The Characterization of Clathrin-mediated Endocytosis in Plants and Its Influence on Cellulose Biosynthesis

Restricted (Penn State Only)
Author:
Bashline, Logan Thomas
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
October 12, 2015
Committee Members:
  • Ying Gu, Dissertation Advisor
  • Ying Gu, Committee Chair
  • Teh Hui Kao, Committee Member
  • Lorraine C Santy, Committee Member
  • Melissa Rolls, Committee Member
  • Gabriele Brigitte Monshausen, Committee Member
Keywords:
  • clathrin-mediated endocytosis
  • trafficking
  • cellulose
  • cell wall
  • Arabidopsis
  • plants
Abstract:
Living cells internalize material from the cell surface using membranous vesicles in a process called endocytosis. The most common mechanism of endocytosis in eukaryotes is clathrin-mediated endocytosis (CME). In CME, many clathrin proteins, in association with numerous other adaptor and accessory proteins, assemble into a cage-like coat that induces the formation of clathrin-coated pits in the plasma membrane. These clathrin-coated pits develop into clathrin-coated membranous vesicles that are released from the plasma membrane into the interior of the cell. The material internalized by CME includes the lipids that comprise the plasma membrane, plasma membrane resident proteins, and other extracellular cargos such as nutrients and hormones. Therefore, CME is crucial to many cellular processes including the maintenance of membrane composition and integrity, the establishment and maintenance of cell polarity, the perception and regulation of cell signaling, and the uptake of nutrients. CME is well characterized in some eukaryotes, such as yeast and mammals, in which many components of the CME machinery have been identified and functionally characterized. Comparatively, the study of CME in plants is in its infancy. The research presented in this dissertation has provided insight into the general CME process in plants, which is similar to CME in other eukaryotes in some regards, but also distinct in many ways. For example, like in yeast and mammals, plants possess a heterotetrameric adaptor protein 2 (AP2) complex, which acts as a core component of CME by interacting with the plasma membrane and recruiting clathrin and specific cargo proteins to the sites of CME. The research presented in Chapters 2 and 3 aided in the original characterization of the AP2 complex in plants. Recent findings show that plants also possess additional CME components that do not exist in yeast or mammals, which could represent the remnants of evolutionarily ancient trafficking machinery. The research presented in iii Chapter 4 explored the function of TWD40-2, a protein that plays a significant role in CME in plants but does not exist in yeast or mammals. Since CME is responsible for the internalization of a large variety of cargo proteins and molecules, CME influences many cellular processes, including the synthesis of the plant cell wall. A majority of the cell wall is comprised of polysaccharides in the form of cellulose, hemicellulose, and pectin. Hemicellulose and pectin are synthesized in the intracellular Golgi apparatus and subsequently delivered to the plasma membrane via vesicular trafficking and discharged into the extracellular cell wall through exocytosis. In contrast, cellulose is synthesized and extruded directly into the cell wall by plasma membrane-resident enzyme complexes, the cellulose synthesis complexes (CSCs). The prerequisite that CSCs must be located at the plasma membrane to function emphasizes the importance of CSC delivery to the plasma membrane and CSC endocytosis from the plasma membrane. In Chapters 2 and 3, the cellulose synthase (CESA) proteins of the CSC were identified as cargos of the CME pathway. In Chapter 4, deficiencies in CME were shown to cause deficiency in the cellulose biosynthesis process by influencing the distribution and behavior of the plasma membrane-localized population of CSCs. Interestingly, without an analogous structure in yeast and mammals, CSCs represent a novel type of CME cargo. Distinct attributes of CSCs, including the localization of CSCs to distinct particles at the plasma membrane, have enabled CSCs to be used as helpful representative cargos of the plant CME pathway. The research presented in this dissertation could be of interest to a broad scientific audience, with implications in the understanding of general processes of the plant cell, such as CME and cellulose biosynthesis, as well as the dissection of specific questions that reside at the crossroads of these two processes.