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Disruption of microtubules uncouples budding and nuclear division in Toxoplasma gondii

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis MO 63110, USA

View larger version (99K): [in a new window]   Fig. 1. Toxoplasma tachyzoites have two discrete populations of microtubules. (A) A diagram of the microtubules of Toxoplasma tachyzoites: the subpellicular microtubules and spindle microtubules. The sequence illustrates the behavior of microtubules during replication by endodyogeny. (A1) The subpellicular microtubules (red) are nucleated from the apical polar ring (green) and are tightly apposed to the inner membrane complex (gray dashed lines) that underlies the plasma membrane. (A2) The spindle microtubules (red) are nucleated from a spindle pole plaque embedded in the nuclear membrane (green). Extranuclear centrioles (red) are closely associated with the spindle pole plaques and daughter buds. (A3) Tachyzoites replicate by endodyogeny, a form of internal budding. The two daughter parasites (each containing a collection of apical organelles) are enclosed by individual sets of inner membrane complex and associated subpellicular microtubules. The mother parasite's subpellicular microtubules and apical complex are retained so that parasites are competent for invasion throughout the cell cycle. (B) Immunofluorescent labeling of the subpellicular microtubules (B1, arrow), spindle microtubules (B2, arrow) and daughter subpellicular microtubules (B3, arrows). The left-hand panels correspond to tubulin labeling and the right-hand panels are phase-contrast images of the parasites growing within primary fibroblasts.

View larger version (132K): [in a new window]   Fig. 2. (A) A thin section of a parasitophorous vacuole (PVM) enclosing two replicating parasites, each tachyzoite contains two daughter buds (marked with *). Half of the spindle is visible in each of the mature parasites and terminates in an invagination of the nuclear membrane (enlarged insets). Scale bar=1 µm. (B) Parallel projection of optical sections obtained with a confocal microscope showing tubulin (green), centrin (red) and DNA (blue) in replicating parasites. As replication proceeds, the dividing nucleus is V-shaped, as is the spindle (arrows), and it is surmounted by the subpellicular microtubules of the forming daughter buds (arrowheads). (C) A thin section of centrioles; the centriolar microtubules are singlet microtubules rather than the triplet microtubules observed in other centrioles. (D) A thin section of daughter parasites emerging from the maternal cell. In the outward-facing areas of the daughter cells, escape involves the coordinated dissociation of the maternal inner membrane complex from the plasma membrane and association of the daughter inner membrane complex onto the plasma membrane (arrow and enlarged inset). Between the two daughter cells, scission involves membrane fusion events to create new plasma membrane (arrow and enlarged inset). Scale bar=1 µm. (E) Optical sections (1-5) and parallel projection of optical sections (P) obtained with a confocal microscope showing tubulin (green) and DNA (blue) in eight tachyzoites that have just completed budding from four maternal cells. The fully intact maternal subpellicular microtubules, apical polar ring and conoid are located at the posterior of the daughter parasites (arrows).

View larger version (183K): [in a new window]   Fig. 3. Toxoplasma subpellicular microtubules and spindle microtubules are differentially sensitive to disruption by oryzalin or colchicine. Phase contrast of Toxoplasma tachyzoites (arrows) grown in HFF fibroblasts in 0.5 µM oryzalin (A) or 2.5 µM oryzalin (B). Electron microscopy of Toxoplasma grown in HFF fibroblasts in 0.5 µM oryzalin (C) or 2.5 µM oryzalin (D). Arrows in (C) indicate incomplete scission between daughter parasites in 0.5 µM oryzalin. Thin sections of tachyzoites in CV2-8 CHO cells in 1.0 mM colchicine (E) or 5.0 mM colchicine (F). In all electron micrographs, the scale bar represents 2 µM. The parasitophorous vacuole membrane is designated PVM and the host cell mitochondria that surround it are labeled M. Parasites are marked *.

