Authors

Discussion

Leishmania mexicana, a parasitic protist, is spread by sand flies and causes cutaneous leishmaniasis in humans and animals. Key to being able to complete its digenetic life cycle and to cause disease in its hosts, is the ability of the parasite to proliferate. It has a complex cell division cycle characterised by the ordered replication of several single-copy organelles, accompanied by cell cycle stage-dependent morphological changes. Understanding this complex process may provide insight into new therapeutic targets. Currently, cell cycle analysis is primarily carried out via flow cytometry, to assess the distribution of cells throughout the cycle, or using microscopy to understand the finer details of cell cycle presentation, such as proportions of cells undergoing mitosis. Both of these methods have limitations, with flow cytometry unable to provide specific cell cycle stage information, and microscopy being time-consuming and labour-intensive.

Here, we present a comprehensive analysis of the L. mexicana promastigote cell cycle in live cells using imaging flow cytometry. IFC boasts the high-throughput and quantitative analysis of flow cytometry with the visual and spatial information of microscopy. While IFC has previously been used to study host-parasite interactions of Leishmania, as well as morphology and viability, its application for cell cycle analysis is limited to mammalian cells (Terrazas et al. (2015) J Immunol Methods, 423, 93-98; Dandugumula et al. (2022) Pathogens, 23;11(9):952; Blasi et al. (2016) Nature, 7, 10256). Using Vybrant™ DyeCycle™ Orange (DCO), we can simultaneously quantify and visualise DNA in live cells. After developing gating and masking strategies, automated analysis of cell cycle distribution was achieved, providing information on the quantity of DNA within a cell, the number of nuclei and kinetoplasts and the length and width of the cell body.

However, DNA staining and morphology alone are insufficient for precise cell cycle stage identification, with late S-phase, G2, and early mitotic cells having similar morphological presentations. Therefore, tagging of the spindle-associated kinesin, LmxKINF, was carried out. By automatically determining the cell cycle-dependent localisation of KINF (single spot, rod shaped or two spots) we were able to improve the resolution of post-S phase cell cycle stage identification, and for the first time, define the proportion and duration of L. mexicana cells in G2.