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Lourido, Sebastian Smith, Tyler A. Massachusetts Institute of Technology. Department of Biology 2022-08-29T16:30:11Z 2022-08-29T16:30:11Z 2022-05 2022-05-23T19:54:32.791Z https://hdl.handle.net/1721.1/145059 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. Ph.D. Massachusetts Institute of Technology In Copyright - Educational Use Permitted Copyright MIT http://rightsstatements.org/page/InC-EDU/1.0/ High-throughput functionalization of the Toxoplasma kinome uncovers a novel regulator of invasion and egress Thesis application/pdf Doctoral Doctor of Philosophy Publication unknown unknown <Publication xmlns="https://www.openaire.eu/cerif-profile/1.1/" id="3a65444f-7f39-4d0b-af47-e7394d9e191c"> <Type xmlns="https://www.openaire.eu/cerif-profile/vocab/COAR_Publication_Types">http://purl.org/coar/resource_type/c_1843</Type> <Title>High-throughput functionalization of the Toxoplasma kinome uncovers a novel regulator of invasion and egress</Title> <PublishedIn> <Publication> </Publication> </PublishedIn> <PublicationDate>2022-05</PublicationDate> <Authors> <Author> <DisplayName>Smith, Tyler A.</DisplayName> <Affiliation> <OrgUnit> </OrgUnit> </Affiliation> </Author> </Authors> <Editors> </Editors> <Publishers> <Publisher> <DisplayName>Massachusetts Institute of Technology</DisplayName> <OrgUnit /> </Publisher> </Publishers> <License>http://rightsstatements.org/page/InC-EDU/1.0/</License> <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> <Access xmlns="http://purl.org/coar/access_right" > </Access> </Publication>