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ABSTRACT The body plan of the human parasite Toxoplasma gondii has a well-defined polarity. The minus ends of the 22 cortical microtubules are anchored to the apical polar ring, which is a putative microtubule-organizing center. The basal complex caps and constricts the parasite posterior end and is crucial for cytokinesis. How this apical–basal polarity is initiated is unknown. Here, we have examined the development of the apical polar ring and the basal complex using expansion microscopy. We found that substructures in the apical polar ring have different sensitivities to perturbations. In addition, apical–basal differentiation is already established upon nucleation of the cortical microtubule array: arc forms of the apical polar ring and basal complex associate with opposite ends of the microtubules. As the nascent daughter framework grows towards the centrioles, the apical and basal arcs co-develop ahead of the microtubule array. Finally, two apical polar ring components, APR2 and KinesinA, act synergistically. The removal of individual proteins has a modest impact on the lytic cycle. However, the loss of both proteins results in abnormalities in the microtubule array and in highly reduced plaquing and invasion efficiency. 

The tubulin-containing cytoskeleton of the human parasite Toxoplasma gondii includes several distinct structures: the conoid, formed of 14 ribbon-like tubulin polymers, and the array of 22 cortical microtubules (MTs) rooted in the apical polar ring. Here we analyze the structure of developing daughter parasites using both 3D-SIM and expansion microscopy. Cortical MTs and the conoid start to develop almost simultaneously, but from distinct precursors near the centrioles. Cortical MTs are initiated in a fixed sequence, starting around the periphery of a short arc that extends to become a complete circle. The conoid also develops from an open arc into a full circle, with a fixed spatial relationship to the centrioles. The patterning of the MT array starts from a “blueprint” with ∼five-fold symmetry, switching to 22-fold rotational symmetry in the final product, revealing a major structural rearrangement during daughter growth. The number of MT is essentially invariant in the wild-type array, but is perturbed by the loss of some structural components of the apical polar ring. This study provides insights into the development of tubulin-containing structures that diverge from conventional models, insights that are critical for understanding the evolutionary paths leading to construction and divergence of cytoskeletal frameworks.