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Revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging

Trypanosoma brucei, the causative agent of human African trypanosomiasis, is highly motile and must be able to move in all three dimensions for reliable cell division. These characteristics make long-term microscopic imaging of live T. brucei cells challenging, which has limited our understanding of...

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Autores principales: Muniz, Richard S., Campbell, Paul C., Sladewski, Thomas E., Renner, Lars D., de Graffenried, Christopher L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8797261/
https://www.ncbi.nlm.nih.gov/pubmed/35041719
http://dx.doi.org/10.1371/journal.ppat.1010218
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author Muniz, Richard S.
Campbell, Paul C.
Sladewski, Thomas E.
Renner, Lars D.
de Graffenried, Christopher L.
author_facet Muniz, Richard S.
Campbell, Paul C.
Sladewski, Thomas E.
Renner, Lars D.
de Graffenried, Christopher L.
author_sort Muniz, Richard S.
collection PubMed
description Trypanosoma brucei, the causative agent of human African trypanosomiasis, is highly motile and must be able to move in all three dimensions for reliable cell division. These characteristics make long-term microscopic imaging of live T. brucei cells challenging, which has limited our understanding of important cellular events. To address this issue, we devised an imaging approach that confines cells in small volumes within cast agarose microwells that can be imaged continuously for up to 24 h. Individual T. brucei cells were imaged through multiple rounds of cell division with high spatial and temporal resolution. We developed a strategy that employs in-well “sentinel” cells to monitor potential imaging toxicity during loss-of-function experiments such as small-molecule inhibition and RNAi. Using our approach, we show that the asymmetric daughter cells produced during T. brucei division subsequently divide at different rates, with the old-flagellum daughter cell dividing first. The flagellar detachment phenotype that appears during inhibition of the Polo-like kinase homolog TbPLK occurs in a stepwise fashion, with the new flagellum initially linked by its tip to the old, attached flagellum. We probe the feasibility of a previously proposed “back-up” cytokinetic mechanism and show that cells that initiate this process do not appear to complete cell division. This live-cell imaging method will provide a novel avenue for studying a wide variety of cellular events in trypanosomatids that have previously been inaccessible.
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spelling pubmed-87972612022-01-29 Revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging Muniz, Richard S. Campbell, Paul C. Sladewski, Thomas E. Renner, Lars D. de Graffenried, Christopher L. PLoS Pathog Research Article Trypanosoma brucei, the causative agent of human African trypanosomiasis, is highly motile and must be able to move in all three dimensions for reliable cell division. These characteristics make long-term microscopic imaging of live T. brucei cells challenging, which has limited our understanding of important cellular events. To address this issue, we devised an imaging approach that confines cells in small volumes within cast agarose microwells that can be imaged continuously for up to 24 h. Individual T. brucei cells were imaged through multiple rounds of cell division with high spatial and temporal resolution. We developed a strategy that employs in-well “sentinel” cells to monitor potential imaging toxicity during loss-of-function experiments such as small-molecule inhibition and RNAi. Using our approach, we show that the asymmetric daughter cells produced during T. brucei division subsequently divide at different rates, with the old-flagellum daughter cell dividing first. The flagellar detachment phenotype that appears during inhibition of the Polo-like kinase homolog TbPLK occurs in a stepwise fashion, with the new flagellum initially linked by its tip to the old, attached flagellum. We probe the feasibility of a previously proposed “back-up” cytokinetic mechanism and show that cells that initiate this process do not appear to complete cell division. This live-cell imaging method will provide a novel avenue for studying a wide variety of cellular events in trypanosomatids that have previously been inaccessible. Public Library of Science 2022-01-18 /pmc/articles/PMC8797261/ /pubmed/35041719 http://dx.doi.org/10.1371/journal.ppat.1010218 Text en © 2022 Muniz et al https://creativecommons.org/licenses/by/4.0/This 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 the original author and source are credited.
spellingShingle Research Article
Muniz, Richard S.
Campbell, Paul C.
Sladewski, Thomas E.
Renner, Lars D.
de Graffenried, Christopher L.
Revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging
title Revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging
title_full Revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging
title_fullStr Revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging
title_full_unstemmed Revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging
title_short Revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging
title_sort revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8797261/
https://www.ncbi.nlm.nih.gov/pubmed/35041719
http://dx.doi.org/10.1371/journal.ppat.1010218
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