Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma
Apicomplexan parasites use Ca²⁺-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca²⁺-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes wi...
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| Format: | Thesis |
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Massachusetts Institute of Technology
2023
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| Online Access: | https://hdl.handle.net/1721.1/151884 |
| _version_ | 1811081096374255616 |
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| author | Chan, Alex Wai |
| author2 | Lourido, Sebastian |
| author_facet | Lourido, Sebastian Chan, Alex Wai |
| author_sort | Chan, Alex Wai |
| collection | MIT |
| description | Apicomplexan parasites use Ca²⁺-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca²⁺-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved.
In this thesis, I describe the discovery and characterization of a regulator of microneme exocytosis in Toxoplasma gondii. In the first chapter, I introduce T. gondii as a model apicomplexan to study motile stages during asexual stages. In the second chapter, I discuss combining sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in T. gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, I identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. In the third chapter, I describe the functional characterization of a target of CDPK1, the putative activating adaptor HOOK. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. I show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired secretion of microneme proteins, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle. |
| first_indexed | 2024-09-23T11:41:33Z |
| format | Thesis |
| id | mit-1721.1/151884 |
| institution | Massachusetts Institute of Technology |
| last_indexed | 2024-09-23T11:41:33Z |
| publishDate | 2023 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/1518842023-08-24T03:33:46Z Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma Chan, Alex Wai Lourido, Sebastian Massachusetts Institute of Technology. Department of Biology Apicomplexan parasites use Ca²⁺-regulated exocytosis to secrete essential virulence factors from specialized organelles called micronemes. Ca²⁺-dependent protein kinases (CDPKs) are required for microneme exocytosis; however, the molecular events that regulate trafficking and fusion of micronemes with the plasma membrane remain unresolved. In this thesis, I describe the discovery and characterization of a regulator of microneme exocytosis in Toxoplasma gondii. In the first chapter, I introduce T. gondii as a model apicomplexan to study motile stages during asexual stages. In the second chapter, I discuss combining sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in T. gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, I identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. In the third chapter, I describe the functional characterization of a target of CDPK1, the putative activating adaptor HOOK. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. I show the FHF complex is partially conserved in T. gondii, consisting of HOOK, an FTS homolog, and two parasite-specific proteins (TGGT1_306920 and TGGT1_316650). CDPK1 kinase activity and HOOK are required for the rapid apical trafficking of micronemes as parasites initiate motility. Moreover, parasites lacking HOOK or FTS display impaired secretion of microneme proteins, leading to a block in the invasion of host cells. Taken together, our work provides a comprehensive catalog of CDPK1 targets and reveals how vesicular trafficking has been tuned to support a parasitic lifestyle. Ph.D. 2023-08-23T16:16:24Z 2023-08-23T16:16:24Z 2023-06 2023-08-04T13:37:47.211Z Thesis https://hdl.handle.net/1721.1/151884 0000-0002-5444-5756 Attribution-ShareAlike 4.0 International (CC BY-SA 4.0) Copyright retained by author(s) https://creativecommons.org/licenses/by-sa/4.0/ application/pdf Massachusetts Institute of Technology |
| spellingShingle | Chan, Alex Wai Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma |
| title | Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma |
| title_full | Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma |
| title_fullStr | Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma |
| title_full_unstemmed | Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma |
| title_short | Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma |
| title_sort | analysis of cdpk1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in toxoplasma |
| url | https://hdl.handle.net/1721.1/151884 |
| work_keys_str_mv | AT chanalexwai analysisofcdpk1targetsidentifiesatraffickingadaptorcomplexthatregulatesmicronemeexocytosisintoxoplasma |