Disruption of microtubules uncouples budding and nuclear division inToxoplasma gondii

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Abstract
The tachyzoite stage of the protozoan parasite Toxoplasma gondiihas two populations of microtubules: spindle microtubules and subpellicular microtubules. To determine how these two microtubule populations are regulated, we investigated microtubule behavior during the cell cycle following treatment with microtubule-disrupting drugs. Previous work had established that the microtubule populations are individually nucleated by two distinct microtubule-organizing centers (MTOCs): the apical polar ring for the subpellicular microtubules and spindle pole plaques/centrioles for the spindle microtubules. When replicating tachyzoites were treated with 0.5 μM oryzalin or 1.0 mM colchicine they retained the capacity to form a spindle and undergo nuclear division. Although these parasites could complete budding,they lost the bulk of their subpellicular microtubules and the ability to reinvade host cells. Both nascent spindle and subpellicular microtubules were disrupted in 2.5 μM oryzalin or 5.0 mM colchicine. Under these conditions,parasites grew in size and replicated their genome but were incapable of nuclear division. After removal from 0.5 μM oryzalin, Toxoplasmatachyzoites were able to restore normal subpellicular microtubules and a fully invasive phenotype. When oryzalin was removed from Toxoplasmatachyzoites treated with 2.5 μM drug, the parasites attempted to bud as crescent-shaped tachyzoites. Because the polyploid nuclear mass could not be correctly segregated, many daughter parasites lacked nuclei altogether although budding and scission from the maternal mass was able to be completed. Multiple MTOCs permit Toxoplasma tachyzoites to control nuclear division independently from cell polarity and cytokinesis. This unusual situation grants greater cell cycle flexibility to these parasites but abolishes the checks for coregulation of nuclear division and cytokinesis found in other eukaryotes.