Analysis of theLeishmania mexicanapromastigote cell cycle using imaging flow cytometry provides new insights into cell cycle flexibility and events of short duration

Abstract

PromastigoteLeishmania mexicanahave a complex cell division cycle characterised by the ordered replication of several single-copy organelles, a prolonged S phase and very 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 ofL. mexicanain live cells. Firstly, we demonstrate that, unlike several other DNA stains, Vybrant™ DyeCycle™ Orange (DCO) is non-toxic and enables quantitative DNA imaging in liveL. mexicanapromastigotes. 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 ofL. mexicanapromastigote cell cycle events that, for the first time, includes the durations of individual G2, mitosis and post-mitosis phases. By applying IFC in this way, we were able, in minutes, to capture tens of thousands of high quality brightfield and fluorescent images of liveL. mexicanacells in solution, and to acquire quantitative data across multiple parameters for every image captured. Our custom-developed masking and gating scheme, allowed us to identify elusive G2 cells, show that cytokinesis commences during early mitosis and continues after mitosis is complete, and identify newly divided cells that were within the first 12 minutes of the new cell cycle. 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 ofLeishmaniacell division.

Impact of flagellated and elongated morphological phenotypes on the focusing behaviours of biological cells in inertial microfluidic devices

“…As various studies have looked at IM devices for sorting cells based on their cell cycle stage, promastigote Leishmania mexicana M379 Cas9 T7 parasites were used as the biological model in this study. When cultured in vitro , this parasite typically forms mixed populations of cells which vary in size and shape according to their stage in the cell cycle (G1, S, G2, mitosis (M) and cytokinesis) [30, 32]. This highly non-homogeneous population is named hereafter the parental cell population and was used as the control for all tests carried out in this study.…”

“…Parental parasites were chemically treated to arrest cells at specific stages (and consequently morphologies) of the cell cycle. Flavopiridol-treated cell populations – mostly arrested in G2 phase [30] – had a similar distribution of length to the untreated parental cells (mean length of 10.0 µm, compared to 9.5 µm for parental with an AUC of 0.54) but were generally wider (mean width of 4.9 µm, compared to 3.3 µm for parental and an AUC of 0.92). Hydroxyurea-treated cell populations on the other hand, reported to arrest other Leishmania spp .…”

“…The subsequent files were analysed using IDEAS® software to extract morphological measurements of individual cells. The cell body mask and the gating strategy outlined in [30] were used to remove images containing speed beads (added to the sample during acquisition for calibration purposes), debris, cell rosettes, two cells and out of focus cells. Area, aspect ratio (the minor axis of a fitted ellipsoid vs the major axis), length and width measurements were calculated for the remaining population of cells and the data was exported from IDEAS® for further analysis in R. Data smoothing was carried out for the length and width measurements to account for the relatively low resolution of the images (length and width measurements are calculated in IDEAS® in increments of 0.33 μm and 0.66 μm, respectively).…”