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3D visualization of macromolecule synthesis
Measuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues. Here we perform in vivo injection of alkyne- or azide-modified analogs of thymidine, uridine, methionine, and glucosamine to label nascent synth...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7669265/ https://www.ncbi.nlm.nih.gov/pubmed/33051003 http://dx.doi.org/10.7554/eLife.60354 |
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author | Duerr, Timothy J Comellas, Ester Jeon, Eun Kyung Farkas, Johanna E Joetzjer, Marylou Garnier, Julien Shefelbine, Sandra J Monaghan, James R |
author_facet | Duerr, Timothy J Comellas, Ester Jeon, Eun Kyung Farkas, Johanna E Joetzjer, Marylou Garnier, Julien Shefelbine, Sandra J Monaghan, James R |
author_sort | Duerr, Timothy J |
collection | PubMed |
description | Measuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues. Here we perform in vivo injection of alkyne- or azide-modified analogs of thymidine, uridine, methionine, and glucosamine to label nascent synthesis of DNA, RNA, protein, and glycosylation. Three-dimensional volumetric imaging of nascent macromolecule synthesis was performed in axolotl salamander tissue using whole-mount click chemistry-based fluorescent staining followed by light sheet fluorescent microscopy. We also developed an image processing pipeline for segmentation and classification of morphological regions of interest and individual cells, and we apply this pipeline to the regenerating humerus. We demonstrate our approach is sensitive to biological perturbations by measuring changes in DNA synthesis after limb denervation. This method provides a powerful means to quantitatively interrogate macromolecule synthesis in heterogenous tissues at the organ, cellular, and molecular levels of organization. |
format | Online Article Text |
id | pubmed-7669265 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-76692652020-11-18 3D visualization of macromolecule synthesis Duerr, Timothy J Comellas, Ester Jeon, Eun Kyung Farkas, Johanna E Joetzjer, Marylou Garnier, Julien Shefelbine, Sandra J Monaghan, James R eLife Developmental Biology Measuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues. Here we perform in vivo injection of alkyne- or azide-modified analogs of thymidine, uridine, methionine, and glucosamine to label nascent synthesis of DNA, RNA, protein, and glycosylation. Three-dimensional volumetric imaging of nascent macromolecule synthesis was performed in axolotl salamander tissue using whole-mount click chemistry-based fluorescent staining followed by light sheet fluorescent microscopy. We also developed an image processing pipeline for segmentation and classification of morphological regions of interest and individual cells, and we apply this pipeline to the regenerating humerus. We demonstrate our approach is sensitive to biological perturbations by measuring changes in DNA synthesis after limb denervation. This method provides a powerful means to quantitatively interrogate macromolecule synthesis in heterogenous tissues at the organ, cellular, and molecular levels of organization. eLife Sciences Publications, Ltd 2020-10-14 /pmc/articles/PMC7669265/ /pubmed/33051003 http://dx.doi.org/10.7554/eLife.60354 Text en © 2020, Duerr et al http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited. |
spellingShingle | Developmental Biology Duerr, Timothy J Comellas, Ester Jeon, Eun Kyung Farkas, Johanna E Joetzjer, Marylou Garnier, Julien Shefelbine, Sandra J Monaghan, James R 3D visualization of macromolecule synthesis |
title | 3D visualization of macromolecule synthesis |
title_full | 3D visualization of macromolecule synthesis |
title_fullStr | 3D visualization of macromolecule synthesis |
title_full_unstemmed | 3D visualization of macromolecule synthesis |
title_short | 3D visualization of macromolecule synthesis |
title_sort | 3d visualization of macromolecule synthesis |
topic | Developmental Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7669265/ https://www.ncbi.nlm.nih.gov/pubmed/33051003 http://dx.doi.org/10.7554/eLife.60354 |
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