Affinity hierarchies and amphiphilic proteins underlie the co-assembly of nucleolar and heterochromatin condensates.

Nucleoli are surrounded by Pericentromeric Heterochromatin (PCH), reflecting a close spatial association between the two largest biomolecular condensates in eukaryotic nuclei. This nuclear organizational feature is highly conserved and is disrupted in diseased states like senescence, however, the mechanisms driving PCH-nucleolar association are unclear. High-resolution live imaging during early Drosophila development revealed a highly dynamic process in which PCH and nucleolar formation is coordinated and interdependent. When nucleolus assembly was eliminated by deleting the ribosomal RNA genes (rDNA), PCH showed increased compaction and subsequent reorganization to a shell-like structure. In addition, in embryos lacking rDNA, some nucleolar proteins were redistributed into new bodies or 'neocondensates,' including enrichment in the core of the PCH shell. These observations, combined with physical modeling and simulations, suggested that nucleolar-PCH associations are mediated by a hierarchy of affinities between PCH, nucleoli, and 'amphiphilic' protein(s) that interact with both nucleolar and PCH components. This result was validated by demonstrating that the depletion of one candidate amphiphile, the nucleolar protein Pitchoune, significantly reduced PCH-nucleolar associations. Together, these results unveil a dynamic program for establishing nucleolar-PCH associations during animal development, demonstrate that nucleoli are required for normal PCH organization, and identify Pitchoune as an amphiphilic molecular link that promotes PCH-nucleolar associations. Finally, we propose that disrupting affinity hierarchies between interacting condensates can liberate molecules to form neocondensates or other aberrant structures that could contribute to cellular disease phenotypes.

Affinity hierarchies and amphiphilic proteins underlie the co-assembly of nucleolar and heterochromatin condensates.

Nucleoli are surrounded by Pericentromeric Heterochromatin (PCH), reflecting a close spatial association between the two largest biomolecular condensates in eukaryotic nuclei. This nuclear organizational feature is highly conserved and is disrupted in diseased states like senescence, however, the mechanisms driving PCH-nucleolar association are unclear. High-resolution live imaging during early Drosophila development revealed a highly dynamic process in which PCH and nucleolar formation is coordinated and interdependent. When nucleolus assembly was eliminated by deleting the ribosomal RNA genes (rDNA), PCH showed increased compaction and subsequent reorganization to a shell-like structure. In addition, in embryos lacking rDNA, some nucleolar proteins were redistributed into new bodies or 'neocondensates,' including enrichment in the core of the PCH shell. These observations, combined with physical modeling and simulations, suggested that nucleolar-PCH associations are mediated by a hierarchy of affinities between PCH, nucleoli, and 'amphiphilic' protein(s) that interact with both nucleolar and PCH components. This result was validated by demonstrating that the depletion of one candidate amphiphile, the nucleolar protein Pitchoune, significantly reduced PCH-nucleolar associations. Together, these results unveil a dynamic program for establishing nucleolar-PCH associations during animal development, demonstrate that nucleoli are required for normal PCH organization, and identify Pitchoune as an amphiphilic molecular link that promotes PCH-nucleolar associations. Finally, we propose that disrupting affinity hierarchies between interacting condensates can liberate molecules to form neocondensates or other aberrant structures that could contribute to cellular disease phenotypes.

Regulation of chromatin microphase separation by binding of protein complexes.

We show evidence of the association of RNA polymerase II (RNAP) with chromatin in a core-shell organization, reminiscent of microphase separation where the cores comprise dense chromatin and the shell, RNAP and chromatin with low density. These observations motivate our physical model for the regulation of core-shell chromatin organization. Here, we model chromatin as a multiblock copolymer, comprising active and inactive regions (blocks) that are both in poor solvent and tend to be condensed in the absence of binding proteins. However, we show that the solvent quality for the active regions of chromatin can be regulated by the binding of protein complexes (e.g., RNAP and transcription factors). Using the theory of polymer brushes, we find that such binding leads to swelling of the active chromatin regions which in turn modifies the spatial organization of the inactive regions. In addition, we use simulations to study spherical chromatin micelles, whose cores comprise inactive regions and shells comprise active regions and bound protein complexes. In spherical micelles the swelling increases the number of inactive cores and controls their size. Thus, genetic modifications affecting the binding strength of chromatin-binding protein complexes may modulate the solvent quality experienced by chromatin and regulate the physical organization of the genome.

Mesoscale, long-time mixing of chromosomes and its connection to polymer dynamics.

