Fig 1.
The morphotypes of Trypanosoma cruzi.
All T. cruzi morphotypes contain a flagellum that is nucleated from a basal body which is docked to the membrane of the flagellar pocket. The basal body is also connected to the kinetoplast, which is the mitochondrial DNA aggregate. The shape of the cell bodies is defined by a set of subpellicular MTs that underlie the plasma membrane (depicted only in the epimastigote form). In the epimastigote and trypomastigote forms, the flagellum emerges onto the cell surface and is attached to the cell body by the flagellum attachment zone (comprising the MtQ and FAZ filament), both of which extend towards the anterior end of the cell body. The primary morphologic difference between epimastigotes and trypomastigotes is the positioning of the kinetoplast-basal body-flagellar pocket, which is posterior to the nucleus in epimastigotes and anterior in trypomastigotes. Amastigote form parasites are small, ovoid-shaped cells with short flagella that extend only 2–3 μm outside of the flagellar pocket.
Fig 2.
T. cruzi epimastigotes incorporate tubulin PTMs at the cell posterior during cell division.
Epimastigote cells carrying a 3xTy1-mNeonGreen::TcFAZ25 allele were fixed and labeled with 1B41 to label the FAZ and posterior punctum (1B41, magenta), anti-Ty1 to label FAZ25 (TcFAZ25, green), and DAPI to label DNA (DNA, blue). Cells were then imaged using epifluorescence and DIC microscopy. (A) Very small 1N1K cells carried a 1B41 punctum at their cell posterior, which was much weaker or absent in larger 1N1K cells (B). (C) The new FAZ appeared on the ventral side of the cell and was FAZ25 and 1B41 positive. The 1B41 signal appeared at the posterior end at this point. (D) As the new FAZ and flagellum extended, the posterior 1B41 punctum increased in brightness. (E) Once the cell has begun cytokinesis, the 1B41 punctum is present at the extreme posterior of the cell body. The new-flagellum daughter cell appears to inherit a much shorter new FAZ and flagellum than the old-flagellum daughter. Asterisks highlight the 1B41 posterior punctum labeling, while the arrowheads identify the new FAZ.
Fig 3.
Defining and quantifying morphogenic intermediates in T. cruzi epimastigote cell division.
(A) Morphogenic intermediates were defined by the presence of a 1B41 punctum and if they were the new flagellum or old flagellum daughter (new-flagellum puncta: NF Puncta; old-flagellum puncta: OF puncta), or their nuclear, kinetoplast, and flagellum state (xNxKxF). Note that all cells that have begun flagellar duplication appear to contain a 1B41 punctum. (B) Cell counts over 9 d were used to calculate the cell division time of the Cas9-T7 epimastigote cells line. (C) Graph depicting the percentages of cells in each state as defined in (A). (D) Using the length of the cell cycle as measured in (B) and the percentages of cells in different cell cycle states measured in (C) allows the calculation of the time that the parasites spend in each state that was defined in (A).
Fig 4.
Quantitation of changes to cell morphology over the course of T. cruzi epimastigote cell division.
(A) Cells were fixed and stained with 1B41 to label the FAZ and posterior punctum and DAPI to label DNA. The cells were then imaged using epifluorescence and DIC microscopy, followed by measurement of all the cell features depicted in this schematic. All graphs were generated as SuperPlots, where each independent experiment is shown in a separate color, with the large circles represent the mean of each independent experiment, and black lines represent the mean and the standard deviation. (B) Measurements of total cell lengths, which includes flagellar overhang, broken down into the cell division categories defined in Fig 3A. (C) Measurements of cell body lengths, broken down into the cell division categories defined in Fig 3A. (D) Measurements of the distance between the cell posterior and the nucleus, broken down into the cell division categories defined in Fig 3A. (E) Measurements of the distance between the cell posterior to the kinetoplast, broken down into the cell division categories defined in Fig 3A. (F) Measurements of the length of the old FAZ, broken down into the cell division categories defined in Fig 3A. (G) Measurements of the length of the old flagellum, broken down into the cell division categories defined in Fig 3A. (H) Measurements of the width of the cell body, broken down into the cell division categories defined in Fig 3A. (I) Table showing significance levels between each pair- wise comparison. Statistical analysis was done by one-way ANOVA. ns = not significant, * p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 5.
The new FAZ does not reach length parity with the old FAZ prior to the completion of cell division.
(A) Cells were fixed and stained with 1B41 to label the FAZ and posterior punctum and DAPI to label DNA. The cells were then imaged using epifluorescence and DIC microscopy, followed by measurement of all the cell features depicted in this schematic. All graphs were generated as SuperPlots, where each independent experiment is shown in a separate color, with the large circles represent the mean of each independent experiment, and black lines represent the mean and the standard deviation. (B) Measurements of the length of the new FAZ as cells progress through cell division. (C) Measurements of the length of the new flagellum as cells progress through cell division. (D) Ratio of the length of the new FAZ to the old FAZ as cells progress through cell division. (E) Ratio of the length of the new flagellum to the old flagellum as cells progress through cell division. Statistical analysis was performed using one-way ANOVA ****p < 0.0001.
Fig 6.
TcSAS-6 levels fluctuate on the basal body during cell division.
