Analyses of Metacaspase 1 and a Potential APAF-1 Orthologue During Heat Stress-Induced Programmed Cell Death in Chlamydomonas reinhardtii
Catumbela, Celso Santos Goncalves
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Purpose The purpose of this thesis was to expand on the knowledge of the PCD machinery of unicellular eukaryotes. To this end, the first half of this thesis employs the single cell green alga, Chlamydomonas reinhardtii, and examines the potential roles of metacaspase type I (MCA1) in modulating the events of programmed cell death (PCD). The second half of this thesis attempts to elucidate the function of a potential orthologue of APAF-1, a key apoptotic protein reported in multicellular eukaryotes, and hypothesized to be present in the unicellular C. reinhardtii. Methodology Using an MCA1 knockout strain of C. reinhardtii (mca1), verified via PCR and DNA sequence analysis, the first half of this thesis utilizes the following techniques to reveal the potential roles of MCA1: fluorescence microscopy and DNA laddering, as well as colony formation and colorimetric assays. All experiments were conducted in triplicate. The data was analyzed via manual cell counts, and qualitative analyses were performed by a single analyst. Statistical software (SAS 9.4 and JMP 13) analyses were conducted by two independent personnel. For the second half of this thesis, proteomic analysis of C. reinhardtii was conducted through usage of whole-cell extraction, sucrose gradient centrifugation, SDS-PAGE, Coomassie blue staining, and Western blotting. Findings Genetic analysis of mca1 revealed that the disruption of MCA1 is linked to the rapid transmission of certain PCD events due to heat stress in C. reinhardtii. Results show that in response to heat stress, MCA1 knockout is associated with significantly increased plasma membrane disruption in C. reinhardtii. Moreover, heat-stressed mca1 cells consistently displayed increased DNA laddering, relative to WT. Furthermore, in response to heat stress, mca1 populations displayed more rapid accumulation of ROS, as well as a significantly greater ROS response with prolonged heat stress exposure. Notably, MCA1 knockout did not alter the rate of phosphatidylserine (PS) externalization, or cell proliferation upon the onset of heat stress. Together, our data suggest the potential that MCA1 acts as a negative regulator of certain heat stress-induced PCD phenotypes within C. reinhardtii. In the second half of this thesis, proteomic analysis revealed that prior published results suggestive of APAF-1 presence in C. reinhardtii could be generated in the absence of antibodies against human APAF-1. Intriguingly, comparable results were obtained through sole usage of goat anti-rabbit IgG, or goat anti-mouse IgG (1:5000, both). Moreover, in silico analysis revealed the primary APAF-1 homologs in C. reinhardtii to lack key conserved domains (CARD and AAA16). Together, the biochemical and in silico data refuted the previous report of a potential APAF-1 orthologue in C. reinhardtii. This thesis provided novel data to aid in the understanding of the unicellular PCD machinery, as well as recommendations for future studies.