Cellulose is the primary structural component of the plant cell wall, and its organized deposition is essential for anisotropic growth and mechanical strength. In Arabidopsis thaliana, the glycosylphosphatidylinositol (GPI)-anchored protein COBRA plays a key but poorly understood role in cellulose biosynthesis. This thesis investigates COBRA’s subcellular localization, trafficking behaviour, and functional domains to elucidate its contribution to cellulose deposition and cell wall integrity. Using live-cell imaging of COBRA-monomeric yellow fluorescent protein (COB-mcYFP), a functional reporter fusion protein, we show that COBRA primarily localizes to motile Golgi bodies and discrete cortical puncta, but not clearly to the cell wall, contradicting earlier immunolabelling studies. Strikingly, COBRA-containing Golgi bodies form dynamic fluorescent "tails" that tether to cortical microtubules and rupture to release vesicles, resembling the delivery of the cellulose synthase complex (CSC). COBRA also partially colocalizes with the CSC-associated protein CSI1/POM2 in dynamic puncta, though differences in cytoskeletal drug responses suggest distinct trafficking routes. Cycloheximide treatments reveal that COBRA undergoes rapid turnover compared to CSCs, suggesting a transient role at the plasma membrane. The structural consequences of cob-1 (G167A) and hulk-1 (G104D) mutations were interrogated using AlphaFold modelling, molecular dynamics simulations, and functional assays, and show that neither mutation induces global misfolding. cob-1 lies adjacent to a cellulose-binding domain (CBD), characterized here as CBM2a-type (carbohydrate-binding module family 2a), and likely impairs cellulose interaction. Consistent with this, the cob-1 mutant shows reduced crystalline cellulose content, as quantified by an Updegraff assay, and both cob-1 and hulk-1 exhibit constitutive root and hypocotyl swelling phenotypes exacerbated by osmotic stress. Notably, galactose and pepstatin A treatments partially rescue these phenotypes, implicating a component that is auxin-dependent. Together, these findings suggest that COBRA is dynamically secreted through microtubule-associated Golgi rupture events, transiently interacts with CSCs or their trafficking intermediates, and potentially facilitates cellulose crystallization via its CBM2a domain. This work provides new insight into how GPI-anchored proteins influence cell wall architecture and highlights COBRA as a key modulator of wall mechanics during stress and growth.
Kamryn Diehl (Thu,) studied this question.