James Watt School of Engineering, University of Glasgow, Glasgow, United Kingdom.
School of Infection and Immunity, University of Glasgow, Glasgow, United Kingdom.
PLoS One. 2024 Oct 3;19(10):e0311367. doi: 10.1371/journal.pone.0311367. eCollection 2024.
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.
墨西哥前鞭毛体利什曼原虫具有复杂的细胞分裂周期，其特征是几个单拷贝细胞器的有序复制、延长的 S 期以及快速的 G2 和胞质分裂期，伴随着与细胞周期阶段相关的形态变化。在这里，我们利用这些形态变化来开发一种高通量和半自动化的成像流式细胞术（IFC）管道，以分析活的 L. mexicana 中的细胞周期。首先，我们证明，与其他几种 DNA 染料不同，Vybrant™ DyeCycle™ Orange（DCO）是无毒的，并能够对活的前鞭毛体进行定量 DNA 成像。其次，通过将孤儿纺锤体驱动蛋白 KINF 标记为 mNeonGreen，我们描述了 KINF 的细胞周期依赖性表达和定位。然后，通过对 DCO DNA 强度曲线进行手动门控，结合对寄生虫图像进行自动掩模和形态测量、对每个细胞的鞭毛数量进行视觉测定，以及对 mNG:KINF 荧光进行自动掩模和分析，我们提供了一种新的详细描述 L. mexicana 前鞭毛体细胞周期事件的方法，该方法首次包括单个 G2、有丝分裂和有丝分裂后阶段的持续时间，并在新细胞周期的前 12 分钟内识别 G1 细胞。我们定制的掩蔽和门控方案使我们能够识别难以捉摸的 G2 细胞，并证明 CDK 抑制剂 flavopiridol 将细胞阻滞在 G2 期而不是有丝分裂期，为 IFC 用于药物作用机制研究提供了原理验证。此外，IFC 的高通量性质允许对前鞭毛体胞质分裂进行密切检查，与相关的动基体寄生虫锥虫和许多其他细胞类型相比，在胞质分裂开始的时间和分裂沟的方向上都显示出相当大的灵活性。我们的新流水线与细胞周期分析的传统方法（如荧光显微镜和流式细胞术）相比具有许多优势，为利什曼原虫细胞分裂的新型高通量分析铺平了道路。
