity of enhancer RNAs to promoters through DNA looping and RNA-mediated formation of membrane-less structures to control chromatin structure and expression. We speculate that linear and spatial proximity between regulatory RNA-encoding genes and gene targets could possibly ease the evolutionary pressure on maintaining regulatory RNA sequence conservation
Calculation of 3D genome structures for comparison of chromosome conformation capture experiments with microscopy: An evaluation of single-cell Hi-C protocols.
Single-cell chromosome conformation capture approaches are revealing the extent of cell-to-cell variability in the organization and packaging of genomes. These single-cell methods, unlike their multi-cell counterparts, allow straightforward computation of realistic chromosome conformations that may be compared and combined with other, independent, techniques to study 3D structure. Here we discuss how single-cell Hi-C and subsequent 3D genome structure determination allows comparison with data from microscopy. We then carry out a systematic evaluation of recently published single-cell Hi-C datasets to establish a computational approach for the evaluation of single-cell Hi-C protocols. We show that the calculation of genome structures provides a useful tool for assessing the quality of single-cell Hi-C data because it requires a self-consistent network of interactions, relating to the underlying 3D conformation, with few errors, as well as sufficient longer-range cis- and trans-chromosomal contacts
Dynamic monitoring of single cell lysis in an impedance-based microfluidic device.
A microfluidic device that is capable of trapping and sensing dynamic variations in the electrical properties of individual cells is demonstrated. The device is applied to the real-time recording of impedance measurements of mouse embryonic stem cells (mESCs) during the process of membrane lysis, with the resulting changes in the electrical properties of cells during this process being quantitatively tracked over time. It is observed that the impedance magnitude decreases dramatically after cell membrane lysis. A significant shift in the phase spectrum is also observed during the time course of this process. By fitting experimental data to physical models, the electrical parameters of cells can be extracted and parameter variations quantified during the process. In the cell lysis experiments, the equivalent conductivity of the cell membrane is found to increase significantly due to pore formation in the membrane during lysis. An increase in the specific capacitance of the membrane is also observed. On the other hand, the conductivity of the cytoplasm is observed to decrease, which may be explained the fact that excess water enters the cell through the gradual permeabilization of the membrane during lysis. Cells can be trapped in the device for periods up to several days, and their electrical response can be monitored by real-time impedance measurements in a label-free and non-invasive manner. Furthermore, due to the highly efficient single cell trapping capacity of the device, a number of cells can be trapped and held in separate wells for concurrent parallel experiments, allowing for the possibility of stepped parametric experiments and studying cell heterogeneity by combining measurements across the array.Biotechnology and Biological Sciences Research Council (Grant ID: BB/K013726/1)This is the final version of the article. It first appeared from Springer via http://dx.doi.org/10.1007/s10544-016-0081-
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Label-Free Live-Cell Imaging of Nucleic Acids Using Stimulated Raman Scattering Microscopy
Imaging of nucleic acids is important for studying cellular processes such as cell division and apoptosis. A noninvasive label-free technique is attractive. Raman spectroscopy provides rich chemical information based on specific vibrational peaks. However, the signal from spontaneous Raman scattering is weak and long integration times are required, which drastically limits the imaging speed when used for microscopy. Coherent Raman scattering techniques, comprising coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy, overcome this problem by enhancing the signal level by up to five orders of magnitude. CARS microscopy suffers from a nonresonant background signal, which distorts Raman spectra and limits sensitivity. This makes CARS imaging of weak transitions in spectrally congested regions challenging. This is especially the case in the fingerprint region, where nucleic acids show characteristic peaks. The recently developed SRS microscopy is free from these limitations; excitation spectra are identical to those of spontaneous Raman and sensitivity is close to shot-noise limited. Herein we demonstrate the use of SRS imaging in the fingerprint region to map the distribution of nucleic acids in addition to proteins and lipids in single salivary gland cells of Drosophila larvae, and in single mammalian cells. This allows the imaging of DNA condensation associated with cell division and opens up possibilities of imaging such processes in vivo.Chemistry and Chemical Biolog
