Maeshima Kazuhiro

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  • Article
    Density imaging of heterochromatin in live cells using orientation-independent-DIC microscopy
    (American Society for Cell Biology, 2017-08-23) Imai, Ryosuke ; Nozaki, Tadasu ; Tani, Tomomi ; Kaizu, Kazunari ; Hibino, Kayo ; Ide, Satoru ; Tamura, Sachiko ; Takahashi, Koichi ; Shribak, Michael ; Maeshima, Kazuhiro
    In eukaryotic cells, highly condensed inactive/silenced chromatin has long been called “heterochromatin.” However, recent research suggests that such regions are in fact not fully transcriptionally silent and that there exists only a moderate access barrier to heterochromatin. To further investigate this issue, it is critical to elucidate the physical properties of heterochromatin such as its total density in live cells. Here, using orientation-independent differential interference contrast (OI-DIC) microscopy, which is capable of mapping optical path differences, we investigated the density of the total materials in pericentric foci, a representative heterochromatin model, in live mouse NIH3T3 cells. We demonstrated that the total density of heterochromatin (208 mg/ml) was only 1.53-fold higher than that of the surrounding euchromatic regions (136 mg/ml) while the DNA density of heterochromatin was 5.5- to 7.5-fold higher. We observed similar minor differences in density in typical facultative heterochromatin, the inactive human X chromosomes. This surprisingly small difference may be due to that nonnucleosomal materials (proteins/RNAs) (∼120 mg/ml) are dominant in both chromatin regions. Monte Carlo simulation suggested that nonnucleosomal materials contribute to creating a moderate access barrier to heterochromatin, allowing minimal protein access to functional regions. Our OI-DIC imaging offers new insight into the live cellular environments.
  • Article
    Flexible and dynamic nucleosome fiber in living mammalian cells
    (Landes Bioscience, 2013-08-12) Nozaki, Tadasu ; Kaizu, Kazunari ; Pack, Chan-Gi ; Tamura, Sachiko ; Tani, Tomomi ; Hihara, Saera ; Nagai, Takeharu ; Takahashi, Koichi ; Maeshima, Kazuhiro
    Genomic DNA is organized three dimensionally within cells as chromatin and is searched and read by various proteins by an unknown mechanism; this mediates diverse cell functions. Recently, several pieces of evidence, including our cryomicroscopy and synchrotron X-ray scattering analyses, have demonstrated that chromatin consists of irregularly folded nucleosome fibers without a 30-nm chromatin fiber (i.e., a polymer melt-like structure). This melt-like structure implies a less physically constrained and locally more dynamic state, which may be crucial for protein factors to scan genomic DNA. Using a combined approach of fluorescence correlation spectroscopy, Monte Carlo computer simulations, and single nucleosome imaging, we demonstrated the flexible and dynamic nature of the nucleosome fiber in living mammalian cells. We observed local nucleosome fluctuation (~50 nm movement per 30 ms) caused by Brownian motion. Our in vivo-in silico results suggest that local nucleosome dynamics facilitate chromatin accessibility and play a critical role in the scanning of genome information.
  • Article
    Single nucleosome imaging reveals loose genome chromatin networks via active RNA polymerase II.
    (Rockefeller University Press, 2019-03-01) Nagashima, Ryosuke ; Hibino, Kayo ; Ashwin, S. S. ; Babokhov, Michael ; Fujishiro, Shin ; Imai, Ryosuke ; Nozaki, Tadasu ; Tamura, Sachiko ; Tani, Tomomi ; Kimura, Hiroshi ; Shribak, Michael ; Kanemaki, Masato T. ; Sasai, Masaki ; Maeshima, Kazuhiro
    Although chromatin organization and dynamics play a critical role in gene transcription, how they interplay remains unclear. To approach this issue, we investigated genome-wide chromatin behavior under various transcriptional conditions in living human cells using single-nucleosome imaging. While transcription by RNA polymerase II (RNAPII) is generally thought to need more open and dynamic chromatin, surprisingly, we found that active RNAPII globally constrains chromatin movements. RNAPII inhibition or its rapid depletion released the chromatin constraints and increased chromatin dynamics. Perturbation experiments of P-TEFb clusters, which are associated with active RNAPII, had similar results. Furthermore, chromatin mobility also increased in resting G0 cells and UV-irradiated cells, which are transcriptionally less active. Our results demonstrated that chromatin is globally stabilized by loose connections through active RNAPII, which is compatible with models of classical transcription factories or liquid droplet formation of transcription-related factors. Together with our computational modeling, we propose the existence of loose chromatin domain networks for various intra-/interchromosomal contacts via active RNAPII clusters/droplets.