Supranucleosomal organization of chromatin fibers in nuclei of Drosophila S2 cells

Earlier, the interphase chromatin structures could not be visualized due to the stickiness of the nuclear material. We have reduced stickiness by the reversal of permeabilization allowing the isolation and microscopic imaging of interphase chromatin structures. By using a high resolution of synchronization, collecting 36 elutriation fractions, we show that major intermediates of chromatin condensation include: (a) decondensed veillike chromatin at the unset of the S phase (2.0-2.2 C-value), (b) polarization of veiled chromatin (2.2-2.6 C), (c) fibrous chromatin (2.6-3.0 C), chromatin bodies (3.0-3.3 C), early precondensed chromosomes (3.3-3.6). The compaction of Drosophila chromosomes did not reach that of the mammalian cells in the final stage of condensation (3.6-4.0 C). Drosophila chromosomes consist of smaller units called rodlets. Results demonstrate that nucleosomal chromatin ("beads on string") does not form a solenoid structure; rather, the topological arrangement consists of meandering and plectonemic loops.     

The layered organization of nucleosomes in 30 nm chromatin fibers

We have used electron microscopy and established methods of three-dimensional reconstruction to obtain structural information on the 30 nm chromatin fibers from sea cucumber sperm and chicken erythrocytes. The fibers show a longitudinal periodicity of 10–11 nm. We have interpreted this periodicity as due to a grouping of nucleosomes into disks, each disk containing about 5–6 nucleosomes. These disks are closely stacked to form the chromatin fiber. We have built a detailed model for four fibers and we have determined the approximate coordinates of all the nucleosomes in them. The average distance found between neighboring nucleosomes has a value close to 11 nm. They may be connected either as a regularly distorted helix or as a layered zigzag. The second model appears more appropriate, since in the constrictions of the fibers the nucleosomes can only be connected as a zigzag.     

CRISPR-based genomic loci labeling revealed ordered spatial organization of chromatin in living diploid human cells

The eukaryotic genome is compacted in the form of chromatin within the nucleus. Whether the spatial distribution of the genome is ordered or not has been a longstanding question. Answering this question would enable us to understand nuclear organization and cellular processes more deeply. Here, we applied a modified CRISPR/dCas9 system to label the randomly selected genomic loci in diploid living cells, which were visualized by high-resolution wide-field imaging. To analyze the spatial positions of three pairs of genomic loci, three sets of parameters were progressively measured: i) the linear distance between alleles; ii) the radial distribution of the genomic loci; and iii) the linear distances between three pairs of genomic loci on nonhomologous chromosomes. By accurate labeling, geometric measuring and statistical analysis, we demonstrated that the distribution of these genomic loci in the 3D space of the nucleus is relatively stable in both late G1 and early S phases. Collectively, our data provided visual evidence in live cells, which implies the orderly spatial organization of chromatin in the nucleus. The combination of orderliness and flexibility ensures the methodical and efficient operation of complex life systems. How the nucleus adopts this ordered 3D structure in living cells is thought-provoking.     

Visualizing chromatin and chromosomes in living cells

The dynamic organization of eukaryotic genomes in cell nuclei recently came into the focus of research interest. The kinetics of genome dynamics can be addressed only by approaches involving live cell microscopy. Different methods are available to visualize chromatin, specific chromatin fractions, or individual chromosome territories within nuclei of living mammalian cells. Appropriate labeling procedures as well as cell chamber systems and important controls for live cell microscopy are described.     

Large-scale cortical functional organization and speech perception across the lifespan

Aging is accompanied by substantial changes in brain function, including functional reorganization of large-scale brain networks. Such differences in network architecture have been reported both at rest and during cognitive task performance, but an open question is whether these age-related differences show task-dependent effects or represent only task-independent changes attributable to a common factor (i.e., underlying physiological decline). To address this question, we used graph theoretic analysis to construct weighted cortical functional networks from hemodynamic (functional MRI) responses in 12 younger and 12 older adults during a speech perception task performed in both quiet and noisy listening conditions. Functional networks were constructed for each subject and listening condition based on inter-regional correlations of the fMRI signal among 66 cortical regions, and network measures of global and local efficiency were computed. Across listening conditions, older adult networks showed significantly decreased global (but not local) efficiency relative to younger adults after normalizing measures to surrogate random networks. Although listening condition produced no main effects on whole-cortex network organization, a significant age group x listening condition interaction was observed. Additionally, an exploratory analysis of regional effects uncovered age-related declines in both global and local efficiency concentrated exclusively in auditory areas (bilateral superior and middle temporal cortex), further suggestive of specificity to the speech perception tasks. Global efficiency also correlated positively with mean cortical thickness across all subjects, establishing gross cortical atrophy as a task-independent contributor to age-related differences in functional organization. Together, our findings provide evidence of age-related disruptions in cortical functional network organization during speech perception tasks, and suggest that although task-independent effects such as cortical atrophy clearly underlie age-related changes in cortical functional organization, age-related differences also demonstrate sensitivity to task domains.     

