Behaviors of nucleosomes with mutant histone H4s in euchromatic domains of living human cells.

Since Robert Feulgen first stained DNA in the cell, visualizing genome chromatin has been a central issue in cell biology to uncover how chromatin is organized and behaves in the cell. To approach this issue, we have developed single-molecule imaging of nucleosomes, a basic unit of chromatin, to unveil local nucleosome behavior in living cells. In this study, we investigated behaviors of nucleosomes with various histone H4 mutants in living HeLa cells to address the role of H4 tail acetylation, including H4K16Ac and others, which are generally associated with more transcriptionally active chromatin regions. We ectopically expressed wild-type (wt) or mutated H4s (H4K16 point; H4K5,8,12,16 quadruple; and H4 tail deletion) fused with HaloTag in HeLa cells. Cells that expressed wtH4-Halo, H4K16-Halo mutants, and multiple H4-Halo mutants had euchromatin-concentrated distribution. Consistently, the genomic regions of the wtH4-Halo nucleosomes corresponded to Hi-C contact domains (or topologically associating domains, TADs) with active chromatin marks (A-compartment). Utilizing single-nucleosome imaging, we found that none of the H4 deacetylation or acetylation mimicked H4 mutants altered the overall local nucleosome motion. This finding suggests that H4 mutant nucleosomes embedded in the condensed euchromatic domains with excess endogenous H4 nucleosomes cannot cause an observable change in the local motion. Interestingly, H4 with four lysine-to-arginine mutations displayed a substantial freely diffusing fraction in the nucleoplasm, whereas H4 with a truncated N-terminal tail was incorporated in heterochromatic regions as well as euchromatin. Our study indicates the power of single-nucleosome imaging to understand individual histone/nucleosome behavior reflecting chromatin environments in living cells.

Chromatin organization and behavior in HRAS-transformed mouse fibroblasts.

In higher eukaryotic cells, a string of nucleosomes, where long genomic DNA is wrapped around core histones, are rather irregularly folded into a number of condensed chromatin domains, which have been revealed by super-resolution imaging and Hi-C technologies. Inside these domains, nucleosomes fluctuate and locally behave like a liquid. The behavior of chromatin may be highly related to DNA transaction activities such as transcription and repair, which are often upregulated in cancer cells. To investigate chromatin behavior in cancer cells and compare those of cancer and non-cancer cells, we focused on oncogenic-HRAS (Gly12Val)-transformed mouse fibroblasts CIRAS-3 cells and their parental 10T1/2 cells. CIRAS-3 cells are tumorigenic and highly metastatic. First, we found that HRAS-induced transformation altered not only chromosome structure, but also nuclear morphology in the cell. Using single-nucleosome imaging/tracking in live cells, we demonstrated that nucleosomes are locally more constrained in CIRAS-3 cells than in 10T1/2 cells. Consistently, heterochromatin marked with H3K27me3 was upregulated in CIRAS-3 cells. Finally, Hi-C analysis showed enriched interactions of the B-B compartment in CIRAS-3 cells, which likely represents transcriptionally inactive chromatin. Increased heterochromatin may play an important role in cell migration, as they have been reported to increase during metastasis. Our study also suggests that single-nucleosome imaging provides new insights into how local chromatin is structured in living cells.

Chromatin organization and DNA damage.

Genomic DNA is organized three-dimensionally in the nucleus as chromatin. Recent accumulating evidence has demonstrated that chromatin organizes into numerous dynamic domains in higher eukaryotic cells, which act as functional units of the genome. These compacted domains facilitate DNA replication and gene regulation. Undamaged chromatin is critical for healthy cells to function and divide. However, the cellular genome is constantly threatened by many sources of DNA damage (e.g., radiation). How do cells maintain their genome integrity when subjected to DNA damage? This chapter describes how the compact state of chromatin safeguards the genome from radiation damage and chemical attacks. Together with recent genomics data, our finding suggests that DNA compaction, such as chromatin domain formation, plays a critical role in maintaining genome integrity. But does the formation of such domains limit DNA accessibility inside the domain and hinder the recruitment of repair machinery to the damaged site(s) during DNA repair? To approach this issue, we first describe a sensitive imaging method to detect changes in chromatin states in living cells (single-nucleosome imaging/tracking). We then use this method to explain how cells can overcome potential recruiting difficulties; cells can decompact chromatin domains following DNA damage and temporarily increase chromatin motion (∼DNA accessibility) to perform efficient DNA repair. We also speculate on how chromatin compaction affects DNA damage-resistance in the clinical setting.

