Daughter microtubules depend on SFA fiber, but the fiber does not depend on microtubules.

<p>(A and B) Immunofluorescence of SFA2-HA parasites treated with oryzalin, a microtubule-disrupting agent that prevents formation of new microtubules in daughter cells, and that has a more moderate effect on existing stable microtubules in the mother <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001444#pbio.1001444-Morrissette3" target="_blank">[34]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001444#pbio.1001444-Morrissette4" target="_blank">[35]</a>. Note that after 12 h of treatment parasite cells fail to assemble buds (B) and compare to the matched control (A). SFA2-HA fibers are nonetheless detected. Note that in untreated parasites, daughter cells are normally detectable when similar fibers are observed. (C) SFA3-YFP parasites were treated with oryzalin and scored for the presence of SFA3-YFP after 0, 12, or 24 h of treatment. Note that treated parasites accumulate fibers. (D) Fiber length was measured in control parasites and parasites treated with oryzalin for 24 h. Data points reflect the mean fiber length per field of view scored (<i>n</i> = 18–81 fiber/field, four fields for each of three independent repeats). Fibers of treated parasites are overall shorter and more uniformly distributed in size. iΔSFA2 parasites were grown in the absence (E) or presence of ATc (F) and stained for acetylated tubulin (red), centrin (green), and DAPI (blue). The anti-acetylated tubulin antibody labels daughter buds strongly (as do antibodies to unmodified tubulin) in untreated mutants. ATc-treated iΔSFA2 parasites exhibit acetyl-tubulin staining exclusively in the mother cell cytoskeleton (note that two mutant cells are shown with microtubules encaging each entire cell; the cells are abnormally large due to the block in budding). No daughter microtubular-skeletons are discernible in these cells despite the fact that each cell shown has two nuclei, both of which are entering mitosis as indicated by the duplicated centrosomes. Scale bars = 5 µm.