Fig 1.
Diagram comparing trypomastigote and epimastigote morphology.
Black oval represents the nucleus; small red circle represents the kinetoplast, an organelle containing the tightly packaged mitochondrial DNA. In trypomastigotes (left) the kinetoplast is posterior to the nucleus, whereas in epimastigotes (right) the kinetoplast is typically anterior to the nucleus (1). In this category, though not as classically defined, we also include trypanosomes with the kinetoplast juxtaposed to the nucleus as shown in examples 2 and 3. The flagellum (thick black line) arises from a basal body that is physically linked to the kinetoplast [37].
Fig 2.
Time course of development of Trypanosoma congolense 1/148 YPFR in vitro.
Trypanosomes were imaged from time zero (T = 0) to 48 hours (T = 48) after seeding cultures from pooled proventricular forms. Each image, except T = 0, represents trypanosomes attached to the coverslip, as free-swimming cells were washed away. Brightfield image merged with the DAPI image (recoloured cyan for clarity). The kinetoplast is indicated by an arrowhead in each trypanosome at T = 0 and is easy to identify in all trypanosomes shown up to T = 20; in the dividing trypanosomes at T = 24 to T = 48, the kinetoplast is often juxtanuclear in the daughter cell and therefore indistinct. Scale bar = 10 μm.
Fig 3.
(A) Images from Trypanosoma congolense 1/148 YPFR time course showing decrease of flagellar length (visualised by YFP::PFR1 fluorescence) over time; details as Fig 2 legend. Note first appearance of PFR1 depot at T = 10 (arrows). The arrowheads indicate that the PFR does not extend to end of cell body (T = 6 to T = 14); white arrowhead, end of PFR; black arrowhead, end of cell body. Brightfield image merged with the YFP image (YFP::PFR1). Scale bar = 10 μm. (B) Length of flagellum (squares) and percentage of trypanosomes with YFP::PFR1 depot (circles) from T = 0 to T = 14. (C) Diagrammatic representation of morphological changes in trypanosomes from T = 0 to T = 14 compiled from morphometric data (S1 Table, S3 Table). The PFR is indicated by the thick black line, with depot shown as a filled rectangle.
Fig 4.
Proventricular trypanosomes from in vitro culture showing accumulation of YFP::PFR1 depot (arrows) in T. congolense WG81 YPFR (A, B) and S104 YPFR (C, D), and of RFP::PFR1 depot (arrows) in WG81 RPFR (E, F). Rows top to bottom: brightfield, DAPI, YFP or RFP, merge. Scale bar = 5 μm.
Table 1.
Presence of a depot of YFP::PFR1 in T. congolense 1/148 proventricular forms during the first cell division.
A total of 355 pairs of dividing cells were scored. Mother and daughter cells were distinguished by their relative positions and cell shape.
Fig 5.
Analysis of flagellar attachment in cytoskeleton preparations.
Cytoskeletons of Trypanosoma congolense 1/148 YPFR were prepared using 0.5% Triton (rows A, C, E and column G) or 0.5% Triton followed by CaCl2 treatment to selectively remove subpellicular microtubules (rows B, D, F and column H) [21]. Rows A and B after 1 hour incubation; rows C and D after 6 hours; rows E and F after 12 hours and columns G and H after 24 hours. Arrows indicate YFP::PFR1 depot; arrowheads indicate PFR of daughter flagellum (columns G and H). Rows A–F, L to R: brightfield, DAPI, merge, YFP, merge. The DAPI staining is more dispersed than usual, because of membrane disruption by detergent. Columns G and H, L to R: brightfield, YFP. Scale bar = 5 μm.
Fig 6.
Principal component analysis of cell morphometrics.
Data from Trypanosoma congolense 1/148 YPFR time course as Fig 2 legend. (A) PC1 and PC2 loading vectors for ten variables; all variables conformed to a normal distribution except for NPost, KPost and NL, which were therefore log transformed. (B) Mean values of PC1 and PC2 for trypanosome population at each time point. Data set T = 0 to T = 14 hours in blue; data set T = 0 to T = 120 minutes in orange. Data sets are in S1 Table and S2 Table.
