ENDOPHILIN B2: AN ENDOCYTIC AND AUTOPHAGIC REGULATOR
Open Access
- Author:
- Serfass, Jacob M
- Graduate Program:
- Pharmacology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 28, 2017
- Committee Members:
- Hong-Gang Wang, Dissertation Advisor/Co-Advisor
Hong-Gang Wang, Committee Chair/Co-Chair
Richard B. Mailman, Committee Member
Gavin P. Robertson, Committee Member
Charles Lang, Outside Member
Oranee Tawatnuntachai, Outside Member - Keywords:
- Endocytosis
Autophagy
Endophilin B proteins
Apoptosis
Cellular Homeostasis
Endosome Maturation
Endocytic trafficking
Endosome Acidification - Abstract:
- This dissertation employed genetic approaches to study the impact of endophilin B2 on apoptosis, autophagy, and endocytic trafficking, which when disrupted can be associated with a variety of disorders including cancer and neurodegeneration. The endophilin family of proteins (including the endophilin A and endophilin B subfamilies) contains an amino-terminal Bin/Amphiphysin/Rvs (N-Bar) domain that induces membrane curvature, and a Src homology 3 (SH3) domain that facilitates protein-protein interactions. Endophilin B1 (SH3GLB1/Bif-1) is a positive regulator of endosome maturation, autophagy, apoptosis, and mitochondrial dynamics, but its major binding partner and fellow family member endophilin B2 (SH3GLB2) has not been as extensively studied. To help understand the physiological role of endophilin B2 in-vivo, we generated knock-out mice. Endophilin B2 was found not to be essential for embryonic development as mice were born at expected Mendelian ratios and did not exhibit any overt physiological defects during development. Regardless of the lack of disease phenotypes, further work revealed that tissue expression of endophilin B2 is similar to that of endophilin B1. We also found that isoforms of endophilin B2 are differentially expressed in a tissue specific manner. We then tested the hypothesis that endophilin B2 is not redundant to the functions of endophilin B1. Using mouse embryonic fibroblasts (MEFs) isolated from knock-out mice, we explored the role of endophilin B2 in mitochondrial apoptosis, endocytosis, endosome maturation, and autophagy. We found that endophilin B2 was not essential for mitochondrial apoptosis resulting from over-expression of pro-apoptotic proteins or treatment of cytotoxic agents. Lack of endophilin B2 also failed to affect MEF mitochondrial dynamics studied via cell fusion assays. Using fluorescently-labeled dextran, we determined that ablation of endophilin B2 had no effect on endocytic internalization, but did decrease the acidification of endosomes assessed by dextran particles conjugated with a pH sensitive fluorescent dye. To further evaluate the role of endophilin B2 as a regulator of endocytic maturation, MEFs were infected with influenza-A virus. Endophilin B2 could localize with incoming viral particles, and decreased endophilin B2 expression reduced the amount of viral genome replication that took place in the nuclei of cells. Finally, we used CRISPR-Cas9 gene editing technology in HeLa cells to delete endophilin B2. This attenuated the degradation of stimulated epithelial growth factor receptors (EGFR) by delaying the delivery of the receptor to lysosomes. Since autophagy is highly dependent upon the endocytic pathway, we further sought to determine if endophilin B2 is an autophagic regulator. We induced autophagic flux through nutrient deprivation in endophilin B2 deficient HeLa cells where loss of endophilin B2 was observed to decrease autophagic flux potentially through the ineffective delivery of material to lysosomes. Re-expression of endophilin B2 in MEFs was observed to restore autophagic flux, confirming the impact of endophilin B2 on the autophagy pathway. In addition, we identified that the endophilin B2 can interact with the class-III phosphoinositide 3-kinase complex involved in phospholipid formation. This observation may explain, at least partially, the observed results with endosome and autophagosome maturation. In summary, this dissertation has found new roles for endophilin B2 in endosome maturation and autophagic flux, while showing that it is not critical for endocytosis, mitochondrial apoptosis, and mitochondrial dynamics.