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1. Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis in Toxoplasma

Author

Alex W Chan, Malgorzata Broncel, Eden Yifrach, Nicole R Haseley, Sundeep Chakladar, Elena Andree, Alice L Herneisen, Emily Shortt, Moritz Treeck, and Sebastian Lourido

Subjects
Toxoplasma gondii, signaling, host–pathogen interactions, vesicular trafficking, CDPK, Medicine, Science, Biology (General), QH301-705.5
Abstract

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. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates in Toxoplasma gondii to identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We 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 microneme protein secretion, 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.

Published
2023
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2. Temporal and thermal profiling of the Toxoplasma proteome implicates parasite Protein Phosphatase 1 in the regulation of Ca2+-responsive pathways

Author

Alice L Herneisen, Zhu-Hong Li, Alex W Chan, Silvia NJ Moreno, and Sebastian Lourido

Subjects
Toxoplasma gondii, calcium, thermal proteome profiling, Medicine, Science, Biology (General), QH301-705.5
Abstract

Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca2+ signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the replicative and lytic phases of the infectious cycle, as well as the transition between them. Despite the presence of conserved Ca2+-responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca2+-responsive proteome of the model apicomplexan Taxoplasma gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca2+ release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca2+-responsive proteins, such as parasite Ca2+-dependent protein kinases. Our approach also identified numerous Ca2+-responsive proteins that are not predicted to bind Ca2+, yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca2+-responsive enzyme that relocalized to the parasite apex upon Ca2+ store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca2+ uptake to promote parasite motility. PP1 may thus be partly responsible for Ca2+-regulated serine/threonine phosphatase activity in apicomplexan parasites.

Published
2022
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3. A positive feedback loop controls Toxoplasma chronic differentiation

Author

M. Haley Licon, Christopher J. Giuliano, Alex W. Chan, Sundeep Chakladar, Julia N. Eberhard, Lindsey A. Shallberg, Sambamurthy Chandrasekaran, Benjamin S. Waldman, Anita A. Koshy, Christopher A. Hunter, and Sebastian Lourido

Subjects
Microbiology (medical), Immunology, Genetics, Cell Biology, Applied Microbiology and Biotechnology, Microbiology
Published
2023
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4. Analysis of CDPK1 targets identifies a trafficking adaptor complex that regulates microneme exocytosis inToxoplasma

Author

Alex W Chan, Malgorzata Broncel, Nicole Haseley, Sundeep Chakladar, Elena Andree, Alice L Herneisen, Emily Shortt, Moritz Treeck, and Sebastian Lourido

Abstract

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. Here, we combine sub-minute resolution phosphoproteomics and bio-orthogonal labeling of kinase substrates inToxoplasma gondiito identify 163 proteins phosphorylated in a CDPK1-dependent manner. In addition to known regulators of secretion, we identify uncharacterized targets with predicted functions across signaling, gene expression, trafficking, metabolism, and ion homeostasis. One of the CDPK1 targets is a putative HOOK activating adaptor. In other eukaryotes, HOOK homologs form the FHF complex with FTS and FHIP to activate dynein-mediated trafficking of endosomes along microtubules. We show the FHF complex is partially conserved inT. 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 microneme protein secretion, 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.

Published
2023
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6. De novo mapping of the apicomplexan Ca2+-responsive proteome

Author

Alice L. Herneisen, Zhu-Hong Li, Alex W. Chan, Silvia NJ Moreno, and Sebastian Lourido

Abstract

Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca2+ signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the transition between the replicative and lytic phases of the infectious cycle. Despite the presence of conserved Ca2+-responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca2+-responsive proteome of the model apicomplexan T. gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca2+ release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca2+-responsive proteins, such as parasite Ca2+-dependent protein kinases. Our approach also identified numerous Ca2+-responsive proteins that are not predicted to bind Ca2+, yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca2+-responsive enzyme that relocalized to the parasite apex upon Ca2+ store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca2+ uptake to promote parasite motility. PP1 may thus be partly responsible for Ca2+-regulated serine/threonine phosphatase activity in apicomplexan parasites.

