Fig 1.
Distinct ultrastructure features were used to define seven cell cycle stages.
(A) 3D reconstructed Stage 1 promastigote cell showing the spatial organisation of organelles within the cell body. Organelles are modelled using the following colours: cell body (opaque green), flagellum (pink), nucleus (blue), kinetoplast (blue), mitochondrion (yellow), acidocalcisomes (dark purple), lipid bodies (red), glycosomes (pale green), contractile vacuole (dark blue), Golgi (purple), ER (pink), PFR (teal), flagellar pocket (deep red). Scale bar = 5 μm. (B) SBF-SEM data slice of a Stage 1 promastigote cell highlighting the ultrastructural features used to identify organelles. Scale bar = 2 μm. (C-D) 3D reconstruction and SBF-SEM data slice of seven distinct cell cycle stages representing the entire promastigote cell cycle. Organelles are modelled using the following colours: cell body (opaque green), old flagellum (pink), new flagellum (orange), nucleus (blue), kinetoplast (blue). (Stage 1) Cell with a single flagellum, kinetoplast and nucleus. (Stage 2) New flagellum (white arrow) wholly within the flagellar pocket. (Stage 3) Cell with a new flagellum that has emerged from the anterior cell tip (white arrow). (Stage 4) Cell with an elongated (dividing) kinetoplast and a single nucleus. (Stage 5) Cell with 2 kinetoplasts and a dividing nucleus. (Stage 6) Cell with two separate kinetoplasts and nuclei, with a division fold forming. (Stage 7) Cells with cytokinesis furrow ingression progressing from the anterior end of the cell (white arrow). Scale bar = 2 μm. (E) The approximate timings using ergodic principles of the seven cell cycle stages during the cell cycle. n = 500. (F) Dot plot showing cell body length across the seven cell cycle stages defined. Stage 7 was split into two categories: nascent daughter cell inheriting the old flagellum (square data points) and nascent daughter cell inheriting the new flagellum (triangle data points). Dot plot showing (G) cell body volume, (H) cell body surface area, (I) old flagellum length and (J) new flagellum length across the seven cell cycle stages defined. Error bars show ±standard deviation. Total number of cells per stage: 6 cells in Stage 1, 9 cells in Stage 2, 4 cells in Stage 3, 6 cells in Stage 4, 6 cells in Stage 5, 6 cells in Stage 6 and 4 cells in Stage 7.
Fig 2.
New flagellum rotation around the old flagellum does not occur.
(A-C) SBF-SEM data slice (scale bar = 1 μm) and two 3D reconstructed models (scale bar = 2 μm) of a Stage 1, 2 and 3 cell illustrating that there is no rotation of the new flagellum (orange) around the old flagellum (opaque pink) during new flagellum growth within and extending out of the flagellar pocket (FP—opaque red) The old flagellum paraflagellar rod (PFR–dark blue) is used as an orientation marker demonstrating the new flagellum is always on the right-hand side of the old flagellum PFR. Mature basal body (MBB), pro basal body (PBB), kinetoplast (K–blue). (D) SBF-SEM data slice showing a cytoplasmic ridge (arrow) within an existing flagellar pocket. Scale bar = 1 μm. (E) Longitudinal SBF-SEM data slice of Stage 2–5 cells showing the flagellar pocket exit (white dashed line). Cross sections of 3D reconstructed Stage 2–5 cell illustrating that the old flagellum (pink) and new flagellum (orange) share the same flagellar pocket (FP) exit (white dashed line) until Stage 5 post cytoplasmic ridge has formed and two separate flagellar pocket exits are present indicating flagellar pocket duplication is complete. Stage 4 shows the positioning of the cytoplasmic ridge (arrow) within the existing flagellar pocket, but the two flagella still share the same exit point. Scale bar = 1 μm. (F) Whole cell negative stain mounts of a cell with two flagella sharing the same flagellar pocket exit point (arrow). Scale bar = 10 μm. (G) Whole cell negative stain mounts of a cell with two flagella with two separate flagellar pocket exit points (arrow). Scale bar = 1 μm. (H) Transmission electron image of a flagellum with a cap at the distal tip (arrowhead). Scale bar = 500 nm. (I) Serial electron tomogram of the L. mexicana flagellar tip in longitudinal and cross-section. There is one annulus that sits within the outer doublets (arrow) and a second annulus that caps the outer doublets (arrowhead). (J) Whole mount detergent extracted L. mexicana cell. Higher magnification micrographs of the flagellum distal tip showing the capping structure (arrowhead). Scale bar = 5 μm. Inset scale bar = 200 nm.
Fig 3.
Increase in multi-copy organelle volumes are seen in early cell cycle stages.
