Project Grant R21AI180525

ASSEMBLY OF THE TOXOPLASMA CORTICAL CYTOSKELETON - SUMMARY FAST ROUNDS OF HOST CELL INVASION AND INTRACELLULAR REPLICATION ARE RESPONSIBLE FOR TISSUE LESIONS CAUSED BY THE TACHYZOITE FORM OF THE OPPORTUNISTIC HUMAN PARASITE TOXOPLASMA GONDII. UNDERLYING THE LYTIC CYCLE IS A PARASITE-SPECIFIC REPLICATION MECHANISM, WHICH FORMS TWO NEW DAUGHTER CELLS IN THE CYTOSOL OF THE MOTHER (ENDODYOGENY). THE PROCESS IS FUNDAMENTALLY DIFFERENT FROM CELL DIVISION OF THE HOST AND RELIES ON A SPECIALIZED CYTOSKELETON, CALLED INNER MEMBRANE COMPLEX (IMC), WHICH IS DEPOSITED ON 22 SUBPELLICULAR MICROTUBULES (SPMTS). THIS UNIQUE CYTOSKELETON IS INVOLVED IN ALL ASPECTS OF THE LYTIC CYCLE AND ACTS AS AN ESSENTIAL SCAFFOLD DURING THE DIVISION PROCESS. THE CURRENT WORKING MODEL SUGGESTS SPMTS NUCLEATE FROM THE APICAL POLAR RING (APR), A PRESUMED SECONDARY MICROTUBULE-ORGANIZING CENTER (MTOC) IN THE PARASITE. HOWEVER, VERY LIMITED EXPERIMENTAL DATA EXISTS THAT CORROBORATES THIS. FURTHERMORE, HOW SPMTS CONNECT TO THE APR EARLY IN DIVISION IS ALSO UNKNOWN. SEVERAL FACTORS HAVE RECENTLY BEEN IDENTIFIED THAT FACILITATE THE ASSOCIATION OF SPMTS AND APR LATER IN THE BUDDING CYCLE, BUT PHENOTYPIC CONSEQUENCES OF PROTEIN DEPLETION INDICATE THAT NONE IMPACT THE EARLY DIVISION STEPS. THIS INDICATES THAT CURRENTLY THE EARLY EVENTS OF PARASITE BUDDING ARE INCOMPLETELY UNDERSTOOD. TO FILL THIS VOID, WE STARTED TO INTERROGATE THE BUDDING CYCLE USING ITERATIVE EXPANSION MICROSCOPY (EXM) AND IDENTIFIED AN INTRIGUING FIVE-FOLD ASSEMBLY OF THE NASCENT SPMTS (NSPMTS) AT THE ONSET OF DIVISION. OUR DATA INDICATE THAT NSPMTS ARE ORGANIZED INTO FIVE "SHEETS" -CONSISTING OF APPROXIMATELY FOUR INDIVIDUAL NSPMTS- GROUPED AROUND THE NASCENT APR. THIS SUGGESTS THAT NUCLEATION UNFOLDS IN DISTINCT AREAS AND A SEPARATE EVENT, LATER IN BUDDING, ARRANGES THE EVEN SPACING OF SPMTS AROUND THE APR AS SEEN IN MATURE PARASITES. TO PROBE FURTHER INTO THE NUCLEATION MECHANISM WE LOCALIZED G-TUBULIN TO THE FORMING APR, SPECIFICALLY AT THE ONSET OF DIVISION. ITS PRESENCE THERE IS SHORT-LIVED, INDICATING A FUNCTION SPECIFICALLY TAILORED TO THE EARLY DAUGHTER SCAFFOLD. WE HYPOTHESIZE THAT G-TUBULIN AND ITS ASSOCIATED COMPLEX (G-TUC) PLAY A PIVOTAL ROLE IN THE EARLY STAGES OF DIVISION. WE WILL TEST THIS UNDER TWO SPECIFIC AIMS. AIM 1 WILL ESTABLISH THE BUDDING-RELATED FUNCTIONS OF G-TUC. WE WILL STUDY THIS BY CONDITIONAL DEPLETION OF G-TUC COMPONENTS IN COMBINATION WITH ITERATIVE EXM. UNDER AIM 2 WE WILL EMPLOY RECIPROCAL PROXIMITY BIOTINYLATION IN COMBINATION WITH CHEMICAL PERTURBATION TO SPECIFICALLY REVEAL THE MOLECULAR MAKE-UP OF G-TUC AT THE FORMING CYTOSKELETON. THIS WILL ALLOW US TO GAIN A MORE COMPLETE UNDERSTANDING OF ITS MOLECULAR FUNCTIONS. TOGETHER, OUR FINDINGS WILL ESTABLISH THE ROLE OF G- TUC IN PARASITE BUDDING, WHICH MAY ALSO BE RELEVANT FOR OTHER APICOMPLEXAN PARASITES EQUIPPED WITH A SIMILAR CYTOSKELETON.