Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress
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...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Springer Science and Business Media LLC
2022
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| Online Access: | https://hdl.handle.net/1721.1/146902 |
| _version_ | 1826194366747639808 |
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| author | Smith, Tyler A Lopez-Perez, Gabriella S Herneisen, Alice L Shortt, Emily Lourido, Sebastian |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Smith, Tyler A Lopez-Perez, Gabriella S Herneisen, Alice L Shortt, Emily Lourido, Sebastian |
| author_sort | Smith, Tyler A |
| collection | MIT |
| description | 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, we 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 a delayed phenotype, was shown to be a regulator of invasion and egress. We named the kinase Store Potentiating/Activating Regulatory Kinase (SPARK), based on its impact on intracellular Ca2+ stores. Despite homology to mammalian 3-phosphoinositide-dependent protein kinase-1 (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. |
| first_indexed | 2024-09-23T09:54:54Z |
| format | Article |
| id | mit-1721.1/146902 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T09:54:54Z |
| publishDate | 2022 |
| publisher | Springer Science and Business Media LLC |
| record_format | dspace |
| spelling | mit-1721.1/1469022022-12-17T03:38:41Z Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress Smith, Tyler A Lopez-Perez, Gabriella S Herneisen, Alice L Shortt, Emily Lourido, Sebastian Massachusetts Institute of Technology. Department of Biology 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, we 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 a delayed phenotype, was shown to be a regulator of invasion and egress. We named the kinase Store Potentiating/Activating Regulatory Kinase (SPARK), based on its impact on intracellular Ca2+ stores. Despite homology to mammalian 3-phosphoinositide-dependent protein kinase-1 (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. 2022-12-16T18:01:17Z 2022-12-16T18:01:17Z 2022 2022-12-16T17:43:41Z Article http://purl.org/eprint/type/JournalArticle https://hdl.handle.net/1721.1/146902 Smith, Tyler A, Lopez-Perez, Gabriella S, Herneisen, Alice L, Shortt, Emily and Lourido, Sebastian. 2022. "Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress." Nature Microbiology, 7 (6). en 10.1038/S41564-022-01104-0 Nature Microbiology Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ application/pdf Springer Science and Business Media LLC PMC |
| spellingShingle | Smith, Tyler A Lopez-Perez, Gabriella S Herneisen, Alice L Shortt, Emily Lourido, Sebastian Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress |
| title | Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress |
| title_full | Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress |
| title_fullStr | Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress |
| title_full_unstemmed | Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress |
| title_short | Screening the Toxoplasma kinome with high-throughput tagging identifies a regulator of invasion and egress |
| title_sort | screening the toxoplasma kinome with high throughput tagging identifies a regulator of invasion and egress |
| url | https://hdl.handle.net/1721.1/146902 |
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