{"id":{"repo_id":"mit","oai_identifier":"oai:dspace.mit.edu:1721.1/145059"},"canonical_url":"https://search.dev.ndltd.org/etd/mit/oai:dspace.mit.edu:1721.1/145059","repository":{"repo_id":"mit","name":"MIT","base_url":"https://dspace.mit.edu/oai/request"},"display":{"title":"High-throughput functionalization of the Toxoplasma kinome uncovers a novel regulator of invasion and egress","abstract":"Protein kinases regulate fundamental aspects of eukaryotic cell biology, making them attractive chemotherapeutic targets in parasites like Plasmodium spp. and Toxoplasma gondii. To systematically examine the parasite kinome, we developed a high-throughput tagging (HiT) strategy to endogenously label protein kinases with an auxin-inducible degron and fluorophore. Hundreds of tagging vectors were assembled from synthetic sequences in a single reaction and used to generate pools of mutants to determine localization and function. Examining 1,160 arrayed clones, I assigned 40 protein localizations and associated 15 kinases with distinct defects. The fitness of tagged alleles was also measured by pooled screening, distinguishing delayed from acute phenotypes. A previously unstudied kinase, associated with delayed death, was shown to be a regulator of invasion and egress. I call the kinase Store Potentiating/Activating Regulatory Kinase (SPARK), based on its impact on intracellular Ca2+ stores. Despite homology to mammalian PDK1, SPARK lacks a lipid-binding domain, suggesting a rewiring of the pathway in parasites. HiT screening extends genome-wide approaches into complex cellular phenotypes, providing a scalable and versatile platform to dissect parasite biology.","abstract_html":"Protein kinases regulate fundamental aspects of eukaryotic cell biology, making them attractive chemotherapeutic targets in parasites like Plasmodium spp. and Toxoplasma gondii. To systematically examine the parasite kinome, we developed a high-throughput tagging (HiT) strategy to endogenously label protein kinases with an auxin-inducible degron and fluorophore. Hundreds of tagging vectors were assembled from synthetic sequences in a single reaction and used to generate pools of mutants to determine localization and function. Examining 1,160 arrayed clones, I assigned 40 protein localizations and associated 15 kinases with distinct defects. The fitness of tagged alleles was also measured by pooled screening, distinguishing delayed from acute phenotypes. A previously unstudied kinase, associated with delayed death, was shown to be a regulator of invasion and egress. I call the kinase Store Potentiating/Activating Regulatory Kinase (SPARK), based on its impact on intracellular Ca2+ stores. Despite homology to mammalian PDK1, SPARK lacks a lipid-binding domain, suggesting a rewiring of the pathway in parasites. HiT screening extends genome-wide approaches into complex cellular phenotypes, providing a scalable and versatile platform to dissect parasite biology.","abstract_has_math":false,"creators":["Smith, Tyler A."],"institution":"Massachusetts Institute of Technology","degree_name":"Doctoral","degree_level":null,"degree_discipline":null,"degree_department":"Massachusetts Institute of Technology. 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To systematically examine the parasite kinome, we developed a high-throughput tagging (HiT) strategy to endogenously label protein kinases with an auxin-inducible degron and fluorophore. Hundreds of tagging vectors were assembled from synthetic sequences in a single reaction and used to generate pools of mutants to determine localization and function. Examining 1,160 arrayed clones, I assigned 40 protein localizations and associated 15 kinases with distinct defects. The fitness of tagged alleles was also measured by pooled screening, distinguishing delayed from acute phenotypes. A previously unstudied kinase, associated with delayed death, was shown to be a regulator of invasion and egress. I call the kinase Store Potentiating/Activating Regulatory Kinase (SPARK), based on its impact on intracellular Ca2+ stores. Despite homology to mammalian PDK1, SPARK lacks a lipid-binding domain, suggesting a rewiring of the pathway in parasites. HiT screening extends genome-wide approaches into complex cellular phenotypes, providing a scalable and versatile platform to dissect parasite biology."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph.D."]},{"key":"dc:title","label":"Title","values":["High-throughput functionalization of the Toxoplasma kinome uncovers a novel regulator of invasion and egress"]}]}],"canonical_facts":{"dc:contributor.advisor":["Lourido, Sebastian"],"dc:contributor.department":["Massachusetts Institute of Technology. Department of Biology"],"dc:creator":["Smith, Tyler A."],"dc:date.accessioned":["2022-08-29T16:30:11Z"],"dc:date.available":["2022-08-29T16:30:11Z"],"dc:date.issued":["2022-05"],"dc:description.abstract":["Protein kinases regulate fundamental aspects of eukaryotic cell biology, making them attractive chemotherapeutic targets in parasites like Plasmodium spp. and Toxoplasma gondii. To systematically examine the parasite kinome, we developed a high-throughput tagging (HiT) strategy to endogenously label protein kinases with an auxin-inducible degron and fluorophore. Hundreds of tagging vectors were assembled from synthetic sequences in a single reaction and used to generate pools of mutants to determine localization and function. Examining 1,160 arrayed clones, I assigned 40 protein localizations and associated 15 kinases with distinct defects. The fitness of tagged alleles was also measured by pooled screening, distinguishing delayed from acute phenotypes. A previously unstudied kinase, associated with delayed death, was shown to be a regulator of invasion and egress. I call the kinase Store Potentiating/Activating Regulatory Kinase (SPARK), based on its impact on intracellular Ca2+ stores. Despite homology to mammalian PDK1, SPARK lacks a lipid-binding domain, suggesting a rewiring of the pathway in parasites. HiT screening extends genome-wide approaches into complex cellular phenotypes, providing a scalable and versatile platform to dissect parasite biology."],"dc:description.degree":["Ph.D."],"dc:identifier.uri":["https://hdl.handle.net/1721.1/145059"],"dc:publisher":["Massachusetts Institute of Technology"],"dc:rights":["In Copyright - Educational Use Permitted","Copyright MIT"],"dc:rights.uri":["http://rightsstatements.org/page/InC-EDU/1.0/"],"dc:title":["High-throughput functionalization of the Toxoplasma kinome uncovers a novel regulator of invasion and egress"],"dc:type":["Thesis"],"thesis:degree_name":["Doctoral","Doctor of Philosophy"]},"updated_at":"2026-07-22T22:21:10Z"}