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First published online 19 October 2004
doi: 10.1242/jcs.01494

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Variability and heritability of cell division pathways in Toxoplasma gondii

¹ Department of Cell and Developmental Biology, University of Pennsylvania, 421 Curie Boulevard, Philadelphia, PA 19104, USA
² Department of Biology, University of Pennsylvania, 421 Curie Boulevard, Philadelphia, PA 19104, USA
³ Laboratorio de Parasitologia Molecular, IIB-INTECH, UNSAM-CONICET, Chascomus, Pcia. de Buenos Aires, Argentina

View larger version (78K): [in a new window]   Fig. 1. Structure of Toxoplasma gondii. The left-hand transparent view of a cell, mid-way through mitosis with two daughter cells forming, shows the conoid (green), inner membrane complex (IMC, red), and subpellicular microtubules (green), which run along the cytoplasmic face of the IMC. The right-hand view shows a longitudinal section of this cell with subpellicular microtubules removed for clarity. Lobes of the dividing nucleus, bordered by ER (yellow-green), Golgi (yellow), and apicoplast (mauve) are surrounded by the developing daughter IMCs (orange). Two rhoptries (secretory organelles, purple) are shown just below the maternal conoid at the apical end of the cell.

View larger version (104K): [in a new window]   Fig. 2. Images of T. gondii expressing histone 2b-YFP fusion protein. The h2b-YFP appears green and parasites were also stained with anti-IMCI antibody to show the inner membrane complex (cyan) and DAPI to visualize DNA (red). The images are the sum of two optical sections close to the middle of the parasites, taken from a deconvolved 3D stack of 15 optical sections 0.3 µm apart. (Upper left panels) Overview DIC and DIC/fluorescence overlay images showing three parasite-containing vacuoles and portions of three host cells. (Upper right and lower panels) Enlarged views of the rightmost vacuole, containing four parasites, of which parasites 1 and 2 are both forming two daughters, parasite 3 is at the very beginning of daughter cell formation where only traces of daughter scaffold can be seen, and parasite 4 is assembling four daughters. The three seemingly separate nuclei of parasite 4 were found to be interconnected in adjacent optical sections (not shown). Chromosome-like aggregations of condensed chromatin (arrowheads) are clearly visible in the segregating nuclei. Apicoplast DNA is also visible (arrows) labeled with DAPI only (red), not with H2b-YFP. In all other regions, the H2b-YFP distribution corresponds precisely to the DNA distribution stained by DAPI.

View larger version (71K): [in a new window]   Fig. 3. Time-lapse microscopy of T. gondii expressing a histone 2b-YFP fusion protein. At time zero, eight parasites in one vacuole have synchronously begun karyokinesis, revealed as a lobulation of the chromatin in the epifluorescence image (also discernible by phase contrast, arrow). Nuclear division is completed in some nuclei by 20 minutes and in all by 30 minutes (not shown). Cytokinesis is discernible by phase contrast approximately 15 minutes after the completion of nuclear division (45 minute time point) and is completed within 15 minutes (not shown).

View larger version (57K): [in a new window]   Fig. 4. Flow cytometry analysis of transgenic T. gondii expressing histone 2b-YFP and stained with Hoechst 3342. Two-dimensional scatter plot showing fluorescence in the YFP and Hoechst channels, on an arbitrary scale with logarithmic detection. Forward scatter intensity and pulse width, and side scatter intensity were used as gates to discriminate between intact single parasites (85.3% of 1.6x105 total events) and cell debris or multiple parasites in a cluster. Discrete peaks for haploid parasites are clearly observed: 1N DNA content, 94% of the parasite population (red background region) and 2N DNA content, 5.4% of the population (green background). A small number of parasites with DNA content 3N (0.1%, yellow background) are also present.

View larger version (91K): [in a new window]   Fig. 5. Multiple-daughter formation in T. gondii. Phase and epifluorescence time-lapse images of a vacuole containing nine mitotic parasites (V1) and a second vacuole containing four interphase parasites (V2). Two parasites in the first vacuole are forming four daughters each (red and yellow ovals), leading eventually (40 minute time point) to a vacuole with 22 parasites.

View larger version (95K): [in a new window]   Fig. 6. Heritability of the multiple-daughter phenotype in T. gondii. Fluorescence images of parasites stained with anti-IMC1 antibody (green) and DAPI (blue). (A) A vacuole containing seven parasites (i.e. the product of at least one multiple-daughter event in previous cycles) all of which are forming two daughters in the current cycle. (B) A vacuole containing six parasites (i.e. at least one prior multiple-daughter event) two of which, numbered 1 and 2, are forming four daughters each. In both A and B, a portion of the host cell nucleus is visible in the DAPI image.

View larger version (26K): [in a new window]   Fig. 7. Histograms of DNA content showing cell-cycle progression in extracellular parasites. Parasites were harvested and analyzed by flow cytometry according to three different protocols: (1) by mechanically inducing escape from host cells, washing to remove debris, staining with Hoechst 33342, followed by flow cytometry analysis (1 hr, red); (2) with a 12 hour delay after induced exit from host cells before washing, staining, and analysis (12 hr, green); and (3) harvested, washed, Hoechst stained and analyzed approximately 24 hours after spontaneous lysis of an entire culture dish of host cells (24 hr, blue). The graph shows superimposed histograms of fluorescence intensity (proportional to DNA content) of approximately 1x105 single cells from each of the three preparations, in arbitrary units on a logarithmic scale.

View larger version (78K): [in a new window]   Fig. 8. Cell division in extracellular parasites. Parasites expressing IMC1-YFP were harvested after lysing out of host cells and suspended in culture medium. A droplet of parasite suspension was put on a coverslip, and a cell in mitosis (orange oval) was identified under epifluorescence observation. Other parasites in suspension moved in and out of the field of view during the observation period. For reference, two additional stationary parasites (interphase) are indicated by the yellow asterisks. Images were recorded every 5 minutes for 30 minutes, during which time the parasite in the orange oval progressed from mid-mitosis through completion of cytokinesis to form two daughter cells.

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