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Visualisation of ribosomes in Drosophila axons using Ribo-BiFC
The distribution of assembled, and potentially translating, ribosomes within cells can be visualised in Drosophila by using Bimolecular Fluorescence Complementation (BiFC) to monitor the interaction between tagged pairs of 40S and 60S ribosomal proteins (RPs) that are close neighbours across inter-s...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Company of Biologists Ltd
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6955225/ https://www.ncbi.nlm.nih.gov/pubmed/31822474 http://dx.doi.org/10.1242/bio.047233 |
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author | Singh, Anand K. Abdullahi, Akilu Soller, Matthias David, Alexandre Brogna, Saverio |
author_facet | Singh, Anand K. Abdullahi, Akilu Soller, Matthias David, Alexandre Brogna, Saverio |
author_sort | Singh, Anand K. |
collection | PubMed |
description | The distribution of assembled, and potentially translating, ribosomes within cells can be visualised in Drosophila by using Bimolecular Fluorescence Complementation (BiFC) to monitor the interaction between tagged pairs of 40S and 60S ribosomal proteins (RPs) that are close neighbours across inter-subunit junctions in the assembled 80S ribosome. Here we describe transgenes expressing two novel RP pairs tagged with Venus-based BiFC fragments that considerably increase the sensitivity of this technique we termed Ribo-BiFC. This improved method should provide a convenient way of monitoring the local distribution of ribosomes in most Drosophila cells and we suggest that it could be implemented in other organisms. We visualised 80S ribosomes in different neurons, particularly photoreceptors in the larva, pupa and adult brain. Assembled ribosomes are most abundant in the various neuronal cell bodies, but they are also present along the full length of axons. They are concentrated in growth cones of developing photoreceptors and are apparent at the terminals of mature larval photoreceptors targeting the larval optical neuropil. Surprisingly, there is relatively less puromycin incorporation in the distal portion of axons in the larval optic stalk, suggesting that some of the ribosomes that have initiated translation may not be engaged in elongation in growing axons. This article has an associated First Person interview with the first author of the paper. |
format | Online Article Text |
id | pubmed-6955225 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | The Company of Biologists Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-69552252020-01-15 Visualisation of ribosomes in Drosophila axons using Ribo-BiFC Singh, Anand K. Abdullahi, Akilu Soller, Matthias David, Alexandre Brogna, Saverio Biol Open Methods & Techniques The distribution of assembled, and potentially translating, ribosomes within cells can be visualised in Drosophila by using Bimolecular Fluorescence Complementation (BiFC) to monitor the interaction between tagged pairs of 40S and 60S ribosomal proteins (RPs) that are close neighbours across inter-subunit junctions in the assembled 80S ribosome. Here we describe transgenes expressing two novel RP pairs tagged with Venus-based BiFC fragments that considerably increase the sensitivity of this technique we termed Ribo-BiFC. This improved method should provide a convenient way of monitoring the local distribution of ribosomes in most Drosophila cells and we suggest that it could be implemented in other organisms. We visualised 80S ribosomes in different neurons, particularly photoreceptors in the larva, pupa and adult brain. Assembled ribosomes are most abundant in the various neuronal cell bodies, but they are also present along the full length of axons. They are concentrated in growth cones of developing photoreceptors and are apparent at the terminals of mature larval photoreceptors targeting the larval optical neuropil. Surprisingly, there is relatively less puromycin incorporation in the distal portion of axons in the larval optic stalk, suggesting that some of the ribosomes that have initiated translation may not be engaged in elongation in growing axons. This article has an associated First Person interview with the first author of the paper. The Company of Biologists Ltd 2020-01-02 /pmc/articles/PMC6955225/ /pubmed/31822474 http://dx.doi.org/10.1242/bio.047233 Text en © 2019. Published by The Company of Biologists Ltd http://creativecommons.org/licenses/by/4.0This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. |
spellingShingle | Methods & Techniques Singh, Anand K. Abdullahi, Akilu Soller, Matthias David, Alexandre Brogna, Saverio Visualisation of ribosomes in Drosophila axons using Ribo-BiFC |
title | Visualisation of ribosomes in Drosophila axons using Ribo-BiFC |
title_full | Visualisation of ribosomes in Drosophila axons using Ribo-BiFC |
title_fullStr | Visualisation of ribosomes in Drosophila axons using Ribo-BiFC |
title_full_unstemmed | Visualisation of ribosomes in Drosophila axons using Ribo-BiFC |
title_short | Visualisation of ribosomes in Drosophila axons using Ribo-BiFC |
title_sort | visualisation of ribosomes in drosophila axons using ribo-bifc |
topic | Methods & Techniques |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6955225/ https://www.ncbi.nlm.nih.gov/pubmed/31822474 http://dx.doi.org/10.1242/bio.047233 |
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