Investigating the roles of beta Heavy-spectrin in epidermal growth factor receptor signaling activity in Drosophila melanogaster
Open Access
- Author:
- Wu, Juan
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- March 30, 2011
- Committee Members:
- Graham Hugh Thomas, Thesis Advisor/Co-Advisor
Graham Hugh Thomas, Thesis Advisor/Co-Advisor - Keywords:
- Trafficking
Spectrin
EGFR
Endocytosis - Abstract:
- The spectrin-based membrane skeleton (SBMS) is a flexible and multifunctional scaffold involved in a wide spectrum of cellular processes including the generation of specialized membrane domains, determination of cell polarity, protein sorting, vesicle transport, endocytosis, protein recycling, morphogenesis, and nuclear positioning. EGFR signaling pathway regulates a variety of important cellular activities, ranging from cell proliferation, growth, differentiation, survival, motility, adhesion and tissue development. It is also increasingly important as a therapeutic target for multiple cancers. The effect of spectrin, the key component of the SBMS, on EGFR signaling remains largely uncharacterized. Drosophila melanogaster is utilized in this thesis as an ideal model for investigating the relationship between beta heavy-spectrin (betaH) and EGFR signaling activity due to the low redundancy in beta-spectrin and EGF receptor isoforms, as well as the linearity of EGFR signaling pathway. The hypothesis of this thesis lies in that loss of betaH enhances EGFR signaling activity. This hypothesis originates from a genetic experiment in which betaH mutant alleles suppressed rhomboidve (rhove) phenotype. Flies homozygous for rhove have incomplete wing veins due to insufficient EGFR activity. Consistent with our hypothesis, we observed increased diphosphorylated Erk (dpErk) levels when the amount of betaH was reduced by RNAi. We then sought to probe the mechanism by which betaH regulates EGFR signaling activity from two perspectives: EGFR ligand activiation and receptor endocytosis. The EGFR ligand mSpitz-GFP accumulated in a perinuclear lobe-like compartment and changed distribution dramatically upon betaH knockdown. However, such distribution change did not seem to correlate with increased ligand activation. Therefore, we instead focus on the endocytic regulation of EGFR and we propose that betaH is associated with EGF receptor endocytosis for the following reasons. betaH was seen associated with EGFR in internal vesicular and tubular structures that were positive for endocytic marker, Eps15. In addition, betaH was concentrated in lots of large cytoplasmic vesicles in Shibire mutant SG exposed at 30oC where clathrin-mediated endocytosis is significantly blocked. Finally, in Annexin B9RNAi SG, betaH was driven into similar internal networks along with Eps15. Based on my current data and other published works, we have proposed a model depicting the mechanism of betaH-associated endocytosis. We suggest that betaH is recruited to the endocytic machinery by Eps15, and both of them would travel with the internalized vesicles. The interaction between betaH-Annexin B9 provides additional means and specificity for vesicle trafficking to MVB. According to this model, efficient endocytic trafficking of EGFR will be compromised upon loss of betaH, which will result in active receptors accumulation at either plasma membrane or earlier endosomal compartments where they continue to signal. This leads to increased EGFR signaling activity. The second half this thesis aims to unraveling the identity of the mSpi-positive lobes. The mSpi lobes are positive for multiple endocytic markers, including Eps15, Hrs and Ubi. Similar detection of such lobes in wild type salivary gland (SG) prompts us to speculate that the lobe is a naturally occurring endocytic compartment potentially involved in endosomal sorting and signaling. We have also uncovered the roles of betaH and Annexin B9 in the formation of such compartment.