Fig 1.
Analysis of L. mexicana promastigote morphology using IFC.
(A) IFC images from live and PFA- or methanol (MeOH)-fixed promastigote L. mexicana C9T7 cells. (B) Schematic of cell body mask-based morphological measurements used for IFC analysis. (C) Images of a cell with and without a mask. (D) Violin plots of the length and width of cell populations from masked IFC images (n >26,499). The box plots show the 25th and the 75th percentiles, and the mean in red. The whiskers represent the median +/- 1.5X interquartile range. Scale bars (dotted lines): 7 μm. See S1A Table for raw data.
Fig 2.
DCO stains DNA quantitatively in live L. mexicana promastigotes.
(A) Brightfield and fluorescence images of L. mexicana promastigote C9T7 cells at different cell cycle stages stained with DCO. N: nucleus; K: kinetoplast; F: flagellum. Scale bars: 7 μm. Note that, consistent with previous fluorescence studies, the nucleus appears to divide before the kinetoplast. (B) The fluorescence intensity profile of C9T7 cells (n = 13920) stained with DCO and analysed with IFC. Peaks are indicated with arrowheads for non-replicated DNA (2C; corresponding to cells in G1 phase), replicated DNA (4C; cells in G2/M phase or cytokinesis) and cells with intermediate DNA content (Int.; cells undergoing S phase). (C) Cell cycle modelling of the DCO DNA intensity profile (panel B) using the FCS Express™ Multicycle engine (Rabinovitch & Bagwell debris subtraction [43, 44] and Dean/Jett/Fox cell cycle modelling [45]. The cell cycle stage of each curve is indicated. (D) Graph comparing the proportions of cells with different DNA contents, as modelled following staining with PI (S2A and S2B Fig) and DCO (panels B and C). Error bars represent the standard deviations of the means of three replicates. See S1B Table for raw data.
Fig 3.
Distinguishing late S, G2, M and cytokinesis cells based on their DNA content and cell body parameters.
L. mexicana C9T7 promastigote cells were stained with DCO and analysed by IFC. (A) The DNA fluorescence profile previously presented in Fig 2B was manually gated using IDEAS™ software (blue boxes) to select cells with 2C, intermediate (Int.; 2C-4C) or 4C DNA content based on the peaks. The percentages of cells in each peak are given on the right. (B) Plot of the cell body lengths of cells within the gated 4C population (blue line). A 10 μm cut-off was applied to separate the two peaks (red and yellow boxes). (C) Plot of the cell body widths of each of the two subpopulations identified in panel B (yellow (length > 10 μm) and red (length ≤ 10 μm), with example images from the respective peaks shown in (D). The distal end of the daughter flagellum in cells with two flagella is indicated by a white arrowhead. Scale bar: 7 μm.
Fig 4.
Cell cycle-dependent expression and localisation of mNG:KINF facilitates the classification of cells in G2, M and cytokinesis.
(A) IFC images of live L. mexicana C9T7 mNG:KINF promastigote cells with differential expression of mNG:KINF (from left to right: brightfield, mNG:KINF (pink), IDEAS™-applied mask (cyan) and brightfield/mNG:KINF merge). The likely cell cycle stage of each example cell is indicated. A white arrowhead indicates the new flagellum that has just emerged from the flagella pocket in G2 phase. (B) Bar graph showing the proportions of cells displaying each expression/localisation pattern of mNG:KINF. (C) Examples of short and wide 1CF cells. Top: cell showing inaccurate masking of the mNG:KINF signal; such cells were subsequently reclassified as 2CF cells. Middle: cell in very early G1 phase (short, with 1F). Bottom: cell in G2 phase (with 2F). (D) % distribution of all cells within the 1CF population after the reclassification of the post-mitotic cells as 2CF. Example images of each cell type are shown. Scale bars: 7 μm. Error bars show standard deviation of the means of 3 replicates. See S1C–S1F Table for raw data.
Table 1.
Properties of C9T7 mNG:KINF cell cycle stage populations.
Fig 5.
Cytokinesis is highly pleiomorphic in L. mexicana promastigotes.
(A) Images of mitotic and post-mitotic C9T7 mNG:KINF cells, as determined from their mNG:KINF fluorescence pattern (spindle and 2CF), were scored according to their cytokinesis stage. Cell images were only included in this analysis if the cell’s A-P axis was visible; cells imaged from their poles were excluded. The cartoon indicates the anterior and posterior poles of a L. mexicana cell. Graph summarises the data from 3 replicate C9T7 mNG:KINF cultures and 146–197 cell images for each spindle and 2CF population. Error bars indicate the standard deviation of the mean. See S1I Table for raw data. (B-F) Example images of spindle and 2CF C9T7 mNG:KINF cells demonstrating different modes of cytokinesis (B: cleavage fold present but no furrow; C: furrowing from anterior pole; D: furrowing from posterior pole; E: furrowing from both poles; F: furrowing commencing internally along the A-P axis). Brightfield and brightfield/mNG:KINF merged images are shown. Closed arrowheads: cleavage fold; open arrowheads: cleavage furrow; arrows: joined posterior cell tips beyond the internal cleavage furrow; asterisks: cells where cleavage furrow is almost complete and cells are approaching abscission. Scale bars: 7 μm.
Fig 6.
Schematic of the L. mexicana cell cycle.
The inner circle shows the five main stages of the cell cycle, scaled according to their duration, along with the DNA content of cells progressing through those stages. The outer circle indicates the cell cycle-dependent expression of mNG:KINF, localising either to circular foci within the nucleus (1 or 2) or to the mitotic spindle. The ‘0’ indicates cells with no mNG:KINF expression. Cartoons of cells at each cell cycle stage showing the cell body and flagella (light purple), nuclei and kinetoplasts (dark purple), and cleavage fold are indicated on the outside. The bracketed cells indicate the diversity of furrowing observed in both mitotic and post-mitotic cells (anterior to posterior, posterior to anterior, bidirectional and internal). mNG:KINF nuclear fluorescence is in green.
Fig 7.
Flavopiridol arrests cells in G2 phase.
(A) L. mexicana promastigote C9T7 mNG:KINF cultures were incubated with flavopiridol or not (control) for one cell cycle (12.4 hrs) before being fixed in MeOH, stained with PI and analysed by flow cytometry. DNA content of individual cells (n > 10,000 per replicate) from duplicate cultures was assessed using FCS Express software and plotted (error bars: standard deviation). (B) The same cultures as in (A) were also analysed live by IFC (unstained), using the image analysis pipeline described above to determine the fluorescence pattern of mNG:KINF. n > 6500. (C) Example IFC images of 1CF cells with two flagella (white arrow heads) that accumulated following flavopiridol treatment. Brightfield, mNG and merged images are shown. Scale bar: 7 μm. Raw data is in S1J Table.
Table 2.
Summary of cell cycle data attainable using IFC pipelines.