(A) Cells carrying a 3xTy1-mNeonGreen::TcSAS-6 allele were fixed and labeled with 1B41 (1B41, magenta) to label the FAZ and posterior punctum, anti-Ty1 (TcSAS-6, green) to label the basal body, and DAPI to label DNA (DNA, blue). The cells were then imaged using epifluorescence and DIC microscopy. Inserts show a 3X enlargement of the basal body region. The empty arrowheads identify the new flagellum; the asterisks highlight the 1B41 posterior punctum. (B) Cells carrying a 3xTy1-mNeonGreen::TcSAS-6 allele were fixed and processed for U-ExM. Samples were labeled with anti-Ty1 (TcSAS-6, green) to label the basal body and TAT1 anti-tubulin antibody (Tubulin, magenta) to label the tubulin-containing structures within the cell. The samples were then imaged using SoRa superresolution microscopy. Boxed insets are a 3X magnification of the basal body region. The empty arrowheads identify the mature basal body, while the filled arrowheads identify the probasal body. Scale bars: 10 μm.
Fig 7.
TcTOEFAZ1 localizes to the dorsal side of the flagellum is expressed during all cell stages.
(A) Cells carrying a 3xTy1-mNeonGreen::TcTOEFAZ1 allele were fixed and labeled with 1B41 to label the FAZ and posterior punctum (1B41, magenta), anti-Ty1 to label TcTOEFAZ1 (TcTF1, green), and DAPI to label DNA (DNA, blue). The cells were then imaged using fluorescence and DIC microscopy. The asterisks identify the 1B41 posterior punctum. (B) Cells carrying a 3xTy1-mNeonGreen::TcTOEFAZ1 allele were fixed and processed for U-ExM. Samples were labeled with anti-Ty1 to label TOEFAZ1 (TcTF1, green) and TAT1 anti-tubulin antibody to label the tubulin-containing structures within the cell (Tubulin, magenta). The samples were then imaged using SoRa super-resolution microscopy. Boxed insets are a 3X magnification of the basal body region. The empty arrowheads point toward the new flagellum. Scale bars: 10 μm.
Fig 8.
TcPLK and TcKLIF localize to the FAZ and the posterior punctum during cell division.
(A) Cells carrying a 3xTy1-mNeonGreen::TcPLK allele were fixed and labeled with 1B41 to label the FAZ and posterior punctum (1B41, magenta), anti-Ty1 to label TcPLK (TcPLK, green), and DAPI to label DNA (DNA, blue). The cells were then imaged using fluorescence and DIC microscopy. Asterisks identify the 1B41 posterior punctum, empty arrowheads show TcPLK recruitment to the cell posterior, and the filled arrowhead shows TcPLK localization to the basal body. (B) Cells carrying a 3xTy1-mNeonGreen::TcKLIF allele were fixed and labeled with 1B41 to label the FAZ and posterior punctum (1B41, magenta), anti-Ty1 to label TcKLIF (TcKLIF, green), and DAPI to label DNA (DNA, blue). Asterisks show the 1B41 posterior punctum, empty arrowheads show TcKLIF location to the cell posterior, filled arrowheads show TcKLIF localization to the FAZ, and arrows show TcKLIF localization to the ingressing furrow.
Fig 9.
T. cruzi daughter cells divide at different rates.
(A) T. cruzi epimastigotes cells were confined in agarose microwells and then imaged using DIC microscopy. Images were taken every 10 min for 2 d. Wells containing one cell were identified and the timing of the first round of division was noted. The time that each daughter cell took to complete a subsequent round of division was recorded. Images show representative still shots of live T. cruzi cells undergoing division. In this example, over 220 minutes (3 h, 40 min) elapsed between the division of the first daughter cell and the second. (B) Staircase plot showing the timing of the first and second daughter cell divisions. T1/2 for division 1 was 12.8 h, while T1/2 for division 2 was 17.7 h. (C) Plot showing direct comparison of daughter cell division rates. Each line represents daughter cell division times from the same progenitor cell. Cell comparisons are color coded to depict cells imaged in the same live-cell imaging run.
Fig 10.
Model describing the different stages of epimastigote T. cruzi cell division described in this work.
(I) At the end of cytokinesis, two types of cells are observed: new flagellum daughters (NF Puncta) that are smaller, have a shorter flagellum and FAZ, and a more intense posterior punctum staining with 1B41 and KLF when compared to the old flagellum daughters (OF Puncta). In both cell types the pro and mature basal bodies are labeled with SAS-6 and TOEFAZ1 is present on the dorsal side of the flagellum. (II) Once the new flagellum daughter has extended its FAZ and flagellum, the two daughter cells are indistinguishable. The posterior punctum labeling disappears entirely. As the cell begins to divide, PLK is present on the basal body. TOEFAZ1 still labels the dorsal region. (III) As the new flagellum is assembled, both PLK and 1B41 labeling become apparent at the cell posterior. PLK is present along the FAZ as well. SAS-6 labeling appears primarily associated with the probasal bodies. The cell begins to widen along its short axis. As the new flagellum and FAZ continue to grow, the KLIF labeling appears at the site where the cleavage furrow will initiate from and is also present at the posterior punctum. TOEFAZ is recruited to cleavage furrow ingression point. Cell widening continues, while the cell posterior shortens along its long axis. (IV) As cytokinesis begins, the posterior punctum is labeled with 1B41, PLK, and KLIF and begins to resolve into two discrete structures. KLIF and TOEFAZ1 are present at the ingressing cleavage furrow, while PLK is present on both FAZ. SAS-6 labeling reappears at the mature basal body.