Liquid-like chromatin in the cell: What can we learn from imaging and computational modeling?

Chromatin in eukaryotic cells is a negatively charged long polymer consisting of DNA, histones, and various associated proteins. With its highly charged and heterogeneous nature, chromatin structure varies greatly depending on various factors (e.g. chemical modifications and protein enrichment) and the surrounding environment (e.g. cations): from a 10-nm fiber, a folded 30-nm fiber, to chromatin condensates/droplets. Recent advanced imaging has observed that chromatin exhibits a dynamic liquid-like behavior and undergoes structural variations within the cell. Current computational modeling has made it possible to reconstruct the liquid-like chromatin in the cell by dealing with a number of nucleosomes on multiscale levels and has become a powerful technique to inspect the molecular mechanisms giving rise to the observed behavior, which imaging methods cannot do on their own. Based on new findings from both imaging and modeling studies, we discuss the dynamic aspect of chromatin in living cells and its functional relevance.

In Vitro Reconstitution of Dynein Force Exertion in a Bulk Viscous Medium.

The forces generated by microtubules (MTs) and their associated motors orchestrate essential cellular processes ranging from vesicular trafficking to centrosome positioning [1, 2]. To date, most studies have focused on MT force exertion by motors anchored to a static surface, such as the cell cortex in vivo or glass surfaces in vitro [2-4]. However, motors also transport large cargos and endomembrane networks, whose hydrodynamic interactions with the viscous cytoplasm should generate sizable forces in bulk. Such forces may contribute to MT aster centration, organization, and orientation [5-14] but have yet to be evidenced and studied in a minimal reconstituted system. By developing a bulk motility assay, based on stabilized MTs and dynein-coated beads freely floating in a viscous medium away from any surface, we demonstrate that the motion of a cargo exerts a pulling force on the MT and propels it in opposite direction. Quantification of resulting MT movements for different motors, motor velocities, over a range of cargo sizes and medium viscosities shows that the efficiency of this mechanism is primarily determined by cargo size and MT length. Forces exerted by cargos are additive, allowing us to recapitulate tug-of-war situations or bi-dimensional motions of minimal asters. These data also reveal unappreciated effects of the nature of viscous crowders and hydrodynamic interactions between cargos and MTs, likely relevant to understand this mode of force exertion in living cells. This study reinforces the notion that endomembrane transport can exert significant forces on MTs.

Analyses of Transient Behaviors of No-Insulation REBCO Pancake Coils During Sudden Discharging and Overcurrent.

Stability margin of a high-temperature superconducting (HTS) coil is two or three orders of magnitude greater than that of a low-temperature superconducting coil. In recent years, many papers have reported test results of turn-to-turn no-insulation (NI) HTS coils having extremely enhanced thermal stability, such that burnout never occurs in an NI coil, even at an operating current exceeding 2.5 times the critical current. Thus, The main goal of this paper is to clarify transient electromagnetic and thermal behaviors and mechanism of the high thermal stability in an NI REBCO coil. A partial element equivalent circuit (PEEC) model is proposed for the numerical simulation of an NI REBCO coil, which considers a local electrical contact resistance between turns, an I-V characteristic of an REBCO tape, and local self and mutual inductances of the NI REBCO coil. Using the PEEC model, we investigate the influence of the turn-to-turn contact resistance on the transient behavior of the NI REBCO coil during sudden discharging. We also perform thermal conduction analyses with the PEEC model to clarify the transient behavior of an NI REBCO coil during an overcurrent operation.