Nucleomeric organization of chromatin

Chromatin in the nuclei fixed in tissue and in the nuclei isolated by low ionic strength solutions in the presence of Mg2+ is represented by globular (nucleomeric) fibrils, 20-25 nm in diameter. The staphylococcal or endogenous nuclear nuclease splits the chromatin fibrils resulting in fragments corresponding to nucleomers and their multimers. Upon removal of firmly bound Mg2+ the nucleomers unfold to form chains consisting of 4-6-8 nucleosomes. Mild hydrolysis of nuclear chromatin by staphylococcal nuclease results in a split-off of mono-, di- and trimers of nucleomers sedimenting in a sucrose density gradient in the presence of EDTA as particles with the sedimentation coefficients of 37, 47 and 55S, respectively. The sedimentation coefficient for the mononucleomer in a sucrose density gradient with MgCl2 is 45S. Determination of the length of DNA fragments of chromatin split-off by staphylococcal nuclease showed that the nucleomer consists of 8 nucleosomes, while the dimer and trimer of the nucleomer consists of 14-16 and 21-24 nucleosomes, respectively. The nucleomeric monomer undergoes structural transition from the compact (45S) to the "loose" state (37S) after removal of Mg2+. This transition is completely reversible, when the nucleomer contains histone H1. The removal of the latter or dialysis of the nucleomer against EDTA in low ionic strength solutions results in a complete unfolding of the nucleomer into a nucleosomal chain fragment. A model for the nucleomer fibril structure in which the helical organization of the nucleosomal chain in the nucleomer (2 turns with 4 nucleosomes in each) is alternated with the impaired helical bonds between the nucleomers is discussed. The functional significance of the nucleomeric organization of chromatin may be an additional restriction of the site-specific recognition of DNA in chromatin with the possibility of local (at the level of one nucleomer) changes in chromatin conformation excluding this restriction.     

Supranucleosomal organization of chromatin

A systematic study of the effect of different ionic conditions on the morphology of the 25–30 nm chromatin fiber from chicken erythrocytes has revealed that, as the ionic strength is increased, knobby fibers with a clear superbead structure are formed in the presence of either Mg⁺⁺ or Na⁺, or both. A further increase in ionic strength results in smooth chromatin fibers due to a tight packing of superbeads. Cross-linking such fibers with formaldehyde and reversal of the ionic conditions, demonstrates the superbead structures underlying the smooth fibers observed at high ionic concentrations. The average size of the superbeads is 34 nm along the length of the fibers, in agreement with the value found in embedded sea cucumber chromatin. A second class of superbeads has an average length of 25 nm and probably corresponds to partially disrupted structures.     

Probing structural stability of chromatin assembly sorted from living cells

Post-translational modifications of the histone tails and other chromatin binding proteins affect the stability of chromatin structure. In this study, we have purified chromatin from live cell nuclei using a fluorescence activated cell sorter (FACS) and studied the structural stability of this self-assembled structure. Using total internal reflection fluorescence (TIRF) microscopy, we map the effect of covalent modifications on the interaction of histone-DNA complex, by measuring the dissociation rates of histones from the chromatin fiber in the presence of different salt concentrations. Dynamic force spectroscopy (DFS) experiments were carried out to measure the structural disintegration of large chromatin globules under force. The characteristic rupture of multiple linkages in the large chromatin globules show differences in the stiffness of the higher order structure of chromatin with altered epigenetic states. Our studies reveal a direct correlation between histone modifications and the structural stability of higher order chromatin assembly.     

Directed immobilization of recombinant staphylococci on cotton fibers by functional display of a fungal cellulose-binding domain

The immobilization of recombinant staphylococci onto cellulose fibers through surface display of a fungal cellulose-binding domain (CBD) was investigated. Chimeric proteins containing the CBD from Trichoderma reesei cellulase Cel6A were found to be correctly targeted to the cell wall of Staphylococcus carnosus cells, since full-length proteins could be extracted and affinity-purified. Furthermore, surface accessibility of the CBD was verified using a monoclonal antibody and functionality in terms of cellulose-binding was demonstrated in two different assays in which recombinant staphylococci were found to efficiently bind to cotton fibers. The implications of this strategy of directed immobilization for the generation of whole-cell microbial tools for different applications will be discussed.     

Mechanical properties of actin stress fibers in living cells

Actin stress fibers (SFs) play an important role in many cellular functions, including morphological stability, adhesion, and motility. Because of their central role in force transmission, it is important to characterize the mechanical properties of SFs. However, most of the existing studies focus on properties of whole cells or of actin filaments isolated outside cells. In this study, we explored the mechanical properties of individual SFs in living endothelial cells by nanoindentation using an atomic force microscope. Our results demonstrate the pivotal role of SF actomyosin contractile level on mechanical properties. In the same SF, decreasing contractile level with 10 μM blebbistatin decreased stiffness, whereas increasing contractile level with 2 nM calyculin A increased stiffness. Incrementally stretching and indenting SFs made it possible to determine stiffness as a function of strain level and demonstrated that SFs have nearly linear stress-stain properties in the baseline state but nonlinear properties at a lower contractile level. The stiffnesses of peripheral and central portions of the same SF, which were nearly the same in the baseline state, became markedly different after contractile level was increased with calyculin A. Because these results pertain to effects of interventions in the same SF in a living cell, they provide important new understanding about cell mechanics.     

Linking chromatin acylation mark-defined proteome and genome in living cells

1. Download : Download high-res image (261KB)
2. Download : Download full-size image