| Clathrin-mediated membrane trafficking is essential for multiple stages of plant development, including the processes of cytokinesis and polarized cell expansion. One key player in clathrin-mediated trafficking in animal systems is the polymerizing GTPase dynamin. In plants, two separate families of dynamin-related proteins are involved in clathrin-mediated endocytosis: the dynamin-related-protein 2 (DRP2) family of classical dynamins as well as the DRP1 family of dynamin-related proteins. In addition to their endocytic roles, members of the DRP1 and possibly the DRP2 family also play a distinct role in formation of the cell plate during cytokinesis. Chapter 2 of this thesis is a study of the fundamental properties of the non-classical dynamin-related protein DRP1A. Bacterially expressed, GTPase active DRP1A forms large homopolymers that can interact with negatively charged liposomes. However, these homopolymers do not form regular spirals, do not undergo dynamic disassembly and do not cause liposome tubulation, suggesting that additional factors or modifications are necessary for DRP1A's in vivo function. Chapter 3 of this thesis examines the role of the DRP2 family in plant development, as illuminated by analysis of drp2a and drp2b double insertional mutants, which display an early arrest during both male and female gametophyte development, demonstrating that the DRP2 family is independently essential for plant development. Unlike pollen from the drp1C-1 mutant, arrested drp2ab gametes do not display defects in plasma membrane morphology, suggesting that the DRP2 family may function in a distinct membrane-trafficking pathway. The appendices to this thesis include preliminary work on putative DRP1A interacting proteins, the generation of a DRP2-GFP construct for live imaging, and the groundwork for an investigation of clathrin light chain localization and function. |
