ABSTRACT
Chromatin modification is traditionally assessed in biochemical assays that provide average measurements of static events given that the analysis requires components from many cells. Microscopy can visualize single cells, but the cell body and organelles can hamper staining and visualization of the nucleus. Normally, chromatin is visualized by immunostaining a fixed sample or by expressing exogenous fluorescently tagged proteins in a live cell. Alternative microscopy tools to observe changes of endogenous chromatin in real-time are needed. Here, we isolated transcriptionally competent nuclei from cells and used antibody staining without fixation to visualize changes in endogenous chromatin. This method allows the real-time addition of drugs and fluorescent probes to one or more nuclei while under microscopy observation. A high-resolution map of 11 endogenous nuclear markers of the histone code, transcription machinery and architecture was obtained in transcriptionally active nuclei by performing confocal and structured illumination microscopy. We detected changes in chromatin modification and localization at the single-nucleus level after inhibition of histone deacetylation. Applications in the study of RNA transcription, viral protein function and nuclear architecture are presented.
This article has an associated First Person interview with the first author of the paper.
Footnotes
Competing interests
The authors declare no competing or financial interests.
Author contributions
Conceptualization: L.S., C.J., Z.A.K.; Methodology: L.S., Z.A.K.; Software: L.S.; Validation: L.S.; Formal analysis: L.S., A.L., S.K., L.B.; Investigation: L.S., S.K., L.R., W.E., T.J., H.V.; Resources: H.S., C.J., Z.A.K.; Writing - original draft: L.S.; Writing - review & editing: L.S., A.L., S.K., H.V., H.S., C.J., Z.A.K.; Visualization: L.S.; Supervision: Z.A.K.; Project administration: Z.A.K.; Funding acquisition: C.J., Z.A.K.
Funding
This work was supported by University of the Sciences internal funds, a National Institutes of Health (NIH)/NIDA Avenir award to Z.A.K., and a grant from the W. W. Smith Charitable Trust to Z.A.K. and C.J. Deposited in PMC for release after 12 months.
Supplementary information
Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.205823.supplemental
- Received May 5, 2017.
- Accepted July 13, 2017.
- © 2017. Published by The Company of Biologists Ltd
Article navigation
Related articles
Cited by...
More in this TOC section
Similar articles
Subject collections
Other journals from The Company of Biologists
In memoriam – Ian Moore
It is with great sadness that Chris Hawes writes the obituary of Ian Moore, Associate Professor in Plant Sciences and Fellow of Wadham College, University of Oxford. Ian succumbed to cancer in August. He made major contributions to plant molecular biology, gene expression systems, the secretory pathway and plant development.
Featured article - Identification and characterization of genes encoding the nuclear envelope LINC complex in the monocot species Zea mays
This Accepted Manuscript is about the plant nuclear envelope in a model grass species. Hank Bass talks about the working model his team propose for the entire maize LINC (Linker of Nucleoskeleton to Cytoskeleton) complex.
Condensins and cohesins generate intramolecular DNA loops. Robert Skibbens explains how condensin rods extrude DNA to compact chromatin during mitosis, whereas structurally adaptive cohesin rings organize the genome during interphase to regulate gene transcription.
Franziska Lautenschläger - credit to Iris Maurer
“Having children forces you to make a clean cut between work and home, and I feel this can be very beneficial because you are forced to think about something else.”
Franziska Lautenschläger’s research focuses on cell migration and polarity, and the links to cell mechanics and the different cytoskeletal networks in cells. Franziska holds a junior professorship in biophysics and is a group leader at both Saarland University and the Leibniz Institute for New Materials in Saarbrücken, Germany.
Want to put organising a Workshop on your CV? We are currently seeking proposals for Workshops to be held during 2021. You can watch this video to hear from some of our previous organisers. Next deadline for applications: 31 May 2019.
Journal of Cell Science is pleased to welcome submissions for an upcoming special issue on 'Cell Biology of the Immune System' edited by Ana-Maria Lennon-Duménil. We invite you to showcase your breakthrough research on all aspects of cell biology as it relates to the immune system. The articles within this issue will receive extensive exposure to a broad audience of cell biologists and immunologists. Submission deadline: 30 June 2019.
First Person interviews
Have you seen our First Person interviews with the early-career first authors of our papers? The authors talk about their work in and out of the lab, the journeys that led them to where they are now and the scientists who inspired them along the way. Recently, we caught up with first authors Yang Li and Chelsea Merkel, Tyler Picariello and Hardeep Gumber.
preLights – A complex containing lysine-acetylated actin inhibits the formin INF2
Laura McCormick highlights a recent preprint from Henry Higgs and team which uses some old-school biochemistry techniques to identify a cellular factor that facilitates INF2 auto-inhibition.
Catch up with recent preLights selected for the cell science community in our latest Table of Contents.
Articles of interest in our sister journals
A crucial role for Arf6 in the response of commissural axons to Slit
Mariko Kinoshita-Kawada, Hiroshi Hasegawa, Tsunaki Hongu, Shigeru Yanagi, Yasunori Kanaho, Ichiro Masai, Takayasu Mishima, Xiaoping Chen, Yoshio Tsuboi, Yi Rao, Junichi Yuasa-Kawada, Jane Y. Wu
Development 2019 146: dev172106
Rab23/Kif17 regulate meiotic progression in oocytes by modulating tubulin acetylation and actin dynamics
Hong-Hui Wang, Yu Zhang, Feng Tang, Meng-Hao Pan, Xiang Wan, Xiao-Han Li, Shao-Chen Sun
Development 2019 146: dev171280