Cell cycle synchronisation of Trypanosoma brucei by centrifugal counter-flow elutriation reveals the timing of nuclear and kinetoplast DNA replication

Figures

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  Figure 1. Counter flow centrifugal elutriation of Trypanosoma brucei. The elutriation chamber is centrifuged at a constant speed with continuous flow of buffer. Cells are introduced and retained in the chamber at low flow rate, and sequential increases in the flow rate forces the cells to elute from the chamber in size order, with smallest cells eluting first.

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  Figure 2. Time-course of early-G1 synchronised Pcf T. brucei. Cells samples were withdrawn hourly, stained with PI and analysed by flow cytometry (n = 50,000). (a) Flow cytometry trace of first cell cycle post-elutriation representing the PI signal (horizontal axis) versus cell count (vertical axis). (b) Bar chart of flow cytometry data for the first cell cycle post elutriation. (c) Bar chart of flow cytometry data for the third cell cycle post-elutriation. (d) Box plot of cell dimensions determined by CASY model TT (n > 1,500). Boxes represent the median and central quartiles, whiskers represent 9 and 91% of data, and * the mode.

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  Figure 3. Flow cytometry time-course of distinct G1 synchronised Pcf T. brucei. (a) Early-G1 cells (97% G1) eluted at 18 ml/min, (b) Mid-G1 cells (92% G1) eluted at 20 ml/min, (c) Late-G1 cells (82% G1) eluted at 22 ml/min. (d) Direct comparison of the proportion of G1 (left) and 1N1K cells (right) over time for the Early-, Mid- and Late-G1 fractions. Left panel - Flow cytometry of PI stained cells at hourly intervals post elutriation (n = 50,000 per time point). Right panel – Microscopy of DAPI stained cells (n > 200 per time point).

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  Figure 4. Cytological markers for cell cycle progression in T. brucei. (a) Schematic of events occurring as cells progress through the cell cycle. (b) Immunofluorescence microscopy of Pcf with BBA4 to detect basal bodies. (c) Observation of basal bodies in early eluting Pcf fractions. (d) Immunofluorescence microscopy of Pcf with α-H3Me2 to detect mitotic and post-mitotic cells. (e) Observation of α-H3Me2 positive cells in early eluting Pcf fractions. F18 - Early-G1 eluted at 18 ml/min, F20 - Mid-G1 cells eluted at 20 ml/min, F22 - Late-G1 cells eluted at 22 ml/min.

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  Figure 5. Model of the T. brucei cell cycle. (a) Model of the relative timing and duration of the nuclear and kinetoplast cell cycle based on temporal observations of early-, mid- and late-G1 Pcf fractions. (b) Model proposed by Woodward & Gull based on BrdU labelling of asynchronous cultures, adapted from29. The cell cycle phases are as follows: G1 –gap 1, SN – nuclear synthesis, SK – kinetoplast synthesis, G2 - gap 2, M - mitosis, C - cytokinesis, D – kinetoplast division and A – after division.

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  Figure 6. Time-course of early-G1 synchronised Bsf T. brucei. Cells samples were withdrawn hourly, stained with PI and analysed by flow cytometry (n = 50,000). (a) Flow cytometry trace of first cell cycle post-elutriation. (b) Bar chart of flow cytometry data post-elutriation. (c) Box plot of cell dimensions determined by CASY model TT (n > 1,500). Boxes represent the median and central quartiles, whiskers represent 9 and 91% of data, and * the mode.

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  Figure 7. Flow cytometry time-course of G1 synchronised Bsf T. brucei fractions. (a) Early-G1 cells (97% G1) eluted at 13 ml/min, (b) Early-G1 cells (96% G1) eluted at 15 ml/min, (c) Late-G1 cells (96% G1) eluted at 17 ml/min. (d) Direct comparison of the proportion of G1 (left) and 1N1K cells (right) over time for fractions eluted at 13, 15 and 17 ml/min. Left panel - Flow cytometry of PI stained cells at hourly intervals post elutriation (n = 50,000 per time point). Right panel – Microscopy of DAPI stained cells (n > 200 per time point).