View larger version (73K): [in a new window]   Fig. 4. In 0.5 µM oryzalin, the subpellicular microtubules are shortened or absent but nuclear division proceeds with correct segregation of the centrioles; in 2.5 µM oryzalin, both spindle and subpellicular microtubules are disrupted, nuclear division and budding cease and centrioles continue to duplicate unchecked. (A) Triple immunofluorescence of microtubules (green), IMC-1 (red) and DNA (blue) in control cells (top row), samples treated with 0.5 µM oryzalin (middle row) or samples treated with 2.5 µM oryzalin (bottom row). Without a drug, the subpellicular microtubules extend along two-thirds of the length of crescent-shaped tachyzoites. In 0.5 µM oryzalin, the subpellicular microtubules are greatly shortened or absent, although nuclear division continues with nuclear segregation. Daughter parasites are round rather than crescent shaped. At 2.5 µM oryzalin, all microtubules are disrupted and the parasite grows as a large round blob. (B) Triple immunofluorescence of tubulin (green), centrin (red) and DNA (blue) in control cells (top row), samples treated with 0.5 µM oryzalin (middle row) or samples treated with 2.5 µM oryzalin (bottom row). Centrioles appear as a single (non-replicated) spot or two (replicated) spots in untreated parasites. Centrioles continue to segregate correctly in 0.5 µM oryzalin, but in 2.5 µM oryzalin they continue to replicate although nuclear division is inhibited. (C) Triple immunofluorescence of microtubules (green), inner membrane complex (red) and DNA (blue) in cells treated with 0.5 µM oryzalin illustrates that although nuclear division occurs in 0.5 µM oryzalin, a subset of the nuclei fail to segregate correctly to the daughter buds and are retained in the residual body. (D) Quantification of nuclear division and segregation in 0.5 µM oryzalin demonstrates that the majority of replicating parasites (60%) correctly divides and segregates their nuclei. A smaller set of parasites undergoes nuclear division but retains one or both nuclei in the residual body (20%). A similar number of parasites either undergo aberrant nuclear division (producing unequally sized nuclei) or arrest prior to nuclear division although daughter buds are formed (20%).

View larger version (66K): [in a new window]   Fig. 5. Triple immunofluorescence of microtubules (green), IMC-1 (red) and DNA (blue) in samples treated for 48 hours with 0.5 µM oryzalin (A,B) or 2.5 µM oryzalin (C-E). Tachyzoites treated with 0.5 µM oryzalin undergo nuclear division and bud as round parasites (A). 48 hours after drug removal, these parasites continue replicating as crescent-shaped parasites with correctly sized and segregating nuclei and fully restored subpellicular microtubules (B). Tachyzoites treated with 2.5 µM oryzalin continue to grow (this is a single parasite) but fail to undergo nuclear division or bud off daughter parasites (C). When these samples are removed from the drug for 48 hours, they attempt to bud as multiple daughter parasites in a process reminiscent of schizogeny (D). The large, polyploid nuclear mass (large arrow) cannot be segregated correctly, so daughter buds (small arrows) are completely devoid of nuclear material or contain the apicoplast. Anucleate zoids (E1-4) can complete budding, escape from the vacuole and in some cases reinvade new cells but are incapable of growth or replication. E1 and 2 show triple-labeled zoids and E3 and 4 show the same cells with the tubulin staining omitted for clarity. The zoid in E1/3 has an apicoplast but lacks a nucleus, as does the left-hand zoid in E2/4. The right-hand zoid in E2/4 lacks both nucleus and apicoplast.

View larger version (38K): [in a new window]   Fig. 6. Drug washout experiments demonstrate that treatment with 0.5 µM oryzalin is fully reversible, but treatment with 2.5 µM oryzalin causes irreparable damage. Quantification of 48-hour oryzalin treatment followed by a 48-hour recovery in the absence of the drug demonstrates that Toxoplasma nuclear division and segregation occur in 0.5 µM oryzalin (left panel). The blue columns enumerate the numbers of normal and abnormal parasitophorous vacuoles that were present at the time of drug treatment (primary vacuoles). The green columns quantify the numbers of secondary vacuoles that were made by parasites that lysed from primary vacuoles and invaded new host cells during the washout and recovery phase. Parasites treated with 0.5 µM oryzalin can recover and go on to make invasive parasites that are capable of replication after drug removal. However, in 2.5 µM oryzalin (right panel), the continued replication of DNA is uncoupled from spindle microtubule-mediated chromosome segregation and produces both aberrant primary vacuoles and second generation parasites that have unbalanced nuclei or lack nuclei and are incapable of continued growth.

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