Revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging.
PLoS Pathog
; 18(1): e1010218, 2022 01.
Article
en En
| MEDLINE
| ID: mdl-35041719
ABSTRACT
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.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Trypanosoma brucei brucei
/
División Celular
/
Microscopía Intravital
Idioma:
En
Revista:
PLoS Pathog
Año:
2022
Tipo del documento:
Article
País de afiliación:
Estados Unidos