Published
2022
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7. A functional genomics screen identifying blood cell development genes in Drosophila by undergraduates participating in a course-based research experience

Author

Ryan Chan, Joshua P Hedtke, Michael T Chang, Kristen M Omi, Namrata Kakade, William Y. Luo, Yurianna Hou, Gloria Chin Young, Sunjong Ji, Mai Abdusamad, Amy Ngo, Maggie M Yung, Carissa Chan, Sharon Lee, Mark R Murata, Ashwin Shinde, Jason P Xu, Angelica C Ayala, Emily Jin Kyung Kim, Elitzander Alegria‐Leal, Phuong Vo, Yuan Wan, Vivian K Chen, Raghav Goyal, Anabel Resendiz, Harin B Parikh, Jaime S Tan, Leah C Dorman, Annisa N Soeboer, Seethim Naicker, Pratyush Kandimalla, Petros S Dertsakyan, Faith A Hobby, Beatrice J Sun, Karen Hoi, Megha Pokhriyal, C Kimberly Tsui, Nguyen Khoi Le, Umar Chaudhary, Patrick Chin, Woo Kyeom Kim, Mengshi He, David A Sanville, Utpal Banerjee, Christine Sutanto, Tiffany Luu, Joshua Lin, Tony Yao, Josephine Liu, Janani Muthaiya, Ravinder Kaur, Steven V. Pham, Andrea Vega-Loza, Mia Lim, Nataliya S Balashova, Adam T Quaal, Hnin Hlaing, Patrick A. Truong, Bryan Stender, Harmanjit Bassi, Erwin Feng, Yuxuan Wang, Sarah Salarkia, Rina H Nguyen, Gloria Lam, Garuem Sin, Seong Ah Kim, Sindhuja Godavarthi, Jialing Li, Eileen Hu, Cindy La, Elaine Sang, Jun Ho Chung, Jinhua Shen, Celesti Hao, Josh Mytych, Elliott Wu, Jin Hee Kim, Duy Tran, Neel Chari, Andie O'Laughlin, Christopher J Smith, Mona Abutouk, Chak Lon Kuang, Pei-Hua Tsai, Alisa Sukhina, Li Li, Chongbin He, Daniel Wong, Jahzeel Paguntalan, Emily R Schoen, Edgar Corona, Vivian Yip, Trisha H Patel, Emily Kim, Ira E. Clark, Andrew Char, Guadalupe Ortega Almazan, Asmar Abdul Ghani, Nadiyah Priasti, Duy Q Ngo, Gabrielle Sibal, Sean P Bennion, Stephanie Pang, Vivien W. Ho, Rachel S Distler, Nateli Sama, Josef Madrigal, Maharshi Panchal, Zijie Cai, Ashley C. Hope, Miriam Beyder, Daphne Jin, Steven C Liu, John M. Olson, Janice Ly, Eric Wang, Irene Louie, Wilson Huang, Justin Rafael de la Fuente, Nathan Stutzman, Bilguudei Naranbaatar, Rose Graf, Jonelle Mungcal, Jason Capili, Ziqi Zhou, Kelly M Gu, Julie Ko, Jeffrey A Kho, Amber J Li, Daniel Tran, Cory J. Evans, Jocelyn Woo, Kristen Schnell, Praptee Chowdhury, Chi Ju Lee, Grace J. Lee, Tracy L. Johnson, Nadezhda Riabkova, David Lopatto, Amanda Phan, Mary C Onglatco, Stephanie Huang, Zhiqiao Dong, Sat Kartar Khalsa, Saurin Kadakia, Maique Vo, Jessica Ye, Eric Lin, Darron Miya, Julia A. Ainsworth, Nicholas James, Jonathan Chang, Chloe C Su, Jeffrey Lin, Leslie Jaworski, Jenna Kovsky, Alice Hsu, Michel Chen, Allysen B Ehresman, Ju-Yeon Lee, Talin Golnazarian, Dana Lee, Kai Y Alexander, Jill Zimmerman, Lauren Johnson, Bosco Q Giap, Jamlah Dalie, Jackson