Corals are cnidarian animals that build the founding structures of tropical reefs, which survival depends upon the obligate symbiotic association to photosynthetic dinoflagellate algae in the family Symbiodiniaceae. As corals are facing increasing environmental and anthropogenic stress, understanding the molecular principles governing this unique symbiotic association is crucial to predict their adaptive potential. Due to logistic, costly, and experimental difficulties of working with corals, we use the sea anemone Aiptasia (sensu Exaiptasia pallida) as a tractable model organism for the molecular study of cnidarian-algal symbiosis. A major advantage of Aiptasia is that it establishes a facultative symbiotic association with Symbiodiniaceae algae, that is, this anemone can be maintained in an aposymbiotic (symbiont-free) state, allowing for comparison of symbiotic and ‘control’ aposymbiotic processes. The main aim of this dissertation was to investigate the signaling pathways involved in the regulation of this symbiotic interaction, and in particular, phosphorylation-mediated protein signaling. Phosphorylation is indeed a major post-translational modification that mediates signal transduction within and across cells. To investigate if protein phosphorylation regulates the complex intercellular signaling that occurs between symbiotic partners, a mass spectrometry-based phosphoproteomic approach was employed. Given the novelty of this application in the field of coral reef biology, the first research chapter details the development and optimization of a protocol that allows quantification of protein phosphorylation in the sea anemone Aiptasia. This chapter includes mass spectrometric analysis in 1) data-dependent acquisition (or shotgun proteomics) for the generation of a so-called assay (spectral) library, a reference dataset that servers for 2) accurate and reproducible label-free quantification of protein phosphorylation in data-independent acquisition (DIA/SWATH-MS). In the second research chapter, the developed protocol was employed to generate a phosphopeptide assay (spectral) library for aposymbiotic and symbiotic Aiptasia, which would allow further quantification of protein phosphorylation across symbiotic conditions. We consistently quantified more than 3,000 phosphopeptides, totaling more than 1,600 phosphoproteins, across biological replicates and symbiotic conditions. Characteristic phosphoproteomic profile distinguished the two symbiotic groups and differential phosphorylation targeted biological processes that have not been previously described in the context of cnidarian-algal symbiosis, namely the phospholipase D signaling pathway and protein processing in the endoplasmic reticulum. We suggest that changes in the phosphorylation status of these signaling pathways may have a potentially relevant role in the control of an established cnidarian-algal association.
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