| Project Detail |
Background and rationale: The infrakingdom of Alveolata groups a large diversity of unicellular eukaryotes with distinct trophic strategies such as phototrophic, predatory or parasitic and unified by the presence of cortical alveoli beneath the plasma membrane. Of significant medical and veterinary relevance, Plasmodium and Cryptosporidium spp. as well as Toxoplasma gondii, are intracellular parasites belonging to the Apicomplexa phylum that are sharing an apical complex tailored for motility and host cell invasion (1-3). It is composed of a set of specialized, regulated secretory organelles termed rhoptries and micronemes and cytoskeletal structures that include the conoid, an enigmatic, dynamic organelle playing a pivotal role in invasion. In motile parasites the conoid extrudes through the apical polar ring (APR) a predicted microtubule organizing center (MTOC) from which the subpellicular microtubules (SPMTs) emerge (4). The conoid is composed of a cone of spiraling tubulin fibers, two preconoidal rings (PCRs) and two intraconoidal microtubules (ICMTs) yet the molecular architecture, composition, function, and regulated coordination of these elements are not well understood. Personal contributions and working hypothesis: Most invasive stages of the Apicomplexa share a unique form of gliding motility powered by an actomyosin system which propels the parasites into host cell and across biological barriers. We and others have identified basic structural, enzymatic, and regulatory components of the gliding machinery which are to a large extent, conserved across the phylum. Instrumental to the invasion process includes the concerted discharge of rhoptry content upon tight contact with a host cell and exocytosis of the micronemes, through the conoid. Our recent findings established that i) components of the glideosome assemble at the PCRs; ii) the ICMTs is critical for organelle, ensuring the positioning of rhoptry neck and microtubule associated vesicles (MVs); iii) the extrusion of the conoid is powered by MyoH and controls motility; iv) RNG2 is a pivotal protein that tethers the conoid to the APR.The main objective of this proposal is to decipher how the molecular architecture and the functional components of the conoid govern a coordinated action leading to the spatio-temporal assembly of the glideosome, the release of rhoptries, the changes in parasites shape and the tight regulation of F-actin flux that control motility. The research plan is articulated around 4 independent and yet highly connected subprojects that are divided in specific aims:- Subproject A. Structure-function relationships of the glideosome-associated connector (GAC). Role of apical lysine methyltransferase (AKMT) in controlling formin 1 (FRM1)-dependent generation of F-actin flux. Determination of the architecture of the PCRs.- Subproject B. Determination of the composition and function of the ICMTs. Biochemical, and functional dissection of ICMAP1, 2, and 3 their role in rhoptry discharge and MVs positioning.- Subproject C. Role the SPMTs in organelle trafficking and change of shape in activated motile parasites. Identification and characterization of kinesins associated with the SPMT and ICMTs. Importance of microtubules posttranslational modifications in tubulin-based structures function. - Subproject D. Composition of the APR and contribution to conoid dynamics. Biochemical and functional dissection of APR proteins (RNG2, RNG6). Structural and molecular architecture of the conoid.Methodologies: This proposal combines reverse genetics with high-resolution imaging, biochemical and structural approaches (HDX-MS and Cryo-EM/ET), global proteomes and imaging (live-video and super-resolution fluorescence microscopy, scanning/dual beam electron microscopy) This work benefits of state-of-the art institutional core facilities as well as one partner and on collaborator. Expected results: The project aims to unveil the molecular architecture of the conoid and to provide a better molecular understanding of the perfectly orchestrated set of events leading to motility and invasion that culminates with the establishment of infection and the dissemination of these deadly parasites.Impact: The project is embracing basic cell biology, parasitology with the potential to fill major gaps in basic biology and to identified novel target to fill the unmet needs for intervention against important human parasites disease. |