Chromosomes are arranged in distinct territories within the nucleus of animal cells. Recent experiments have shown that these territories overlap at their edges, suggesting partial mixing during interphase. Experiments that knock-down of condensin II proteins during interphase indicate increased chromosome mixing, which demonstrates control of the mixing. In this study, we use a generic polymer simulation to quantify the dynamics of chromosome mixing over time. We introduce the chromosome mixing index, which quantifies the mixing of distinct chromosomes in the nucleus. We find that the chromosome mixing index in a small confinement volume (as a model of the nucleus), increases as a power-law of the time, with the scaling exponent varying non-monotonically with self-interaction and volume fraction. By comparing the chromosome mixing index with both monomer subdiffusion due to (non-topological) intermingling of chromosomes as well as even slower reptation, we show that for relatively large volume fractions, the scaling exponent of the chromosome mixing index is related to Rouse dynamics for relatively weak chromosome attractions and to reptation for strong attractions. In addition, we extend our model to more realistically account for the situation of the Drosophila chromosome by including the heterogeneity of the polymers and their lengths to account for microphase separation of euchromatin and heterochromatin and their interactions with the nuclear lamina. We find that the interaction with the lamina further impedes chromosome mixing.

The LINC Complex Inhibits Excessive Chromatin Repression.

The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex transduces nuclear mechanical inputs suggested to control chromatin organization and gene expression; however, the underlying mechanism is currently unclear. We show here that the LINC complex is needed to minimize chromatin repression in muscle tissue, where the nuclei are exposed to significant mechanical inputs during muscle contraction. To this end, the genomic binding profiles of Polycomb, Heterochromatin Protein1 (HP1a) repressors, and of RNA-Pol II were studied in Drosophila larval muscles lacking functional LINC complex. A significant increase in the binding of Polycomb and parallel reduction of RNA-Pol-II binding to a set of muscle genes was observed. Consistently, enhanced tri-methylated H3K9 and H3K27 repressive modifications and reduced chromatin activation by H3K9 acetylation were found. Furthermore, larger tri-methylated H3K27me3 repressive clusters, and chromatin redistribution from the nuclear periphery towards nuclear center, were detected in live LINC mutant larval muscles. Computer simulation indicated that the observed dissociation of the chromatin from the nuclear envelope promotes growth of tri-methylated H3K27 repressive clusters. Thus, we suggest that by promoting chromatin-nuclear envelope binding, the LINC complex restricts the size of repressive H3K27 tri-methylated clusters, thereby limiting the binding of Polycomb transcription repressor, directing robust transcription in muscle fibers.

Containerized Architecture Performance Analysis for IoT Framework Based on Enhanced Fire Prevention Case Study: Rwanda.

Nowadays, building infrastructures are pushed to become smarter in response to desires for the environmental comforts of living. Enhanced safety upgrades have begun taking advantage of new, evolving technologies. Normally, buildings are configured to respond to the safety concerns of the occupants. However, advanced Internet of Things (IoT) techniques, in combination with edge computing with lightweight virtualization technology, is being used to improve users' comfort in their homes. It improves resource management and service isolation without affecting the deployment of heterogeneous hardware. In this research, a containerized architectural framework for support of multiple concurrent deployed IoT applications for smart buildings was proposed. The prototype developed used sensor networks as well as containerized microservices, centrally featuring the DevOps paradigm. The research proposed an occupant counting algorithm used to check occupants in and out. The proposed framework was tested in different academic buildings for data acquisition over three months. Different deployment architectures were tested to ensure the best cases based on efficiency and resource utilization. The acquired data was used for prediction purposes to aid occupant prediction for safety measures as considered by policymakers.

Speed Management Strategy: Designing an IoT-Based Electric Vehicle Speed Control Monitoring System.

Road accidents represent the greatest public health burden in the world. Road traffic accidents have been on the rise in Rwanda for several years. Speed has been identified as a core factor in these road accidents. Therefore, understanding road accidents caused by excessive speeding is critical for road safety planning. In this paper, input and out pulse width modulation (PWM) was used to command the metal-oxide-semiconductor field-effect transistor (MOSFET) controller which supplied voltage to the motor. A structural speed control and Internet of Things (IoT)-based online monitoring system was developed to monitor vehicle data in a continuous manner. Two modeling techniques, multiple linear regression (MLR) and random forest (RF) models, were evaluated to find the best model to estimate the required voltage to be supplied to the motors in a particular zone. The built models were evaluated based upon the coefficient of determination R2. The RF performs better than the MLR as it reveals a higher R2 value and it is found to be 98.8%. Based on the results, the proposed method was proven to significantly reduce the supplied voltage to the motor and consequently increase safety.

Live imaging of chromatin distribution reveals novel principles of nuclear architecture and chromatin compartmentalization.