(A) 3D reconstructed Stage 1–4 cells highlighting the duplication timing and movement of the Golgi (purple) and contractile vacuole (dark blue) as the flagellar pocket duplicates (opaque red). Scale bar = 1 μm. (B) SBF-SEM data slices of the appearance of an additional contractile vacuole (CV) and Golgi next to a CV and Golgi in a Stage 3 cell. Scale bar = 500 nm. (C) Dot plot graph showing contractile vacuole volumes across the seven cell cycle stages defined. Error bars show ±standard deviation; n = 41. (D-F) 3D reconstructed Stage 1 and Stage 6 cell showing the positioning of the lipid bodies (red), glycosomes (green) and acidocalcisomes (purple) within the cell body. White dotted line along the Stage 6 cell indicates where the division fold lies and the separation of the multi-copy organelles away from this fold in the cell membrane. Scale bar = 2 μm. Dot plots showing (G-I) the number of lipid bodies, glycosomes and acidocalcisomes per cell and (J-L) the total volume of lipid bodies, glycosomes and acidocalcisomes per cell across the seven cell cycle stages defined. n = 41. Key for 3C, 3G-L: Whole cells (circle data points), nascent daughter cell inheriting the old flagellum (square data points) and nascent daughter cell inheriting the new flagellum (triangle data points). Error bars show ±standard deviation. Total number of cells per stage: 6 cells in Stage 1, 9 cells in Stage 2, 4 cells in Stage 3, 6 cells in Stage 4, 6 cells in Stage 5, 6 cells in Stage 6 and 4 cells in Stage 7.
Fig 4.
Nuclear pores are excluded from the nuclear bridge during mitosis.
Dot plots showing (A) kinetoplast disk membrane volume, n = 56, (B) nucleus volume and (C) nucleus surface area per cell across the seven cell cycle stages defined. n = 52. (D) SBF-SEM dataset slices and 3D reconstruction of two nuclei (blue) and membrane bound structures (green—arrow). Scale bar = 1 μm. (E) SBF-SEM data slice of a Stage 1 nucleus showing the positioning of nuclear pores (arrows) within the nuclear envelope. Scale bar = 500 nm. (F) 3D reconstructed Stage 1 nucleus showing the distribution of nuclear pores (pink—white arrow) throughout the nuclear envelope. Scale bar = 500 nm. (G) Dot plot showing the number of nuclear pores (NP) per nucleus. n = 50. (H) Dot plot showing the nuclear pore (NP) density per nucleus. n = 50. (I) 3D reconstructed nuclei at different stages of mitosis illustrating the absence of nuclear pores (pink) along the mitotic nuclear bridge (arrow) as the nucleus progresses through mitosis. Scale bar = 500 nm. (J) 3D reconstruction of the ER (pink—white arrow) positioning in a Stage 1 and 5 cell. Scale bar = 2 μm. (K) SBF-SEM data slice showing the ER connected to the nuclear envelope (dashed box) in a Stage 1 cell. Scale bar = 1 μm. (L) SBF-SEM data slice showing the ER close to the sub-pellicular microtubules (dashed boxes). Scale bar = 1 μm. (M-N) Serial section tomograms of the ER (arrow) intercalating between the sub-pellicular microtubules (MTs). Scale bar = 50 nm. (O) 3D reconstructed Stage 1–7 cells showing the complex mitochondrion network throughout the cell cycle (yellow). Scale bar = 1 μm. Dot plots showing (P) the mitochondrion volume and (Q) mitochondrion surface area across the seven cell cycle stages defined. Stage 6 and 7 are split into 3 and 2 categories, respectively: cells with the mitochondrion not yet segregated (circle data points), nascent daughter cell inheriting the old flagellum (square data points) and nascent daughter cell inheriting the new flagellum (triangle data points). n = 50. (R) 3D reconstructed Stage 5 cell showing the branching of the mitochondrion network (yellow). Scale bar = 1 μm. Key for 4A-C, 4F, 4G, 4K: Whole cells (circle data points), nascent daughter cell inheriting the old flagellum (square data points) and nascent daughter cell inheriting the new flagellum (triangle data points). Error bars show ±standard deviation. Total number of cells per stage: 6 cells in Stage 1, 9 cells in Stage 2, 4 cells in Stage 3, 6 cells in Stage 4, 6 cells in Stage 5, 6 cells in Stage 6 and 4 cells in Stage 7.
Fig 5.
Sub-pellicular microtubule organisation from whole cell electron tomography reveals a cytokinesis-derived seam.
