Identification and Characterization of the IMC Protein Family in Toxoplasma gondii

Thesis advisor: Marc-Jan GubbelsThe apicomplexan parasite Toxoplasma gondii divides rapidly and asexually through a unique process of internal daughter budding. The physical infrastructure for this process is the cytoskeleton, which is composed of subpellicular microtubules, flattened vesicles (alveoli), and a meshwork of intermediate filament-like proteins. This meshwork is composed of a family of 14 inner membrane complex (IMC) proteins that were identified based on the presence of a repeat sequence shared across the Alveolata, the alveolin-repeat. All 14 proteins were cloned as YFP fusions to study their subcellular localization and antibodies were generated against several representative IMC proteins. Each IMC displays unique spatio-temporal dynamics throughout development, but four physically distinct localizations were identified: eight IMCs localize to the alveoli, four IMCs localize to a structure known as the basal complex, IMC11 localizes to the apical cap in mature parasites, and IMC15 localizes primarily to the centrosomes and early buds. IMC15 is of particular interest because its appearance before membrane occupation and recognition nexus 1 (MORN1) in the early bud suggests that it is the first cytoskeletal component to associate with the buds. A conditional knockdown of this protein using the destabilization domain (DD) reveals IMC15 has a strong affinity for the centrosomes that overcomes targeting of the DD fusion protein to the proteasome and the presence of IMC15 in the early bud may not be necessary for the division process. Conditional knockdowns using a tetracycline repressible promoter reveal that a minimal amount of IMC15 is sufficient for parasite survival. In order to further characterize IMC15, dominant negative constructs based on mutating putative palmitoylation sites or overexpression of deletion constructs are being pursued. Collectively, the IMC family is being incorporated into the temporal and spatial dynamics of cytoskeletal development through the creation of a comprehensive timeline of daughter bud assembly. These findings are contributing unprecedented detail to the cell division process.Thesis (PhD) — Boston College, 2011.Submitted to: Boston College. Graduate School of Arts and Sciences.Discipline: Biology