Functional studies of membrane trafficking and secretion –phosphoinositides in plant abiotic stress responses

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Functional characterization of clathrin adaptors and Rab-binding proteins in Arabidopsis thaliana

Publication . Marchese, Dario; Malhó, Rui Manuel dos Santos; Hussey, Patrick J.

In plant cells, the dynamic interplay between endocytosis and vacuolar trafficking within the cell endomembrane system is essential for maintaining cellular integrity, regulating plant growth and development, and responding to environmental stimuli. The endomembrane system, comprising the plasma membrane, endosomes, Golgi apparatus, endoplasmic reticulum and vacuoles, orchestrates the movement and sorting of molecules throughout the cell. Clathrin-mediated endocytosis facilitates the internalization of plasma membrane components, nutrients and signalling molecules, which are then sorted in endosomes for either recycling back to the plasma membrane or degradation in vacuoles. Vacuolar trafficking ensures the delivery of these sorted cargoes to vacuoles, thus playing a crucial role in cellular metabolism and stress responses. Clathrin adaptor proteins and Rab effectors are crucial in mediating vesicular trafficking within plant cells. Despite their known roles, their specific contributions to clathrin-mediated endocytosis and maintenance of vacuolar function in pollen remain largely unexplored. This doctoral work investigates the molecular mechanisms underlying these processes in Arabidopsis thaliana by combining the experimental analyses conducted in the context of two main research projects, aimed at characterizing these distinct yet interconnected protein families. This thesis examines the roles of key clathrin adaptor proteins ECA4, CAP1, EPSIN3, and the RabG3-binding proteins IRQ2 and IRQ3 in pollen development. Reverse-genetic analysis, gene expression profiling, in vivo subcellular localization studies and protein-protein interaction screens were employed to elucidate the functions of these proteins. The first set of studies revealed that the disruption of ECA4 and EPSIN3 resulted in significant morphological abnormalities in pollen tubes, including larger apical tube diameters and increased frequency of aberrant tip shapes. Double mutants of eca4 or epsin3 with phosphoinositide-related genes exhibited enhanced phenotypic abnormalities and an early flowering phenotype, highlighting the critical relationship between phosphoinositide levels and these adaptor proteins. Additionally, confocal microscopy studies demonstrated that ECA4, CAP1 and EPSIN3 localize to the plasma membrane and endosomes, indicating their broader role in vesicle formation and membrane curvature. The second set of studies focused on the roles of the RabG3-binding proteins IRQ2 and IRQ3. Reverse-genetic analysis of mutations in IRQ2 and IRQ3 genes revealed reduced pollen germination rates, highlighting their roles in the early stages of pollen germination. IRQ2 was confirmed to localize at the tonoplast of the growing pollen tubes, while IRQ3 exhibited a punctate pattern partially decorating the actin filaments during pollen tube elongation, indicating its involvement in intracellular transport mechanisms. Colocalization studies additionally confirmed the association of IRQ2 with Rab GTPases, specifically with the active, GTP-bound form of RabG3A, highlighting their cooperative role in vacuolar trafficking and morphology regulation. The findings presented in this thesis revealed key functional roles for these pollen-specific proteins: ECA4, CAP1 and EPSIN3 are central members of clathrin adaptor complexes, driving the formation of clathrin-coated vesicles and regulating the internalization of cellular components, while IRQ2 and IRQ3, through their specific interactions with RabG3 GTPases, are essential for the proper trafficking and fusion of vesicles to the vacuole.

2024-11-22Doctoral thesis

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