Fig 7.
First cell division of proventricular forms of T. congolense.
Cultured proventricular forms from three different strains of T. congolense: WG81 (A), Gam2 (B), S104 (C). Rows top to bottom: brightfield, DAPI, merge. The white arrowheads indicate the kinetoplast; where the kinetoplast of the daughter cell is not indicated, it is presumably juxtanuclear and therefore hidden (columns 2 and 4). In column 2, the kinetoplast of the mother cell is elongated, indicating that a further round of cell replication has begun. Scale bar = 5 μm.
Fig 8.
Sequence of events in formation of first and second daughter cells.
Cultured proventricular forms of T. congolense 1/148 YPFR. (A) Cell prior to division, 1K1N; (B) elongated kinetoplast, 1K1N; (C, D) enlarged kinetoplast and growth of new flagellum, 1K1N; (E) nuclear division, 2K1N; (F, G) formation of cleavage furrow, 2K2N; (H) mother cell with attached daughter; enlarged kinetoplast and new flagellum indicate that mother cell is dividing again; (I) rosette of three cells. White arrows indicate YFP::PFR1 depot; black arrowheads indicate daughter cells. Rows top to bottom: brightfield, DAPI, merge, YFP, merge. Scale bar = 5 μm.
Fig 9.
Sequence of events during first division by SEM.
Sequence of events during division of proventricular cells of T. congolense 1/148. (A) emergence of the new flagellum (arrow) on the posterior side of the old flagellum. (B) separation of the origins of the two flagella. The mid-region of the old flagellum appears to contact the substrate in a pool of matter (arrow). The old flagellum does not extend to the tip of the mother cell body (arrowhead). (C) Formation of the cleavage furrow between the two flagella. The origin of the old flagellum sinks into the groove and is no longer visible. (D) The cleavage furrow extends further to the posterior and the nascent posterior ends of mother (M) and daughter (D) trypanosomes are visible (arrow). The mid-regions of the M and D flagella are in contact with the substrate in separate pools of matter. The old flagellum does not extend to the tip of the mother cell body (arrowhead). (E-G) The shape of the small daughter cell becomes increasingly well-defined during ingression of the cleavage furrow. The nascent posterior ends of mother (M) and daughter (D) trypanosomes are visible in E (arrow). The old flagellum does not extend to the tip of the mother cell body in G (arrowhead). (H) Preabscission. The anterior-posterior cleavage furrow is arrowed. (I) Preabscission. A broad cytoplasmic bridge remains (arrow). The old flagellum does not extend to the tip of the mother cell body (arrowhead). (J) Abscission. The tip of the daughter flagellum appears to be stuck in matter (arrow). (K) Mother cell, M, with two sequentially produced daughter cells, D1, D2. To the left is another dividing cell. Scale bar = 2 μm.
Fig 10.
SEM images of proventricular cells of T. congolense 1/148 during first cell division. (A) The posterior-to-posterior conformation with a narrow cytoplasmic bridge connecting the two trypanosomes (arrowed); both cells appear to be trypomastigotes, judging by the exit position of the flagellum, rather than a proventricular cell and daughter; scale bar = 2 μm. (B) Proventricular trypanosome with daughter cell (top) joined by a cytoplasmic bridge between the posterior of the mother cell and the mid-region of the daughter; judging by the length of the free flagellum of the daughter, these two cells are about to separate; scale bar = 2 μm. (C) Close up of the cytoplasmic bridge; scale bar = 1 μm. (D) Proventricular trypanosome with daughter cell (bottom) joined by a cytoplasmic bridge between the posterior of the mother cell and the mid-region of the daughter; scale bar = 2 μm. (E) Close up of the cytoplasmic bridge; scale bar = 1 μm.
Fig 11.
Comparison of the development of proventricular trypanosomes in Trypanosoma congolense and T. brucei.
The development of T. congolense is summarized from the current work, while that for T. brucei is from [3,38].