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Combining optogenetics with sensitive FRET imaging to monitor local microtubule manipulations
Optogenetic methods for switching molecular states in cells are increasingly prominent tools in life sciences. Förster Resonance Energy Transfer (FRET)-based sensors can provide quantitative and sensitive readouts of altered cellular biochemistry, e.g. from optogenetics. However, most of the light-i...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7138840/ https://www.ncbi.nlm.nih.gov/pubmed/32265472 http://dx.doi.org/10.1038/s41598-020-62874-3 |
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author | Van Geel, Orry Cheung, Stephanie Gadella, Theodorus W. J. |
author_facet | Van Geel, Orry Cheung, Stephanie Gadella, Theodorus W. J. |
author_sort | Van Geel, Orry |
collection | PubMed |
description | Optogenetic methods for switching molecular states in cells are increasingly prominent tools in life sciences. Förster Resonance Energy Transfer (FRET)-based sensors can provide quantitative and sensitive readouts of altered cellular biochemistry, e.g. from optogenetics. However, most of the light-inducible domains respond to the same wavelength as is required for excitation of popular CFP/YFP-based FRET pairs, rendering the techniques incompatible with each other. In order to overcome this limitation, we red-shifted an existing CFP/YFP-based OP18 FRET sensor (COPY) by employing an sYFP2 donor and mScarlet-I acceptor. Their favorable quantum yield and brightness result in a red-shifted FRET pair with an optimized dynamic range, which could be further enhanced by an R125I point mutation that stimulates intramolecular interactions. The new sensor was named ROPY and it visualizes the interaction between the microtubule regulator stathmin/OP18 and free tubulin heterodimers. We show that through phosphorylation of the ROPY sensor, its tubulin sequestering ability can be locally regulated by photo-activatable Rac1 (PARac1), independent of the FRET readout. Together, ROPY and PARac1 provide spatiotemporal control over free tubulin levels. ROPY/PARac1-based optogenetic regulation of free tubulin levels allowed us to demonstrate that depletion of free tubulin prevents the formation of pioneer microtubules, while local upregulation of tubulin concentration allows localized microtubule extensions to support the lamellipodia. |
format | Online Article Text |
id | pubmed-7138840 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-71388402020-04-11 Combining optogenetics with sensitive FRET imaging to monitor local microtubule manipulations Van Geel, Orry Cheung, Stephanie Gadella, Theodorus W. J. Sci Rep Article Optogenetic methods for switching molecular states in cells are increasingly prominent tools in life sciences. Förster Resonance Energy Transfer (FRET)-based sensors can provide quantitative and sensitive readouts of altered cellular biochemistry, e.g. from optogenetics. However, most of the light-inducible domains respond to the same wavelength as is required for excitation of popular CFP/YFP-based FRET pairs, rendering the techniques incompatible with each other. In order to overcome this limitation, we red-shifted an existing CFP/YFP-based OP18 FRET sensor (COPY) by employing an sYFP2 donor and mScarlet-I acceptor. Their favorable quantum yield and brightness result in a red-shifted FRET pair with an optimized dynamic range, which could be further enhanced by an R125I point mutation that stimulates intramolecular interactions. The new sensor was named ROPY and it visualizes the interaction between the microtubule regulator stathmin/OP18 and free tubulin heterodimers. We show that through phosphorylation of the ROPY sensor, its tubulin sequestering ability can be locally regulated by photo-activatable Rac1 (PARac1), independent of the FRET readout. Together, ROPY and PARac1 provide spatiotemporal control over free tubulin levels. ROPY/PARac1-based optogenetic regulation of free tubulin levels allowed us to demonstrate that depletion of free tubulin prevents the formation of pioneer microtubules, while local upregulation of tubulin concentration allows localized microtubule extensions to support the lamellipodia. Nature Publishing Group UK 2020-04-07 /pmc/articles/PMC7138840/ /pubmed/32265472 http://dx.doi.org/10.1038/s41598-020-62874-3 Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Van Geel, Orry Cheung, Stephanie Gadella, Theodorus W. J. Combining optogenetics with sensitive FRET imaging to monitor local microtubule manipulations |
title | Combining optogenetics with sensitive FRET imaging to monitor local microtubule manipulations |
title_full | Combining optogenetics with sensitive FRET imaging to monitor local microtubule manipulations |
title_fullStr | Combining optogenetics with sensitive FRET imaging to monitor local microtubule manipulations |
title_full_unstemmed | Combining optogenetics with sensitive FRET imaging to monitor local microtubule manipulations |
title_short | Combining optogenetics with sensitive FRET imaging to monitor local microtubule manipulations |
title_sort | combining optogenetics with sensitive fret imaging to monitor local microtubule manipulations |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7138840/ https://www.ncbi.nlm.nih.gov/pubmed/32265472 http://dx.doi.org/10.1038/s41598-020-62874-3 |
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