K Uriu, Amala John, Osvaldo Acosta, Tebogo Mokwena, Sanan Venkatesh, Brandon L. Tsai, Haris M Akram, Seo-Kyung Oh, Andrea T Fua, Mark A Douglass, Rebecca Yang, Jessica M. Lee, Derek Spitters, Carmen Javier, Alex W Chan, Richard Wong, Ivy H Fu, Connie Y Liu, Parich Tangmatitam, Sena Ji, Claire Park, Arash Ghaffari-Rafi, Vincent T Nguyen, Dorothy Yim, Kush V. Bhatt, Tonatiuh Montoya, Rayna Tian, Vivian Chiu, Malcolm Phung, Victoria Lee, Chinmay Bhoot, Purvi D Patel, Jensen Pak, Puja Yogi, Zhouyang Shen, Nhat-Thi Vo, Bama Charan Mondal, Davis W Popovich, Mane Williams, Oscar M A Del Rio, Alexander J Chassiakos, Ruth Ryu, Zijun Zhao, Renae L Cruz, Yishan Mai, Michael K Wang, Kenneth Chang Chien, Kayla Patel, Jun Wang, Courtney J Cruz, Nicholas J Gills, Alexis Baranoff, Dinali Wijiewarnasurya, Yancey Y Cashell, Iman Hamid, Ashley Hanson, Olivia L Lee, Jasmine Sjarif, Patrick W Chang, Jasmin Johal, Monica Ghaly, Momoko Ishii, Alexandra M Libro, Boyu Cao, Paras Shah, Jennifer Hsueh, Aaron W Bradshaw, Jagteshwar Singh Khatra, Tien M Phan-Everson, Jonathan Lai, David J. Lim, Na Eun Jeon, Caleb Kim, Hyun Wook Kim, Hannah Kim, Sean Full, Tierney G Brannigan, Inho Chang, Suhas P Dasari, Emily R Dennis, Ceejay Lee, Alexander Zai, Christine Zhang, Ayse Elif Kesaf, Ralph Albert, Rebecca M Barber, Shijia W Lu, Austin Ma, Spencer S Hu, Ipsita Dey, Hee Jong Kim, Nancy Ruano, Timothy C Voros, Sarah M. Kamel, Kimberly L Yan, Gah-Eun Jang, Dallas L Mould, Eduardo Hernandez, Sahra M Hosseinian, Iris Chen, Chon Ao, Cindy K Trac, Eric Y Du, Sam Law, Ysrael K Hernandez, Jeffrey S. Zhao, Angad Beniwal, Patricia Yeo, Vina Tran, James R. Lee, Manny Sosa, Zheying Chen, Alec Estrada, Cristian Sosa, Kevin Herrera, Mina Mohammadi, Rina Watanabe, Timothy Chai, Huy Nguyen, Dong Yeon Lee, Vishaal Yepuri, Melanie Huynh, Kyle Marik, Thao T T Nguyen, Xiao X Zhang, Jamie A Ngo, Anh Nguyen, Colton Bracken, Brandon Wei, Brian Aguirre, Sayonika Mohanta, Jeffrey C. Wang, Casey Shapiro, Sonia Gill, Fred Rong, Lauren Petersen, Pingdi Huang, Alexander Grunfeld, Sang Kuk Lee, An N Ha, Kevin Nishida, Shraddha Kumar, Terrence C Lee, Ariano Abbasi, Jenny Summapund, Karl Querubin, Siman Wei, Hanning Xing, Evan Yang, Liset Contreras, Sahar Yaftaly, Matthew Wilson, Ken Siangchin, Brian Kit, Mallory R Neebe, Irvin C Lien, Julia E Petersen, Jonathan Woo, Kaitlyn Honeychurch, Jesse Juarez, Stephen Chang, Raymond Zhou, Cristian Medina, Sean Louise G Laput, Jason T. Lee, Nguon L Tan, Sharon Choi, Natalia C Gutierrez, Zafar S Gill, Kenneth Kim, Jefferson M Dang, Tanveer Salim, Marc Levis-Fitzgerald, Dan Huynh, Joyce Wang, Marsha R Cheng, Lillian Xie, Hannah Markovic, Conny Tran, Mimi Chiang, Vivian Lam, Nick Waite, Ricardo Gray, and Jo M Huang

