Exploring the Role of Glutamate Signaling in the Regulation of the Aiptasia-Symbiodiniaceae Symbiosis Thesis by Migle Kotryna Konciute In Partial Fulfillment of the Requirements For the Degree of Master of Science King Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi Arabia April, 2020 Migle Kotryna Konciute 2 EXAMINATION COMMITTEE PAGE The thesis of student Migle Kotryna Konciute is approved by the examination committee. Committee chairperson: Assoc. Prof. Manuel Aranda Committee Members: Asst. Prof. Kyle J. Lauersen, Assoc. Prof. Xose Anxelu G. Moran 3 COPYRIGHT PAGE ©April, 2020 Migle Kotryna Konciute All Rights Reserved 4 5 ABSTRACT Exploring the Role of Glutamate Signaling in the Regulation of the Aiptasia- Symbiodiniaceae Symbiosis Migle Kotryna Konciute The symbiotic relationship between cnidarians and their photosynthetic dinoflagellate symbionts underpins the success of coral reef communities in oligotrophic, tropical seas. Despite several decades of study, the cellular and molecular mechanisms that regulate the symbiotic relationship between the dinoflagellate algae and the coral hosts are still not clear. One of the hypotheses on the metabolic interactions between the host and the symbiont suggests that ammonium assimilation by the host can be the underlying mechanism of this endosymbiosis regulation. An essential intermediate of the ammonium assimilation pathway is glutamate, which is also known for its glutamatergic signaling function. Interestingly, recent transcriptomic level and DNA methylation studies on sea anemone Aiptasia showed differences in metabotropic glutamate signaling components when comparing symbiotic and non-symbiotic animals. The changes in this process on transcriptional and epigenetic levels indicate the importance of glutamate signaling in regard to cnidarian symbiosis. In this study, I tested glutamatergic signaling effect on symbiosis in sea anemone Aiptasia using a broad-spectrum glutamate receptor inhibitor 7- CKA and glutamate. Significantly decreased cell density was observed in animals with inhibitor treatment suggesting a possible correlation between glutamate signaling and the establishment or maintenance of symbiosis. Using RNA-Seq, I was able to obtain transcriptional profiles of the animals under inhibitor and glutamate treatment. Differential gene expression and gene ontology analyses indicated changes in amino acid metabolism, lipid metabolism and such signaling pathways as MAPK, NF-kappa B and phospholipase 6 C. Although amino acid and lipid metabolism could be a result of the reduced symbiotic state of inhibitor treated Aiptasia, the signaling pathways which are related to apoptosis and immune response provide an exciting venue for direct regulatory interaction between symbiosis and glutamatergic signaling. However, as these signaling pathways mainly act via signal transduction through protein phosphorylation, further studies looking at changes on a post-translational level might provide further insight into the mechanisms underlying the observed phenotype. 7 ACKNOWLEDGMENTS First of all, I would like to thank my supervisor Professor Manuel Aranda who guided me through this project and who taught me science with patience. Thank you for supporting and leading me when I was lost in details. I am thankful to Professors Xose Anxelu G. Moran and Kyle J. Lauersen for their time and agreement to be part of my defense committee. I am truly grateful to Guoxin Cui, who mentored me throughout all stages of this experiment, taught all lab techniques I can now do, and performed the computational analysis of this project’s sequencing data. Thank you for never stopping believing in this project or my capabilities, even when I was unable to do so. Thank you to all Coral Symbiomics laboratory members for your support and friendly working atmosphere. Special thanks go to Lucia and Sandy, who did the initial trials for this project, which provided me with a starting point and Octavio consulting with me. Also, thank you to my friends Jess, Alessandro, Sandy, Sergio, and Anieka, for always cheering me up and joining the afternoon tea breaks. Lastly, I am extremely grateful to my parents Elona and Andrius, my brother Ignas and grandma Leokadija. Without their support, I would not be able to pursue higher education. Thank you for your advice and encouragement. 8 TABLE OF CONTENTS EXAMINATION COMMITTEE PAGE ............................................................................ 2 COPYRIGHT PAGE ........................................................................................................... 3 ABSTRACT ........................................................................................................................ 5 ACKNOWLEDGMENTS ................................................................................................... 7 TABLE OF CONTENTS .................................................................................................... 8 LIST OF ABBREVIATIONS ........................................................................................... 10 LIST OF FIGURES ........................................................................................................... 11 LIST OF TABLES ............................................................................................................ 12 1 Introduction ............................................................................................................... 13 1.1 Objectives and contributions ............................................................................. 17 2 Materials and methods .............................................................................................. 19 2.1 Maintenance of Aiptasia animals and Symbiodiniaceae cell cultures .............. 19 2.2 Inhibitor treatment ............................................................................................. 20 2.3 Symbiodiniaceae cell density measurement ...................................................... 21 2.4 Symbiodiniaceae cell density data analysis ...................................................... 23 2.5 RNA extraction and library preparation ............................................................ 23 2.6 Differential gene expression analysis ................................................................ 24 3 Results ....................................................................................................................... 26 3.1 Cell density measurements ................................................................................ 26 3.2 Differential expression analysis ........................................................................ 28 3.2.1 Amino acid metabolism associated genes ................................................. 29 3.2.2 Genes affecting cell adhesion .................................................................... 30 3.2.3 Gene expression regulating lipid metabolism ........................................... 31 3.2.4 Apoptosis regulation related genes ........................................................... 32 4 Discussion ................................................................................................................. 34 4.1 7-CKA and glutamate effect on cell density ..................................................... 34 4.2 Differential expression analysis ........................................................................ 35 4.2.1 The target of glutamate inhibitor 7-CKA in Aiptasia ............................... 36 4.2.2 Cell adhesion in regard to symbiont expulsion ......................................... 37 4.2.3 Calcium importance in apoptosis .............................................................. 38 4.2.4 Calcium and protein phosphorylation ....................................................... 39 9 4.2.5 Lipid signaling ........................................................................................... 41 5 Conclusions ............................................................................................................... 42 REFERENCES .................................................................................................................. 45 Appendices ........................................................................................................................ 51 Appendix A. List of differentially expressed genes. ..................................................... 51 Appendix B. Gene ontology enrichment analysis results. ............................................ 79 7-CKA vs. control dataset ......................................................................................... 79 7-CKA+Glutamate vs. control dataset ...................................................................... 85 Glutamate vs. control dataset .................................................................................... 89 10 LIST OF ABBREVIATIONS ↑ Upregulation of gene expression ↓ Downregulation of gene expression 7-CKA 7-Chloro-4-hydroxyquinoline-2-carboxylic acid AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ASW Autoclaved seawater DEG Differentially expressed genes DL-AP4 DL-2-Amino-4-phosphonobutyric acid DL-AP5 DL-2-Amino-5-phosphonopentanoic acid gamma-DGG (R)-4-(Carboxymethylcarbamoyl)-2-aminobutanoic acid GS/GOGAT Glutamine synthase/glutamate synthetase pathway MAPK mitogen-activated protein kinase NF-kappa B Nuclear factor kappa-light-chain-enhancer of activated B cells NMDA N-methyl-D-aspartate PCA Principal component analysis