Morphological events during the cell cycle of Leishmania major

Abstract

The morphological events involved in the Leishmania major promastigote cell cycle have been investigated in order to provide a detailed description of the chronological processes by which the parasite replicates its set of single-copy organelles and generates a daughter cell. Immunofluorescence labeling of β-tubulin was used to follow the dynamics of the subcellular cytoskeleton and to monitor the division of the nucleus via visualization of the mitotic spindle, while RAB11 was found to be a useful marker to track flagellar pocket division and to follow mitochondrial DNA (kinetoplast) segregation. Classification and quantification of these morphological events were used to determine the durations of phases of the cell cycle. Our results demonstrate that in L. major promastigotes, the extrusion of the daughter flagellum precedes the onset of mitosis, which in turn ends after kinetoplast segregation, and that significant remodelling of cell shape accompanies mitosis and cytokinesis. These findings contribute to a more complete foundation for future studies of cell cycle control in Leishmania.

Fig. 1.

Western blot of L. major promastigote cell extract with anti-β-tubulin KMX1 and anti-RAB11 antibodies. Promastigote cells were lysed in 0.5% NP-40, fractionated by centrifugation at 250 × g for 30 min into a soluble fraction (S) and an insoluble fraction (I), blotted, and probed with T. brucei anti-RAB11 and KMX1 antibodies. Two forms of L. major RAB11 were detected (black and red arrowheads).

Fig. 2.

Immunofluorescence analysis of β-tubulin during the L. major cell cycle. Fixed cells were labeled with mouse KMX1 anti-β-tubulin monoclonal antibody and Alexa 488-conjugated anti-mouse antibody (green). The nuclear and kinetoplast DNAs were stained with DAPI (blue). (A to I) Representative pictures of the 1,500 cells examined are shown. (Left) DIC images. (Right) Merged images of β-tubulin (green) and DAPI (blue) staining. The white arrowheads indicate the microtubules of the mitotic spindle. The white arrows indicate an area of constriction where β-tubulin accumulates along the division plane. The black arrowheads indicate the presence of a growing daughter flagellum. Bar = 10 μm.

Fig. 3.

RAB11 expression in L. major promastigotes. (A) Immunofluorescence labeling of L. major RAB11 by use of anti-TbRAB11 antibody. Fixed cells were labeled with rabbit anti-TbRAB11 and Alexa 594-conjugated anti-rabbit (red) antibodies. Cells were incubated with FITC-conjugated ConA (green) prior to fixation to allow labeling of the flagellar pocket (fp) and the endosomal compartment (e). The nucleus and kinetoplast are shown in blue (DAPI). (B) Western blot analysis of whole-cell extract of L. major expressing GFP-RAB11 probed with anti-TbRAB11 antibody. Proteins of the expected sizes for endogenous RAB11 and GFP-RAB11 are shown by red and green arrowheads, respectively. (C to F) Live promastigotes expressing GFP-RAB11 (green). (C and D) Examples of the GFP-RAB11 signals observed for 1N1K1F cells. The inset panel in the merged picture (right) shows a ×2 magnification of the kinetoplast area. (E and F) Cells were incubated with FM4-64 for 15 min at 4°C and for 2 min at room temperature to label the flagellar pocket and the early endosomal compartment. The cell presented in panel F, which possesses a growing daughter flagellum (black arrowhead), has a nascent second GFP-RAB11-positive structure (white arrowheads). Bar = 10 μm for all images.

Fig. 4.

Immunofluorescence analysis of RAB11 during kinetoplast division in L. major. Fixed cells were labeled with rabbit anti-TbRAB11 and Alexa 488-conjugated anti-rabbit (green) antibodies. The nuclear and kinetoplast DNAs were stained with DAPI (blue). The inset panels in the merged images (right) show ×2 magnifications of the kinetoplast area. The black arrowheads in the DIC pictures (left) indicate the presence of a daughter flagellum. (A to F) See the text for descriptions. Bar = 10 μm.

Fig. 5.

Events during cell division in L. major promastigotes detected by immunofluorescence microscopy. The nuclear and kinetoplast DNAs were stained with DAPI (blue). Microtubules associated with nuclear division and shape remodelling were visualized with mouse KMX1 anti-β-tubulin monoclonal antibody and Alexa 488-conjugated anti-mouse antibody (green). Events associated with kinetoplast segregation, daughter flagellar pocket production, and flagellum growth were visualized with anti-TbRAB11 and Alexa 594-conjugated anti-rabbit (red) antibodies. (Left) DIC images. (Right) Merged images of β-tubulin (green), RAB11 (red), and DAPI (blue) staining. The black arrowheads indicate daughter flagella. For each cell, the numbers of nuclei (N), kinetoplasts (K), and flagella (F) are shown. Asterisks notate organelles in S phase or G₂ phase prior to segregation, with M for nuclear mitosis and D for kinetoplast division. The white arrow indicates a cleavage furrow. The colored letter code refers to the nomenclature in Fig. 6. Bar = 10 μm.

Fig. 6.

Schematic representation of the L. major promastigote cell cycle. The results presented are the means for 3 independent experiments performed on wild-type L. major promastigotes in which 1,500 cells were counted. For each cell, the numbers of nuclei (N), kinetoplasts (K), and flagella (F) were scored. Asterisks notate organelles in S phase or G₂ phase, with M for nuclear mitosis and D for kinetoplast division. Cells with similar configurations were then grouped (configurations a to h′). The percentages of these populations within the total cell number are shown as means ± standard deviations. Cells classified as “others” possess a rare configuration or are cells in doublets or clusters. Panel A shows a simplified version of the promastigote cell cycle where only the most abundant configurations of cells are shown. Panel B shows the configurations that were omitted in panel A due to their low abundance.

Fig. 7.

Timing of the various phases of the L. major promastigote cell cycle. (A) Growth curve for L. major promastigotes in culture. Cell density was measured at hourly intervals. The generation time was calculated to be 10.2 h (r² = 0.9737). (B) Summary of the calculated durations (shown in hours [top line] and as fractions of 1 [bottom line]) and sequence of pre-M, M, and C phases for the nucleus and of pre-D, D, and A phases for the kinetoplast, represented as a linear map of the L. major promastigote cell cycle. Durations of phases were calculated using Williams analysis (33).