Figure 1.
Estimation of generation time and the profile of organelle segregation in L. amazonensis promastigotes.
Panel A) Typical growth curve of L. amazonensis promastigotes in M199 medium at 28 °C. Promastigotes grew logarithmically in the range 1 x 106 to 3.5 x 107 cells ml-1. Panel B) Cell density was measured hourly over 12 h. The generation time was calculated to be 7 h (r2 = 0.983). Errors bars indicate SD of three independent assays. Panel C) Distinct morphological patterns observed in exponentially growing L. amazonensis promastigote cultures. The data were obtained from 1,186 cells counted from three independent axenic cultures of wild-type L. amazonensis promastigotes. Images are representative of DAPI-stained cells showing different organelle segregation, 1K1N are cells not in division, 1K2N and 2K1N are cells in division, and 2K2N are cells in post-M/post-D. Images were captured using a Nikon 80i and NIS element v.3.0 Software. K = kinetoplast, N = nucleus. Bars = 2 µm.
Figure 2.
DNA replication occurs simultaneously in the nucleus and in the kinetoplast of L. amazonensis promastigotes.
Panel A) EdU incorporation was revealed by click chemistry using azide labeled with Alexa 594. DAPI was used to stain DNA in the kinetoplast (K) and in the nucleus (N). The images represent cells in early, mid and late S phase, determined by EdU incorporation and the morphology of DAPI-stained nucleus and kinetoplast. The column on the right shows the percentage of cells in the population that had the same characteristics. Bars, 2 µm. Panel B) The amount of EdU-incorporation was estimated by fluorescence intensity using NIS elements v.3.0 software. The duration of S phase for each organelle was determined by morphology and increased fluorescence intensity, which was directly proportional to increased EdU incorporation. The maximum fluorescence intensity/incorporation (signaled as EdU saturation) indicated the end of S-phase for each DNA-containing organelle (approximately 120,000 a.u for nuclear DNA and approximately 10,000 a.u for kinetoplast DNA).
Figure 3.
Morphological patterns and flagellum emergence during the cell cycle of L. amazonensis promastigotes.
Panel A) The panel shows that a new flagellum emerges in S/G2 phases of 1K1N cells before the segregation of both kinetoplast and nucleus. EdU label was used as mark of DNA replication in both DNA-containing organelles. Panel B) The panel shows nuclear (blue) and kinetoplast (pink) events and demonstrates differences in the percentage of cells that segregate the kinetoplast before and after the nucleus. We can also observe that organelle segregation takes place after the appearance of a new flagellum in S and G2 phases of both organelles. In panels A and B, nuclear (blue) and kinetoplast (pink) events are, respectively labeled as G1, S, G2, M, D (kinetoplast division) and Post-M/Post-D (or cytokinesis). DIC (differential interference contrast), DAPI staining (blue), EdU labeling (green) and flagellum labeling with monoclonal antibody MAbAC (red). N = nucleus, K = kinetoplast. Bars = 2 µm. Panel C) The diagram represents a summary of the results shown in A and B.
Figure 4.
Cell cycle periods and timings for Leishmania amazonensis promastigotes.
Summary of the calculated durations and sequences of the nuclear and kinetoplast events are separately represented for 65% of cells that divide the kinetoplast before the nucleus (top) and for the remaining 35% that do the opposite (bottom). The estimated timing for the appearance of a new flagellum is also noted. These calculations were based on the generation time for L. amazonensis promastigotes (7 h), which corresponds to one unit of the cell cycle.
Figure 5.
HU-synchronized L. amazonensis promastigotes have the same behavior of organelle segregation as wild-type parasites.
Panel A) Histograms show DNA content per cell after propidium iodide staining and FACS analysis (20,000 total events counted per timepoint). The positions of G1, S, and G2/M, respectively, derived from the data using CellQuest software. Samples were harvested hourly after release from HU. Panel B) Graph shows a comparison of the timing estimated for nuclear + kinetoplast cell cycle phases (G1, S, G2/M/post-M) between non-synchronized and HU-synchronized cells. Panel C) Percentage (%) of HU-synchronized and of non-synchronized cells with differences in organelle segregation. In this assay we analyzed 1,186 non-synchronized cells used in most assays of this article and 1,020 synchronized cells which were harvested and analyzed (using DAPI staining) each 30 minutes after HU release up to the end of the cell cycle (around 4,5h after HU release). The percentage showed is related only to cells in division.