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Density imaging of heterochromatin in live cells using orientation-independent-DIC microscopy

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...

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Autores principales: Imai, Ryosuke, Nozaki, Tadasu, Tani, Tomomi, Kaizu, Kazunari, Hibino, Kayo, Ide, Satoru, Tamura, Sachiko, Takahashi, Koichi, Shribak, Michael, Maeshima, Kazuhiro
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society for Cell Biology 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5687035/
https://www.ncbi.nlm.nih.gov/pubmed/28835378
http://dx.doi.org/10.1091/mbc.E17-06-0359
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author Imai, Ryosuke
Nozaki, Tadasu
Tani, Tomomi
Kaizu, Kazunari
Hibino, Kayo
Ide, Satoru
Tamura, Sachiko
Takahashi, Koichi
Shribak, Michael
Maeshima, Kazuhiro
author_facet Imai, Ryosuke
Nozaki, Tadasu
Tani, Tomomi
Kaizu, Kazunari
Hibino, Kayo
Ide, Satoru
Tamura, Sachiko
Takahashi, Koichi
Shribak, Michael
Maeshima, Kazuhiro
author_sort Imai, Ryosuke
collection PubMed
description 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.
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spelling pubmed-56870352018-01-22 Density imaging of heterochromatin in live cells using orientation-independent-DIC microscopy Imai, Ryosuke Nozaki, Tadasu Tani, Tomomi Kaizu, Kazunari Hibino, Kayo Ide, Satoru Tamura, Sachiko Takahashi, Koichi Shribak, Michael Maeshima, Kazuhiro Mol Biol Cell Articles 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. The American Society for Cell Biology 2017-11-07 /pmc/articles/PMC5687035/ /pubmed/28835378 http://dx.doi.org/10.1091/mbc.E17-06-0359 Text en © 2017 Imai et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.
spellingShingle Articles
Imai, Ryosuke
Nozaki, Tadasu
Tani, Tomomi
Kaizu, Kazunari
Hibino, Kayo
Ide, Satoru
Tamura, Sachiko
Takahashi, Koichi
Shribak, Michael
Maeshima, Kazuhiro
Density imaging of heterochromatin in live cells using orientation-independent-DIC microscopy
title Density imaging of heterochromatin in live cells using orientation-independent-DIC microscopy
title_full Density imaging of heterochromatin in live cells using orientation-independent-DIC microscopy
title_fullStr Density imaging of heterochromatin in live cells using orientation-independent-DIC microscopy
title_full_unstemmed Density imaging of heterochromatin in live cells using orientation-independent-DIC microscopy
title_short Density imaging of heterochromatin in live cells using orientation-independent-DIC microscopy
title_sort density imaging of heterochromatin in live cells using orientation-independent-dic microscopy
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5687035/
https://www.ncbi.nlm.nih.gov/pubmed/28835378
http://dx.doi.org/10.1091/mbc.E17-06-0359
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