MSc Exit Seminar
Tuesday, January 21th, 2020 at 10:10am – Ramsay Wright Building, Room 432
Angelica Mae Miraples (Yoshioka Lab)
The systematic investigation of the regulation and function of cyclic nucleotide-gated ion channels in Arabidopsis
Plant survivability is dependent on the coordination of developmental processes and environmental stress responses at the cellular level. Calcium ion (Ca2+) is a secondary messenger crucial to signal transduction associated with development and stress, however much less is known about channels involved in Ca2+ signaling. The Arabidopsis cyclic nucleotide-gated ion channel (CNGC) family are non-selective Ca2+ permeable channels and has 20 members. Extensive expansion of this family in higher plants suggest events of gene duplication over time, indicating redundancy and neofunctionalization within this family. In this thesis, I present an analysis of Arabidopsis clade I CNGCs that demonstrate functional redundancy during early development and hormone signaling alluding to potential subunit binding partners. To further address channel composition, I have generated a screening method to explore subunit binding partners of CNGC12 using the chimeric gene CNGC11/12, which causes lesion formation. Finally, to expand on CNGC12-mediated Ca2+ signaling, implicated in immunity, I have attempted to confirm an interaction between CNGC12 and a Ca2+ sensory C2 domain protein, and generate its knockout mutant using CRISPR- Cas system.
Investigation of proteasome architecture, activity, and interactors in Arabidopsis thaliana under abiotic stresses
The 26S proteasome plays a critical role in protein homeostasis via turnover of cellular proteins. Changes in subunit expression, assembly/disassembly of the holoenzyme, and association of non-canonical activators or inhibitors can fine-tune proteasome activity, although such pathways have not been explored in plants. This thesis examines the influence of abiotic stresses on structure, function, and interactors of the Arabidopsis thaliana proteasome. I found that oxidation and salinity decreased the cellular ratio of 26S to 20S proteasomes, altered proteasome subunit composition, and reduced binding of proteasome-associated proteins (PAPs) such as heat-shock and assembly chaperones. Additionally, oxidative stress specifically activated the 20S proteasome; a process which might be promoted by ATP deficiency. Further characterization of PAPs revealed that PBAC1, a 20S assembly chaperone, dampens oxidative damage to the germinating seed. Overall, findings suggest that regulation of proteasome activity, either through PAPs or ATP availability, is necessary for appropriate stress responses in Arabidopsis.
(Zhao & Gazzarrini Lab)
Characterizing the Microtubule Organizing Centres in Osteoclasts
The skeleton is a metabolically active organ that undergoes continuous remodeling in order to uphold structural integrity and to repair bone following injury. Osteoclasts are highly specialized, multinucleated cells responsible for the selective resorption of bone matrix components, however, they are also responsible for the pathological bone destruction found in microgravity environments, periodontitis, and osteoporosis. Our study investigates the origin of the microtubule cytoskeleton during differentiation and bone resorption. Microtubule nucleation is generally restricted to specific subcellular sites called microtubule organizing centres (MTOCs) and is primarily fulfilled by the centrosome in mitotic animal cells. While mononuclear osteoclast precursor cells contain centrosomal MTOCs, previous research has suggested that functional centrosomal MTOCs do not exist in osteoclasts. To revisit and characterize the MTOCs in osteoclasts, both cell line and primary cell-derived murine osteoclasts were subjected to high-resolution imaging to track centrosome behaviour and their ability to organize microtubules. Through live-cell imaging, fixed immunofluorescence, and ultrastructural analyses, we observed that most, if not all centrosomes donated from precursor cells clustered early in osteoclastogenesis and persisted post-differentiation in non-resorbing and resorbing osteoclasts. Drug-induced microtubule regrowth assays revealed that centrosomes remained individually functional post-differentiation but clustered in a microtubule-dependent manner in order to organize microtubules. Quantification of microtubules emanating from centrosome clusters showed that they were capable of nucleating more microtubules compared to lone centrosomes. Finally, by visualizing Golgi reorganization and the nucleation of Golgi-derived microtubules, we identified the Golgi as a possible non-centrosomal MTOC that potentially facilitates the production of polarized microtubule arrays in osteoclasts. Together these findings show that multinucleated osteoclasts employ unique centrosomal and non-centrosomal MTOCs to organize microtubules.
Supervisor: Prof. Rene Harrison