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Single Molecule Imaging of Transcription Factor Binding to DNA in Live Mammalian Cells
Imaging single fluorescent proteins in living mammalian cells is challenged by out-of-focus fluorescence excitation. To reduce out-of-focus fluorescence we developed reflected light-sheet microscopy (RLSM), a fluorescence microscopy method allowing selective plane illumination throughout the nuclei of living mammalian cells. A thin light sheet parallel to the imaging plane and close to the sample surface is generated by reflecting an elliptical laser beam incident from the top by 90° with a small mirror. The thin light sheet allows for an increased signal-to-background ratio superior to that in previous illumination schemes and enables imaging of single fluorescent proteins with up to 100-Hz time resolution. We demonstrated the single-molecule sensitivity of RLSM by measuring the DNA-bound fraction of glucocorticoid receptor (GR) and determining the residence times on DNA of various oligomerization states and mutants of GR and estrogen receptor-α (ER), which permitted us to resolve different modes of DNA binding of GR. We demonstrated two-color single-molecule imaging by observing the spatiotemporal colocalization of two different protein pairs. Our single-molecule measurements and statistical analysis revealed dynamic properties of transcription factors.The Classic
Citrullination of HP1γ chromodomain affects association with chromatin.
BACKGROUND: Stem cell differentiation involves major chromatin reorganisation, heterochromatin formation and genomic relocalisation of structural proteins, including heterochromatin protein 1 gamma (HP1γ). As the principal reader of the repressive histone marks H3K9me2/3, HP1 plays a key role in numerous processes including heterochromatin formation and maintenance. RESULTS: We find that HP1γ is citrullinated in mouse embryonic stem cells (mESCs) and this diminishes when cells differentiate, indicating that it is a dynamically regulated post-translational modification during stem cell differentiation. Peptidylarginine deiminase 4, a known regulator of pluripotency, citrullinates HP1γ in vitro. This requires R38 and R39 within the HP1γ chromodomain, and the catalytic activity is enhanced by trimethylated H3K9 (H3K9me3) peptides. Mutation of R38 and R39, designed to mimic citrullination, affects HP1γ binding to H3K9me3-containing peptides. Using live-cell single-particle tracking, we demonstrate that R38 and R39 are important for HP1γ binding to chromatin in vivo. Furthermore, their mutation reduces the residence time of HP1γ on chromatin in differentiating mESCs. CONCLUSION: Citrullination is a novel post-translational modification of the structural heterochromatin protein HP1γ in mESCs that is dynamically regulated during mESC differentiation. The citrullinated residues lie within the HP1γ chromodomain and are important for H3K9me3 binding in vitro and chromatin association in vivo.Cancer Research UK (grant reference RG17001)
Wellcome Trust (Core Grant reference WT203144)
Cancer Research UK (grant reference C6946/A24843).
Wellcome Trust (206291/Z/17/Z)
Medical Research Council (MR/P019471/1 and MR/M010082/1).
Royal Society Professorship (RP150066)
Medical Research Council (MR/K015850/1
Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.
Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.We thank the Wellcome Trust for the PhD studentship of MP (093756/B/10/Z), and the Royal Society for the University Research Fellowship of SFL (UF120277). The work by SB and DL was also funded by the Wellcome Trust (082010/Z/07/Z). UE and MH acknowledge funding by the German Science Foundation (grants EXC 115 and SFB 902). SB is funded by a BBSRC grant (BB/K013726/1). AMC acknowledges ERC Award 268788-SMI-DDR. We also thank the European Commision for support through the 4DCellFate project (EC FP7 CP 277899).This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pone.012543
FRET-enhanced photostability allows improved single-molecule tracking of proteins and protein complexes in live mammalian cells.
