Author: Kaija Jo Goodman

Publisher:

Published: 2020

Total Pages: 0

ISBN-13:

Endocytosis and endosomal trafficking control the abundance and distribution of key membrane proteins at the cell surface and in other cellular compartments. The Endosomal Sorting Complex Required for Transport (ESCRT) mediates the formation of vesicles to sequester ubiquitinated membrane proteins (cargo) into late endosomes en route to the vacuole for degradation. We previously found that the late-acting ESCRT components, ISTL1 and LIP5 are important for plant development, proper defense responses, and multivesicular endosome formation in root cells. Using genetic, confocal, and electron microscopy approaches, I found a unique role for ISTL1 and LIP5 in endosomal trafficking in tapetal cells, which surround and nourish developing pollen grains within anthers. Together, ISTL1 and LIP5 are required in the tapetum to maintain plasma membrane localization of ABCG9, a transporter important for pollen wall development. Plants lacking ISTL1 and LIP5 showed defects in exocytosis of the sec-RFP secretion marker in anthers. Pollen in the istl1 lip5 mutant developed abnormal pollen wall patterns, leading to partial pollen lethality. Finally, istl1 lip5 tapetal cells exhibited ultrastructural defects in both multivesicular endosome morphology and trans-Golgi Networks, indicating that besides endosomal sorting, late-acting ESCRTs have a role in the early endosomal pathway and/or exocytosis. The ESCRT machinery mediates negative membrane deformation (i.e. membrane bending away from the cytoplasm). Exactly how the ESCRT proteins orchestrate membrane deformation and fission is not well known. The current models for how intralumenal vesicles form assume that vesicles are formed one at a time. However, in plants intralumenal vesicles form as concatenated networks, establishing a novel mechanism to entrap cargo more efficiently. In Chapter 3, I use electron tomography to analyze vesicle network architecture from plant endosomes. I developed quantitative tools to evaluate the complexity of individual vesicle networks and applied them to compare intralumenal vesicle networks from different cell types. In addition, I developed rules and tools to recapitulate how these intralumenal vesicle networks form and infer the activity of budding sites on the endosomal surface.