(A) 3D reconstructed Stage 5 cell showing the division fold occurring bi-directionally and along the anterior-posterior axis of the cell body. Scale bar = 1 μm. (B) SBF-SEM data slice of a Stage 5 cell showing the in-pushing of the cell body membrane to form the division furrow (arrows). Scale bar = 1 μm. (C) 3D reconstructed Stage 6 and 7 cell showing the twisting (arrows) of the old flagellum (pink) and the new flagellum (orange) away from each other as the furrow ingresses towards the posterior end of the cell. Scale bar = 1 μm. (D) 3D reconstructed Stage 7 cell showing the double indentation of the cell membrane to form the ridge along the anterior-posterior axis. Scale bar = 1 μm. (E) 3D reconstructed Stage 1 cell showing the presence of the ridge post abscission. Scale bar = 1 μm. (F) 3D reconstructed sub-pellicular microtubules of Stage 1 and 2 cells, from serial tomograms. In the two Stage 1 cells, a unique ‘seam’ of converging microtubules is visible (dashed circle). Scale bar = 1 μm. (G) Box whisker plot of sub-pellicular microtubule length distribution in each cell (Stage, S) analysed in 5F. Boxes represent the 25th, 50th and 75th percentiles and whiskers represent the 5th and 95th percentile. (H) Bar graph of number of sub-pellicular microtubules identified in each cell (Stage, S) analysed in 5F. This does not include the flagellar axoneme, microtubule quartet, or pocket and lysosome associated microtubules. Data points represent estimated minimum possible number of microtubules, from maximum cell radius. (I) Quantitative schematic of the microtubule organisation in each cell analysed in 5F, produced by ‘unwrapping’ the sub-pellicular microtubules around a line running along the midline of the long axis of the cell from anterior to posterior. In each plot, each line represents a microtubule and where it starts and ends along the cell midline, plotted, from top to bottom, clockwise around the cell looking posterior to anterior. The unique seams in the two Stage 1 cells are visible as neighbouring microtubules with ends far from the cell posterior. A problematic region for microtubule tracing near the Stage 1 cell posterior is indicted. (J) Plot of radial position, in 30 degree bins, where microtubules end in the Stage 1 and Stage 2 cells.
Fig 6.
Culture and sand fly derived promastigotes differ in organelle volume and spatial organisation.
(A) SBF-SEM data slice showing the cross section of the sand fly stomodeal valve. Scale bar = 5 μm. (B) SBF-SEM data slice of sand fly derived cells highlighting the key organelles identified. Scale bar = 1 μm. (C) 3D model of leptomonad promastigote cells showing the spatial organisation of organelles within the cell body. Organelles are modelled using the following colours: cell body (green), flagellum (pink), nucleus (blue), kinetoplast (blue), mitochondrion (yellow), acidocalcisomes (dark purple), lipid bodies (red) and the flagellar pocket (opaque deep red). Scale bar = 1 μm. (D) Scatter plot of cell body length against flagellum length. Each data point represents one cell. n = 16. Dot plot showing (E) cell body volume (F) cell body surface area (G) nucleus volume and (H) cell body surface area to volume ratio between the sand fly derived cells and culture derived (Stage 1) cells. n = 16, t-Test. (I) 3D models of leptomonads showing the spatial organisation of acidocalcisomes (purple) and lipid bodies (red) throughout the cell body. Scale bar = 1 μm. Dot plot showing (J) number of acidocalcisomes per cell (K) total acidocalcisomes volume per cell and (L) ratio of acidocalcisomes volume in cell between the sand fly derived cells and culture derived (Stage 1) cells. n = 16. Dot plot showing (M) number of lipid bodies per cell (N) total lipid body volume per cell and (O) ratio of lipid body volume in cell between the sand fly derived cells and culture derived (Stage 1) cells. n = 16. Error bars show ±standard deviation. 6 Stage 1 culture derived cells and 10 sand fly derived cells were analysed.
Fig 7.
Illustration of the morphological events occurring during the cell cycle of L. mexicana promastigote cells as defined in Fig 1.
Fig 7 does not represent the approximate timing of the cell cycle events from Fig 1E. Organelles are modelled using the following colours: cell body (grey), old flagellum (black), new flagellum (orange), nucleus (blue), kinetoplast (blue) mitochondrion (yellow), acidocalcisomes (dark purple), lipid bodies (red), glycosomes (pale green), contractile vacuole (dark blue), Golgi (purple), ER (pink), cytoplasmic spur in the flagellar pocket (red). Black dashed line represents the division fold and furrow forming along the anterior-posterior axis of the cell body. Blue dashed lines on the nuclear envelope represent nuclear pores. Stage 1 represents a G1 cell. The cell enters S-phase in Stage 2 and a new flagellum is assembled within the flagellar pocket and the contractile vacuole duplicates. The new flagellum grows in length and extends out of the flagellar pocket in Stage 3. The Golgi duplicates and the nucleus enters mitosis. In Stage 4 the kinetoplast begins to undergo division. During Stage 5, the kinetoplast has divided and the nucleus is in the process of nuclear division. A cytoplasmic spur forms within the existing flagellar pocket to divide the flagellar pocket between the old and new flagellum. One contractile vacuole and Golgi appears either side of the dividing flagellar pocket. A division fold forms along the anterior-posteriors axis, pinching the cell body into two nascent daughter cell morphologies. In Stage 6, two separate nuclei and kinetoplast are present and the flagellar pocket has divided. Lipid bodies, acidocalcisomes, glycosomes and the endoplasmic reticulum segregate between the two nascent daughter cells. The mitochondrion is the last organelle is segregate. During Stage 7, the cell body undergoes cytokinesis which gives rise to one daughter cell which inherits the old flagellum and one daughter cell inheriting the new flagellum.