A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods of time in the dense environments found in living cells. Here, we introduce the concept of using FRET to enhance the photophysical properties of photo-modulatable (PM) fluorophores commonly used in such studies. By developing novel single-molecule FRET pairs, consisting of a PM donor fluorophore (either mEos3.2 or PA-JF549) next to a photostable acceptor dye JF646, we demonstrate that FRET competes with normal photobleaching kinetic pathways to increase the photostability of both donor fluorophores. This effect was further enhanced using a triplet-state quencher. Our approach allows us to significantly improve single-molecule tracking of chromatin-binding proteins in live mammalian cells. In addition, it provides a novel way to track the localization and dynamics of protein complexes by labeling one protein with the PM donor and its interaction partner with the acceptor dye
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Study of Chromatin Structure Using Stimulated Raman Scattering Microscopy in Living Mammalian Cells
DNA is packaged into the nucleus of a mammalian cell as a nucleoprotein complex called chromatin. Changes in chromatin structure occur during processes that are critical to an understanding of mammalian cell biology such as cell division. Existing fixed-cell techniques have provided insight into chromatin organization in the mammalian nucleus. In addition, fluorescence microscopy techniques have allowed us to study changes in chromatin structure in living cells. However, most of these fluorescence techniques cannot be used for tissue imaging or long-term imaging due to photobleaching. In this thesis, we demonstrate that a label-free technique called Stimulated Raman Scattering (SRS) microscopy can be used to solve these problems and study chromatin structure in living mammalian cells both in culture and in tissue. SRS is a vibrational microscopy technique that takes advantage of intrinsic contrast arising from specific chemical bonds in a molecule. Nucleic acids have specifc phosphate and CH vibrations that can be used to determine their cellular distributions. Imaging at specific phosphate peaks using fingerprint SRS microscopy allows the detection of polytene chromosomes in Drosophila salivary gland cells and condensed chromatin in metaphase mammalian cells. In addition, we develop a technique called multicolor SRS microscopy, in which we image at several wavelengths across the CH vibrational band, and then use linear combination to simultaneously determine the nucleic acid, lipid and protein distributions in living mammalian cells. This technique achieves greater contrast than imaging at the phosphate vibrational peak due to the stronger SRS signal in the high wavenumber CH band and so allows us to determine chromatin structure in interphase mammalian cells. This technique also allows long-term imaging of living mammalian cells and the imaging of tissue such as mouse skin. The technique is used to monitor mammalian cell division in culture and paves the way for similar studies in living tissue. This technique will provide insight into cell division, differentiation and apoptosis during development and in disease models such as cancer
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RNA, Genome Output and Input
RNA, the transcriptional output of genomes, not only templates protein synthesis or directly engages in catalytic functions, but can feed back to the genome and serve as regulatory input for gene expression. Transcripts affecting the RNA abundance of other genes act by mechanisms similar to and in concert with protein factors that control transcription. Through recruitment or blocking of activating and silencing complexes to specific genomic loci, RNA and protein factors can favor transcription or lower the local gene expression potential. Most regulatory proteins enter nuclei from all directions to start the search for increased affinity to specific DNA sequences or to other proteins nearby genuine gene targets. In contrast, RNAs emerge from spatial point sources within nuclei, their encoding genes. A transcriptional burst can result in the local appearance of multiple nascent RNA copies at once, in turn increasing local nucleic acid density and RNA motif abundance before diffusion into the nuclear neighborhood. The confined initial localization of regulatory RNAs causing accumulation of protein co-factors raises the intriguing possibility that target specificity of non-coding, and probably coding, RNAs is achieved through gene/RNA positioning and spatial proximity to regulated genomic regions. Here we review examples of positional cis conservation of regulatory RNAs with respect to target genes, spatial proximity of enhancer RNAs to promoters through DNA looping and RNA-mediated formation of membrane-less structures to control chromatin structure and expression. We speculate that linear and spatial proximity between regulatory RNA-encoding genes and gene targets could possibly ease the evolutionary pressure on maintaining regulatory RNA sequence conservation