Biology subjects

Minami, K.

Publications and source records attributed to Minami, K..

7 recordsLinked to original sources

bioRxiv2026

Machine learning-assisted Repli-Histo labeling reveals distinct transcription-dependent constraints on chromatin motion in living cells

Genomic DNA is wrapped around core histones to form nucleosomes, which are organized in cells from euchromatin to heterochromatin with distinct genome functions. Although transcription is known to shape chromatin behavior in live cells, it remains unclear how different transcription systems shape chromatin classes and nuclear subcompartments. We developed machine learning-assisted Repli-Histo labeling to classify euchromatin and heterochromatin classes (Classes IA, IB, II, and III) and combined it with single-nucleosome imaging in live cells. Nucleosome motion was progressively constrained from euchromatin to heterochromatin. RNA polymerase II inhibition by THZ1, DRB, or -amanitin increased nucleosome motion in euchromatic Classes IA and IB and in heterochromatin around nucleoli, but not at the nuclear periphery. In contrast, RNA polymerase I inhibition by CX-5461 selectively increased nucleosome motion in Class III heterochromatin around nucleoli. Our study reveals that Pol II and Pol I transcription shape chromatin behavior in distinct chromatin classes and nuclear subcompartments. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/736477v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@18fd1eeorg.highwire.dtl.DTLVardef@11f85fdorg.highwire.dtl.DTLVardef@95a59eorg.highwire.dtl.DTLVardef@ac4725_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Cohesin prevents local mixing of condensed euchromatic domains in living human cells

The human genome is folded into chromatin loops by the cohesin complex, forming functional chromatin domains that underlie transcription and DNA replication/repair. However, how cohesin organizes these domains in living cells, especially in active euchromatin, remains elusive. To address this question, we combined single-nucleosome imaging/tracking and super-resolution 3D-structured illumination microscopy (3D-SIM) with euchromatin-specific labeling of histone H3.3. Using this nanoscopic approach, we revealed that euchromatin forms condensed domains that are constrained by cohesin-mediated loops. This organization refines the classical textbook view of euchromatin as largely open, in line with emerging evidence. Transcription machinery appears to be located near the condensed domain surfaces/borders. Cohesin loss increased nucleosome fluidity within these domains without altering their overall compaction, leading to local mixing of domains and compromising transcriptional insulation. These findings uncover an unexpected physical role of cohesin in maintaining the integrity of condensed euchromatic domains and ensuring proper higher-order regulation of gene expression.

cell biology↗

bioRxiv2025

Liquid-like transcription condensates locally constrain chromatin in living human cells

The organization and dynamics of chromatin play important roles in transcriptional regulation. The transcription machinery is known to constrain chromatin dynamics. Recently, transcription condensates formed via liquid-liquid phase separation (LLPS) or other mechanisms have emerged as key regulators of gene expression. What is the physical nature of such condensates in the cell? Do they interact with and constrain chromatin? To address these questions, we focused on BRD4-NUT, a fusion oncoprotein found in NUT carcinoma. Using single-molecule dual-color imaging, we found that individual BRD4-NUT molecules diffuse within condensates like a viscous liquid in live human cells. Single-nucleosome imaging specific to euchromatin shows that these liquid-like condensates restrict the movement of chromatin through BRD4 bromodomain-dependent crosslinking of neighboring acetylated nucleosomes. Our findings uncover a previously unrecognized mechanism by which LLPS-based condensates modulate chromatin dynamics, suggesting that condensates contribute to genome regulation through physical, and not only biochemical, control.

cell biology↗

bioRxiv2024

Replication-dependent histone (Repli-Histo) labeling dissects the physical properties of euchromatin/heterochromatin in living human cells.

A string of nucleosomes, where genomic DNA is wrapped around histones, is organized in the cell as chromatin. Chromatin in the cell varies greatly, from euchromatin to heterochromatin, in its genome functions. It is important to understand how heterochromatin is physically different from euchromatin. However, their specific labeling methods in living cells are limited. To address this, we have developed replication-dependent histone labeling (Repli-Histo labeling) to label nucleosomes in euchromatin and heterochromatin based on DNA replication timing. We investigated local nucleosome motion in the four chromatin classes from euchromatin to heterochromatin of living human and mouse cells. We found that more euchromatic regions (earlier replicated regions) show greater nucleosome motion. Notably, the motion profile in each chromatin class persists throughout interphase. Genome chromatin is essentially replicated from regions with greater nucleosome motions, even though the replication timing program is perturbed. Our findings, combined with computational modeling, suggest that earlier replicated regions have more accessibility and local chromatin motion can be a major determinant of genome-wide replication timing.

cell biology↗

bioRxiv2020

IN VITRO RECONSTITUTION OF DYNEIN FORCE EXERTION IN BULK CYTOPLASM

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 force exertion from motors anchored on 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 size 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 places endomembrane transport as a significant mode of MT force exertion with far-reaching consequences for cellular organization.

biophysics↗

bioRxiv2020

Implementation of single molecule FRET for visualizing intramolecular movement in CRISPR-Cas9

SHORT ABSTRACTThis paper summarizes how to visualize the flexible inter-domain movements of CRISPR-associated protein Cas9 using single molecule FRET LONG ABSTRACTThe CRISPR-associated protein Cas9 is widely used as a genome editing tool because of its ability to be programmed to cleave any DNA sequence that is followed by a protospacer adjacent motif. The continuing expansion of Cas9 technologies has stimulated studies regarding the molecular basis of the Cas9 catalytic process. Here we summarize methods for single molecule FRET (smFRET) to visualize the inter-domain movements of Cas9 protein. Our measurements and analysis demonstrate flexible and reversible movements of the Cas9 domains. Such flexible movements allow Cas9 to adopt transient conformations beyond those solved by crystal structures and play important roles in the Cas9 catalytic process. In addition to the smFRET measurement itself, to obtain precise results, it is necessary to validate Cas9 catalytic activity. Also, fluorescence anisotropy data are required to interpret smFRET data properly. Thus, in this paper, we describe the details of these important additional experiments for smFRET measurements.

biophysics↗

bioRxiv2018

Estimating fish population abundance by integrating quantitative data on environmental DNA and hydrodynamic modelling

We propose a general framework of abundance estimation based on spatially replicated quantitative measurements of environmental DNA in which production, transport, and degradation of DNA are explicitly accounted for. Application to a Japanese jack mackerel (Trachurus japonicus) population in Maizuru Bay revealed that the method gives an estimate of population abundance comparable to that of a quantitative echo sounder method. These findings indicate the ability of environmental DNA to reliably reflect population abundance of aquatic macroorganisms and may o[ff]er a new avenue for population monitoring based on the fast, cost-e[ff]ective, and non-invasive sampling of genetic information.

ecology↗