Analysis of the Leishmania mexicana promastigote cell cycle using imaging flow cytometry provides new insights into cell cycle flexibility and events of short duration.

Autor: Howell J; James Watt School of Engineering, University of Glasgow, Glasgow, United Kingdom., Omwenga S; School of Infection and Immunity, University of Glasgow, Glasgow, United Kingdom., Jimenez M; Biomedical Engineering Department, University of Strathclyde, Glasgow, United Kingdom., Hammarton TC; School of Infection and Immunity, University of Glasgow, Glasgow, United Kingdom.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2024 Oct 03; Vol. 19 (10), pp. e0311367. Date of Electronic Publication: 2024 Oct 03 (Print Publication: 2024).
DOI: 10.1371/journal.pone.0311367
Abstrakt: Promastigote Leishmania mexicana have a complex cell division cycle characterised by the ordered replication of several single-copy organelles, a prolonged S phase and rapid G2 and cytokinesis phases, accompanied by cell cycle stage-associated morphological changes. Here we exploit these morphological changes to develop a high-throughput and semi-automated imaging flow cytometry (IFC) pipeline to analyse the cell cycle in live L. mexicana. Firstly, we demonstrate that, unlike several other DNA stains, Vybrant™ DyeCycle™ Orange (DCO) is non-toxic and enables quantitative DNA imaging in live promastigotes. Secondly, by tagging the orphan spindle kinesin, KINF, with mNeonGreen, we describe KINF's cell cycle-dependent expression and localisation. Then, by combining manual gating of DCO DNA intensity profiles with automated masking and morphological measurements of parasite images, visual determination of the number of flagella per cell, and automated masking and analysis of mNG:KINF fluorescence, we provide a newly detailed description of L. mexicana promastigote cell cycle events that, for the first time, includes the durations of individual G2, mitosis and post-mitosis phases, and identifies G1 cells within the first 12 minutes of the new cell cycle. Our custom-developed masking and gating scheme allowed us to identify elusive G2 cells and to demonstrate that the CDK-inhibitor, flavopiridol, arrests cells in G2 phase, rather than mitosis, providing proof-of-principle of the utility of IFC for drug mechanism-of-action studies. Further, the high-throughput nature of IFC allowed the close examination of promastigote cytokinesis, revealing considerable flexibility in both the timing of cytokinesis initiation and the direction of furrowing, in contrast to the related kinetoplastid parasite, Trypanosoma brucei and many other cell types. Our new pipeline offers many advantages over traditional methods of cell cycle analysis such as fluorescence microscopy and flow cytometry and paves the way for novel high-throughput analysis of Leishmania cell division.
Competing Interests: The authors have declared that no competing interests exist.
(Copyright: © 2024 Howell et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
Databáze: MEDLINE
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