The three-dimensional organization of chromatin contributes to transcriptional control, but information about native chromatin distribution is limited. Imaging chromatin in live Drosophila larvae, with preserved nuclear volume, revealed that active and repressed chromatin separates from the nuclear interior and forms a peripheral layer underneath the nuclear lamina. This is in contrast to the current view that chromatin distributes throughout the nucleus. Furthermore, peripheral chromatin organization was observed in distinct Drosophila tissues, as well as in live human effector T lymphocytes and neutrophils. Lamin A/C up-regulation resulted in chromatin collapse toward the nuclear center and correlated with a significant reduction in the levels of active chromatin. Physical modeling suggests that binding of lamina-associated domains combined with chromatin self-attractive interactions recapitulate the experimental chromatin distribution profiles. Together, our findings reveal a novel mode of mesoscale organization of peripheral chromatin sensitive to lamina composition, which is evolutionary conserved.

Mesoscale phase separation of chromatin in the nucleus.

Intact-organism imaging of Drosophila larvae reveals and quantifies chromatin-aqueous phase separation. The chromatin can be organized near the lamina layer of the nuclear envelope, conventionally fill the nucleus, be organized centrally, or as a wetting droplet. These transitions are controlled by changes in nuclear volume and the interaction of chromatin with the lamina (part of the nuclear envelope) at the nuclear periphery. Using a simple polymeric model that includes the key features of chromatin self-attraction and its binding to the lamina, we demonstrate theoretically that it is the competition of these two effects that determines the mode of chromatin distribution. The qualitative trends as well as the composition profiles obtained in our simulations compare well with the observed intact-organism imaging and quantification. Since the simulations contain only a small number of physical variables we can identify the generic mechanisms underlying the changes in the observed phase separations.

Irregular Chromatin: Packing Density, Fiber Width, and Occurrence of Heterogeneous Clusters.

How chromatin is folded on the length scale of a gene is an open question. Recent experiments have suggested that, in vivo, chromatin is folded in an irregular manner and not as an ordered fiber with a width of 30 nm that is expected from theories of higher order packaging. Using computational methods, we examine how the interplay between DNA-bending nonhistone proteins, histone tails, intrachromatin electrostatic, and other interactions decide the nature of the packaging of chromatin. We show that although the DNA-bending nonhistone proteins make the chromatin irregular, they may not alter the packing density and size of the fiber. We find that the length of the interacting region and intrachromatin electrostatic interactions influence the packing density, clustering of nucleosomes, and the width of the chromatin fiber. Our results suggest that the heterogeneity in the interaction pattern will play an important role in deciding the nature of the packaging of chromatin.

Effect of ionic size compensation by Ag+ incorporation in homogeneous Fe-substituted ZnO: studies on structural, mechanical, optical, and magnetic properties.

Substituting an ion of different size from that of the host element introduces lattice strain and defects. However, this mismatch may be significantly reduced by substituting an additional ion with a compensating size relative to the dopant. Such a double substitution might offer better solubility irrespective of the local distortions as well as the formation of defects in the valence states. Fe-substituted ZnO has been widely reported with conflicting results primarily arising from lack of chemical and structural homogeneity originating from preparation techniques, compositional fluctuations, and equivocal comprehension of actual solubility limits of the dopants. In this study, Ag ion has been incorporated in Fe-substituted ZnO to compensate the ionic size of Zn1-x [Fe0.8Ag0.2] x O (0 ≤ x ≤ 0.03125) by determining the solubility limit of the homogeneous material and their corresponding structural, mechanical, optical and magnetic properties have been investigated thoroughly. Co-substitution rearranges the lattice and leads to better crystal structures with tunable properties related to the amount of substitution.

Correction: Effect of ionic size compensation by Ag+ incorporation in homogeneous Fe-substituted ZnO: studies on structural, mechanical, optical, and magnetic properties.

[This corrects the article DOI: 10.1039/C8RA02393J.].

Binding of DNA-bending non-histone proteins destabilizes regular 30-nm chromatin structure.

Why most of the in vivo experiments do not find the 30-nm chromatin fiber, well studied in vitro, is a puzzle. Two basic physical inputs that are crucial for understanding the structure of the 30-nm fiber are the stiffness of the linker DNA and the relative orientations of the DNA entering/exiting nucleosomes. Based on these inputs we simulate chromatin structure and show that the presence of non-histone proteins, which bind and locally bend linker DNA, destroys any regular higher order structures (e.g., zig-zag). Accounting for the bending geometry of proteins like nhp6 and HMG-B, our theory predicts phase-diagram for the chromatin structure as a function of DNA-bending non-histone protein density and mean linker DNA length. For a wide range of linker lengths, we show that as we vary one parameter, that is, the fraction of bent linker region due to non-histone proteins, the steady-state structure will show a transition from zig-zag to an irregular structure-a structure that is reminiscent of what is observed in experiments recently. Our theory can explain the recent in vivo observation of irregular chromatin having co-existence of finite fraction of the next-neighbor (i + 2) and neighbor (i + 1) nucleosome interactions.