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

WeNMR : the tale of virtual research community in NMR and structural biology

Structural biology and life sciences in general, and NMR in particular, have always been associated with advanced computing. The current challenges in the post-genomic era call for virtual research platforms that provide the worldwide research community with both user-friendly tools, platforms for data analysis and exchange, and an underlying e-Infrastructure. WeNMR, a three-year European Commission co-funded project started in November 2010, groups different research teams into a worldwide virtual research community. It builds on the established eNMR e-Infrastructure and its steadily growing virtual organisation, which is currently the second largest VO in the area of life sciences. WeNMR provides an e-Infrastructure platform and Science Gateway for structural biology. It involves researchers from around the world and will build bridges to other areas of structural biology

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-

Quantitative single-molecule microscopy reveals that CENP-A(Cnp1) deposition occurs during G2 in fission yeast

The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and 'counts' individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-A(Cnp1) with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-A(Cnp1) levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-A(Cnp1) is deposited solely during the G2 phase of the cell cycle

Metal ion co-ordination in the DNA binding domain of the yeast transcriptional activator GAL4

AbstractThe structure of the DNA binding domain of the yeast transcriptional activator GAL4 was investigated by extended X-ray fine structure (e.x.a.f.s.). Two samples of GAL4 were studied, one containing cadmium as a structural probe (Cd(II)GAL4) and the other containing the ‘native’ zinc (Zn(Il)-GAL4). The results suggest that the structure of the DNA binding domain of GAL4 contains a two metal ion cluster distinguishing it from the ‘zinc finger’ proteins typified by the Xenopus laevis transcription factor TFIIIA

Recruitment of Polo kinase to the spindle midzone during cytokinesis requires the Feo/Klp3A complex

Our results reveal that the Feo/Klp3A complex is necessary for Polo recruitment to the spindle midzone. A similar finding has also been recently reported in mammalian cells [1], suggesting that this basic mechanism has been conserved during evolution, albeit with some differences. Finally, since cleavage furrow formation and ingression are unaffected following feo RNAi, our data imply that Polo recruitment to the central spindle is not required for furrowing, but some other aspect of cytokinesis

Elucidating the mechanisms underlying GATA-1 activity

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Interaction between Anillin and RacGAP50C connects the actomyosin contractile ring with spindle microtubules at the cell division site

Anillin, one of the first factors recruited to the cleavage site during cytokinesis, interacts with actin, myosin II and septins, and is essential for proper organization of the actomyosin contractile ring. We employed affinity-purification methodology coupled with mass spectrometry to identify Anillin-interacting molecules in Drosophila cells. We isolated several actin and myosin proteins, three of the five Drosophila septins and RacGAP50C (Tum), a component of the centralspindlin complex. Using drug and RNA interference (RNAi) treatments we established that F-actin is essential for Anillin cortical localization in prometaphase but not for its accumulation at the cleavage furrow after anaphase onset. Moreover, septins were not recruited to the cleavage site in cells in which Anillin was knocked down by RNAi, but localized to central-spindle microtubules, suggesting that septins travel along microtubules to interact with Anillin at the furrow. Finally, we demonstrate that RacGAP50C is necessary for Anillin accumulation at the furrow and that the two proteins colocalize in vivo and interact in vitro. Thus, in addition to its role in activating RhoA signalling, RacGAP50C also controls the proper assembly of the actomyosin ring by interacting with Anillin at the cleavage furrow

Interaction between Anillin and RacGAP50C connects the actomyosin contractile ring with spindle microtubules at the cell division site

Anillin, one of the first factors recruited to the cleavage site during cytokinesis, interacts with actin, myosin II and septins, and is essential for proper organization of the actomyosin contractile ring. We employed affinity-purification methodology coupled with mass spectrometry to identify Anillin-interacting molecules in Drosophila cells. We isolated several actin and myosin proteins, three of the five Drosophila septins and RacGAP50C (Tum), a component of the centralspindlin complex. Using drug and RNA interference (RNAi) treatments we established that F-actin is essential for Anillin cortical localization in prometaphase but not for its accumulation at the cleavage furrow after anaphase onset. Moreover, septins were not recruited to the cleavage site in cells in which Anillin was knocked down by RNAi, but localized to central-spindle microtubules, suggesting that septins travel along microtubules to interact with Anillin at the furrow. Finally, we demonstrate that RacGAP50C is necessary for Anillin accumulation at the furrow and that the two proteins colocalize in vivo and interact in vitro. Thus, in addition to its role in activating RhoA signalling, RacGAP50C also controls the proper assembly of the actomyosin ring by interacting with Anillin at the cleavage furrow