CLIC FUNCTIONS TO REGULATE MOESIN PHOSPHORYLATION DURING DROSOPHILA RHABDOMERE FORMATION A Thesis Presented to The College of Arts and Sciences Ohio University In Partial Fulfillment of the Requirements for Graduation with Honors from the College of Arts and Sciences with the degree of Bachelor of Science in Molecular and Cellular Biology By Kara J. Finley May 2015 1 ABSTRACT ERM family proteins have a critical role in the formation of actin-rich structures through the process of linking the cytoskeleton to the plasma membrane. In Drosophila melanogaster, Moesin, the sole ERM protein, is responsible for the development of the stacks of thousands of actin-based microvilli that form the rhabdomeres of photoreceptors in the compound eye. When phosphorylated, active Moe can bind both F-actin and the phospholipid PIP2; dephosphorylation inactivates Moe and leads to the dissociation of F-actin. The phosphoregulation of Moe is accomplished through the activities of various proteins. Moe binds to the phospholipid PIP2, which is produced through the action of Sktl. Slik kinase then phosphorylates Moe, thereby activating it and promoting binding of the actin cytoskeleton. This Moe- actin complex is next connected to a membrane-bound protein via the protein Sip1 (Drosophila homolog of EBP50). Dephosphorylation of Moe can then occur. The chloride intracellular channel protein Clic is implicated to have a role in this pathway. Our study focuses on determining the function and location of Clic in this cycle. We overexpressed Slik, both individually and in combination with Sip1 or Sktl, with a GMR driver and compared these in the wild type and a Clic deficient background by means of external morphology and internal eye morphology using a transmission electron microscope. Excess Slik kinase or Sktl, and the combination of both increases the phosphorylation of Moe, leading to larger rhabdomere formations; loss of Clic enhances these effects. Clic deficiency appears to result in a slight rescue of the phenotype of offspring with excess Slik kinase and Sip1, leading to a more regular 2 rhabdomere organization. Our results suggest that Clic functions at multiple points in Moe phosphoregulation. 3 ACKNOWLEDGMENTS First and foremost, I would like to thank Dr. Tanda of the Ohio University Department of Biological Sciences for being a great instructor and mentor. It was through his mentoring and patience that I was able to pursue an honors thesis. I am grateful for everything he has taught me in the lab, as well as for his advice about my plans for the future. My time at Ohio University would not have been the same without him. Furthermore, I would like to thank Dr. Berryman of the Ohio University Department of Biomedical Sciences for his help and advice, as well as for his previous research on this topic. I would also like to thank Dr. Robert Hikida of the Ohio University Department of Biomedical Sciences for his contributions to this project, including embedding, staining, and sectioning of tissue samples, along with his instruction on how to use the Ultramicrotome. His expertise in the use of transmission electron microscopy contributed to the informative electron micrographs used in my thesis. This work was supported by the Provost’s Undergraduate Research Fund (PURF) at Ohio University. Additionally, I would like to thank Dr. John Kopchick for his donations to form the John J. Kopchick Molecular and Cellular Biology/Translational Biomedical Sciences Undergraduate Student Support Fund, from which I received funding for this project. 4 TABLE OF CONTENTS Introduction...................................................................................................................7 I. Drosophila melanogaster photoreceptor differentiation........................8 II. Rhabdomere formation.........................................................................10 III. ERM proteins........................................................................................13 IV. Moesin regulation.................................................................................14 V. Chloride intracellular channels.............................................................15 VI. Chloride intracellular channels and Moesin.........................................17 VII. Rho signaling........................................................................................18 VIII. Rho, ERM proteins, and Moe...............................................................18 Experimental Design..................................................................................................21 I. Use of Drosophila melanogaster as a model organism........................21 II. Research questions and hypotheses......................................................23 Materials and Methods..............................................................................................24 I. Fly strains and genetics.........................................................................24 II. Maintenance of fly cultures..................................................................26 III. External eye imaging.............................................................................27 IV. Fixation, staining, and embedding........................................................27 V. Sectioning..............................................................................................28 VI. Western blot..........................................................................................29 VII. Immunofluorescence.............................................................................31 5 Results..........................................................................................................................34 I. Clic is involved in rhabdomere morphogenesis....................................34 II. Clic functions antagonistically to Moe phosphorylation......................37 III. Clic functions synergistically to Moe phosphorylation........................43 Discussion....................................................................................................................47 Future Directions........................................................................................................52 Literature Cited..........................................................................................................55 6 INDEX OF FIGURES Figure 1. Schematic drawing of the Drosophila adult ommatidium............................12 Figure 2. EM picture of normal ommatidium..............................................................12 Figure 3. Current model for Moesin activation............................................................15 Figure 4. Stereocilia in jitterbug mice.........................................................................16 Figure 5. Western blot of larval salivary glands..........................................................17 Figure 6. Model of ERM, Clic, and Rho complex.......................................................19 Figure 7. RNAi gene knockdown schematic...............................................................26 Figure 8. EM of WT vs. Clic109 ommatidia..................................................................34 Figure 9. EM of GMR control ommatidium................................................................35 Figure 10. EM of MoeRNAi WT vs. MoeRNAi Clic109 ommatidia....................................36 Figure 11. Co-localization assay in pupal retinas........................................................37 Figure 12. LM of UAS-Sktl WT vs. UAS-Sktl Clic109 eye tissue sections.................38 Figure 13. EM of UAS-Slik WT vs. UAS-Slik Clic109 ommatidia..............................39 Figure 14. EM of UAS-Sktl + UAS-Slik in WT vs. Clic109ommatidia.......................40 Figure 15. EM of UAS-Slik + UAS-MoeWT.myc in WT vs. Clic109 ommatidia..........41 Figure 16. Western blot of adult head tissue................................................................43 Figure 17. EM of UAS-Slik + UAS-Sip1 in WT vs. Clic109 ommatidia......................45 Figure 18. LM of UAS-Sip1 in WT vs. Clic109 adult eye exterior...............................45 Figure 19. EM of UAS-Pp1-87B (x2) in WT vs. Clic109 ommatidia...........................46 Figure 20. Model of Clic activity in Moe phosphorylation.........................................51 Figure 21. Mosaic analysis with a repressible marker schematic................................53 7 INTRODUCTION Hearing loss affects up to 360 million people worldwide (Atkinson et al., 2014). When exposed to loud noise, damage occurs to the stereocilia in the ears, which can lead to cell death of sensory cells. Often, this occurs over a long period of exposure to noise and is typically found in the elderly; however, hearing loss is becoming more prevalent in the younger generations due to damaging sounds, such as loud music, construction noise, and gunfire. According to the National Institute on Deafness and Other Communication Disorders, of those affected by hearing loss in America, about 26 million cases are caused by exposure to harmful noises. Common treatments include cochlear implants and hearing aids; however, hearing aid production currently only covers 10% of the global need (Disorders, 2010). Since there is a drastic gap between supply and demand, alternative methods for treating deafness must be studied. An understanding of the genetic and protein pathways responsible for the development and functioning of the stereocilia, which are