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%).