Thursday, April 3, 2025
4/3/2025
Investigating PEX11 roles in shaping peroxisome architecture and dynamics - Investigating the role of PEX11 in peroxisome architecture Peroxisomes are critical organelles housing numerous metabolic reactions including fatty acid β-oxidation and reactive oxidative species decomposition. Although peroxisomes are classically thought to be single membrane-bound organelles enclosing an aqueous lumen, it has been recently established that peroxisomes can contain pervasive intralumenal vesicles (ILVs). How these suborganellar compartments form and impact peroxisome function remains unclear. The applicant discovered that mutants lacking two isoforms of PEX11, a membrane-shaping protein implicated in peroxisomal division, have major defects in generating ILVs. This new role for PEX11 in ILV formation raises questions about other PEX11 functions. This project will elucidate the roles of PEX11 isoforms in all aspects of peroxisome membrane shaping events, including ILV formation, peroxisome division, and de novo peroxisome biogenesis. The experiments will utilize Arabidopsis because its large peroxisomes allow facile visualization of peroxisome architecture using confocal microscopy, and its specialized peroxisomal metabolism enables robust whole-organism readouts of peroxisome function. In the first aim, null mutations in all PEX11 isoforms (PEX11A-E) will be generated using a CRISPR-based approach in a line that allows visualization of peroxisome membranes and lumen. Because ILV formation is implicated in peroxisomal fatty acid catabolism, fatty acid β-oxidation defects will be assessed by monitoring triacylglycerol content and seedling growth. Additionally, a unique tri- fluorescent reporter system that separately marks the peroxisome membrane, lumen, and individual PEX11 isoforms will be employed to visualize PEX11 dynamics and function within the context of ILVs. The second aim will revisit the canonical function of PEX11 in peroxisome division and a recent suggested function in de novo peroxisome biogenesis. Peroxisome division will be examined by quantifying the number and size of peroxisomes in various pex11 mutants compared to wild type and established peroxisome division mutants. Peroxisome biogenesis will be observed by visualizing the transition of pre-peroxisomes to mature peroxisomes and monitoring peroxisome maturation rates. The evolutionary conservation of PEX11 roles in these processes will be probed using heterologous expression of human and yeast isoforms. Beyond advancing our understanding of cell biology and organellar evolution, illuminating the cryptic peroxisomal processes of ILV formation, peroxisome division, and de novo biogenesis could enhance agricultural sciences and reveal the etiology of human pathologies, such as peroxisome biogenesis disorders. In addition, the accomplishment of these aims will be a core component of a comprehensive training plan to enable the applicant to progress towards an independent academic research career.
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