Subjects
AcademicSubjects/SCI01140, Candidate gene, Cell type, Universities, AcademicSubjects/SCI00010, Computational biology, Biology, AcademicSubjects/SCI01180, 03 medical and health sciences, 0302 clinical medicine, blood, RNA interference, Genetics, Animals, Humans, Students, Molecular Biology, Gene, Genetics (clinical), Loss function, CURE, 030304 developmental biology, Investigation, education, 0303 health sciences, Blood Cells, Genomics, hematopoiesis, Undergraduate research, RNAi, AcademicSubjects/SCI00960, Drosophila, Functional genomics, 030217 neurology & neurosurgery, Function (biology)
Abstract

Undergraduate students participating in the UCLA Undergraduate Research Consortium for Functional Genomics (URCFG) have conducted a two-phased screen using RNA interference (RNAi) in combination with fluorescent reporter proteins to identify genes important for hematopoiesis in Drosophila. This screen disrupted the function of approximately 3500 genes and identified 137 candidate genes for which loss of function leads to observable changes in the hematopoietic development. Targeting RNAi to maturing, progenitor, and regulatory cell types identified key subsets that either limit or promote blood cell maturation. Bioinformatic analysis reveals gene enrichment in several previously uncharacterized areas, including RNA processing and export and vesicular trafficking. Lastly, the participation of students in this course-based undergraduate research experience (CURE) correlated with increased learning gains across several areas, as well as increased STEM retention, indicating that authentic, student-driven research in the form of a CURE represents an impactful and enriching pedagogical approach.

Published
2021
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8. Zebrafish expression reporters and mutants reveal that the IgSF cell adhesion molecule Dscamb is required for feeding and survival

Author

Alvaro Sagasti, Joshua P. Barrios, Donald P. Julien, Jaffna Mathiaparanam, Marc A. Wolman, Adam D. Douglass, and Alex W Chan

Subjects
0301 basic medicine, Neurite, 1.1 Normal biological development and functioning, Transgene, Clinical Sciences, Mutant, Genetically Modified, Biology, Article, Animals, Genetically Modified, 03 medical and health sciences, Cellular and Molecular Neuroscience, DSCAM, 0302 clinical medicine, Genome editing, Underpinning research, Genetics, genome editing, Animals, Zebrafish, Neurology & Neurosurgery, behavior, Cell adhesion molecule, Neurosciences, Feeding Behavior, Zebrafish Proteins, biology.organism_classification, Cell biology, 030104 developmental biology, Dscam, expression pattern, Neurological, Axon guidance, Generic health relevance, Cell Adhesion Molecules, feeding, 030217 neurology & neurosurgery
Abstract

Down syndrome cell adhesion molecules (DSCAMs) are broadly expressed in nervous systems and play conserved roles in programmed cell death, neuronal migration, axon guidance, neurite branching and spacing, and synaptic targeting. However, DSCAMs appear to have distinct functions in different vertebrate animals, and little is known about their functions outside the retina. We leveraged the genetic tractability and optical accessibility of larval zebrafish to investigate the expression and function of a DSCAM family member, dscamb. Using targeted genome editing to create transgenic reporters and loss-of-function mutant alleles, we discovered that dscamb is expressed broadly throughout the brain, spinal cord, and peripheral nervous system, but is not required for overall structural organization of the brain. Despite the absence of obvious anatomical defects, homozygous dscamb mutants were deficient in their ability to ingest food and rarely survived to adulthood. Thus, we have discovered a novel function for dscamb in feeding behavior. The mutant and transgenic lines generated in these studies will provide valuable tools for identifying the molecular and cellular bases of these behaviors.

Published
2018
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