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A Novel Component of the Apical Barrier Formed Between Hair Cells and Supporting Cells in the Inner Ear Sensory Epithelia

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A Novel Component of the Apical Barrier Formed Between Hair Cells and Supporting Cells in the Inner Ear Sensory Epithelia PISP: A Novel Component of the Apical Barrier Formed Between Hair Cells and Supporting Cells in the Inner Ear Sensory Epithelia By Harshita Gupta Submitted in partial fulfillment of the requirements For the degree of Master of Science Thesis Advisor: Dr. Brian McDermott Department of Biology CASE WESTERN RESERVE UNIVERSITY May, 2012 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Harshita Gupta candidate for the Master’s of Science degree *. (signed) Robin Snyder (chair of the committee) Heather Broihier Brian M. McDermott Emmitt R. Jolly (date) February 1, 2012 *We also certify that written approval has been obtained for any proprietary material contained therein. 2 Table of Contents Introduction ................................................................................................................ 5 Part 1. The Inner Ear.................................................................................................................................................5 1.1.1 Sensory Epithelia.............................................................................................................................................6 1.1.2 Auditory System: Organ of Corti...............................................................................................................9 1.1.3 Mechanotransduction of Hair Cells......................................................................................................11 Part 2. Reticular Lamina.......................................................................................................................................12 1.2.1 Molecular Components of TJs & AJs in the Reticular Lamina ...................................................14 1.2.2 Specialized Junctional Adaptations in OHC-DC Region of the Reticular Lamina.............14 1.2.3 Development of the Junctional Complex of the Reticular Lamina ..........................................16 Part 3. Model Organisms......................................................................................................................................18 1.3.1 Mouse as a Model Organism....................................................................................................................19 1.3.2 Chicken as a Model Organism.................................................................................................................19 1.3.3 Zebrafish as a Model Organism..............................................................................................................21 Part 4. PISP (plasma membrane calcium ATPase-interacting single-PDZ protein) Structure and Function..............................................................................................................................................................22 1.4.1 PMCAs and Hair Cells..................................................................................................................................23 Materials and Methods ............................................................................................. 25 2.1 Reverse Transcription - Polymerase Chain Reaction (RT-PCR)..................................................25 2.2 Protein Immunoblotting...............................................................................................................................25 2.3. Immunocytochemistry.................................................................................................................................26 2.4 Whole Mount Zebrafish Staining ..............................................................................................................27 2.5 Interaction of Pisp and Pmca2b using the Yeast Two-Hybrid System .....................................28 2.6 Expression and Purification of Polyhistidine-Tagged PISP Protein ..........................................30 2.7 Over-Expression of Pisp in Zebrafish......................................................................................................31 Results ...................................................................................................................... 33 3.1 PISP is Expressed in the Inner Ear of Mouse and Zebrafish .........................................................33 3.2 Zebrafish Pisp and Pmca2b Interact in vitro........................................................................................35 3.3 Over-Expression Pattern of PISP in Hair Cells of Zebrafish..........................................................38 3.4 PISP Protein Localizes Towards the Apical Portion of the Mouse and Chicken Sensory Epithelium..................................................................................................................................................................40 3.5 Characterization of the Human Polyclonal Antibody Against PISP ...........................................42 3.6 PISP is a Junctional Protein Candidate in Chicken Sensory Epithelium ..................................43 Discussion ................................................................................................................. 46 4.1 PISP is a Single PDZ Domain-Containing Protein Conserved Between Mouse, Chicken, and Zebrafish ............................................................................................................................................................46 4.2 In vitro Interaction of PISP with the C-terminus of PMCA2b........................................................47 4.3 Zebrafish PISP Localized Towards the Apical Portion of the Hair Cells ..................................48 4.4 PISP Localizes towards the Apical Portion of the Inner Ear Sensory Epithelium ...............49 4.5 Possible Role of PISP......................................................................................................................................52 Bibliography.............................................................................................................. 54 List of Figures Figure 1. .............................................................................................................................. 4 Figure 2. .............................................................................................................................. 9 Figure 3 ............................................................................................................................... 8 Figure 4 ............................................................................................................................. 13 Figure 5 ............................................................................................................................. 16 Figure 6 ............................................................................................................................. 17 Figure 7. ............................................................................................................................ 20 Figure 8 ............................................................................................................................. 21 Figure 9 ............................................................................................................................. 34 Figure 10. .......................................................................................................................... 35 Figure 11 ........................................................................................................................... 37 Figure 12 ........................................................................................................................... 38 Figure 13 ........................................................................................................................... 41 Figure 14 ........................................................................................................................... 42 Figure 15 ........................................................................................................................... 44 Figure 16 ........................................................................................................................... 53 2 Acknowledgements I would like to thank the people who made this research possible. Dr. Brian McDermott for having the confidence in me and allowing me to independently explore my research topic, having patience and always giving an encouraging word through the difficult phases of this study. The lab, all the lab members made the lab atmosphere an excellent environment to work in. It has always been fun to look forward to coming into lab, to work with this group. The lab is a testament of how people are the most important resource. Gustavo Gomez, Lana Pollock, Megan West, Victoria (Shih Chou), Carol Fernando, and Phil Hwang – thank you for maintaining a friendly and helpful work atmosphere. Dr. Heather Broihier and Dr. Emmitt Jolly, for being a supportive committee and guiding me through the process of my defense. I have been fortunate to work with some amazing people around campus. I owe my deepest thanks to Dr. Mark Parker. He has been a constant source of support, helping me learn several techniques, getting in touch with other people having relevant areas of expertise and someone with whom I could talk about my research. I would also like to thank Summer Watterson, Dr. Polly Phillips, Dr. Suhasini Gopal for their help. No lab research is possible without thinking of
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