A more complex basal complex: novel components mapped to the Toxoplasma gondii cytokinesis machinery portray an expanded hierarchy of its assembly and function

Abstract

AbstractThe basal complex (BC) of Toxoplasma gondii has an essential role in cell division but details on the mechanism are lacking. To promote insights in this process, reciprocal proximity based biotinylation was used to map the basal complex proteome. An assembled protein map was interrogated by spatiotemporal characterization of critical components as well as functionally by disrupting the expression of the components. Spatially, this revealed four proteins sub-complexes with distinct sub-structural BC localization. Temporally, several patterns were differentiated based on their first appearance and/or disappearance from the BC corresponding with different steps in BC development (initiation, expansion, constriction, maturation). We also identified a protein pre-ceding BC formation (BCC0) laid out in a 5-fold symmetry. This symmetry marks the apical annuli and site of alveolar suture formation. From here, it was determined that the apical cap is assembled in the apical direction, whereas the rest of the IMC expands in the basal direction, inspiring a new bi-directional daughter budding process. Furthermore, we discovered BCC4, an essential protein exclusively localizing to the BC during cell division. Although depletion of BCC4 did not prevent BC formation, it led to BC fragmentation at the mid-point of cell division. Based on these data, a model is presented wherein BCC4 and MORN1 stabilize each other and form a rubber band that implies an essential role for the BC in preventing the fraying of the basal end of the assembling daughter cytoskeleton scaffolds. Furthermore, one new component of the Myosin J and Centrin2 cluster was BCC1, a hypothetical protein whose depletion prevents the non-essential last step of BC constriction. Overall, the BC is a highly dynamic, multi-functional structure that is critical to the hierarchical assembly of the daughter parasites.