Date of Award
Doctor of Philosophy
Biochemistry and Cellular and Molecular Biology
Daniel M. Roberts
Tessa Burch-Smith, Andreas Nebenführ, Brad Binder, Elizabeth Fozo
The central focus of this work is to understand how rgsCaM homologs regulate mRNP bodies under cellular stress. While rgsCaM is best known as an endogenous suppressor of gene silencing in tobacco, our findings for a homologous protein in Arabidopsis, CML38, suggest that a broader function in the regulation of mRNP bodies may be common to this family of calmodulin-like proteins. CML38 is induced by hypoxia stress, contributes to plant survival under hypoxia, and localizes to stress-induced mRNP bodies called stress granules (SGs), as well as to processing bodies (PBs). Members of this protein family target viral and endogenous proteins for degradation through the autophagy pathway. We propose that rgsCaM and its relatives may localize to stress-induced mRNP bodies and target them for autophagic degradation (granulophagy). In our investigations for rgsCaM, we used hypoxia stress as a means to induce stress granule formation and found that rgsCaM localizes to hypoxia-induced cytosolic granules which are both independent of and bound to SGs and PBs. We further show that rgsCaM colocalizes with the autophagosome cargo-binding protein ATG8e, and interacts with ATG8e in planta by BiFC assay. Mutations disrupting the N-terminus of rgsCaM, or ones affecting the ability of ATG8 to bind cargo adaptors caused a loss in BiFC interaction. This suggests that the N-terminus contains a site for binding to the cargo binding protein ATG8e, and which might mediate the targeting of rgsCaM and bound cargo to autophagosomes as part of the granulophagy process. For future studies, a FRET-based approach for probing rgsCaM interactions in vivo is demonstrated.In a second trust, we report the development of a novel, fluorescence-based, quantitative oxygen biosensor to facilitate the non-invasive assessment of the oxygen status of cells of living cells by fluorescent imaging.
Conner, W. Craig, "Regulator of Gene Silencing Calmodulins: Components of RNA granules and autophagy during plant stress. " PhD diss., University of Tennessee, 2020.