-r- A Hair Bundle Proteomics Approach to Discovering Actin Regulatory Proteins in Inner Ear Stereocilia by Anthony Wei Peng SEP 17 2009 B.S. Electrical and Computer Engineering LIBRARIES Cornell University, 1999 SUBMITTED TO THE HARVARD-MIT DIVISION OF HEALTH SCIENCES AND TECHNOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN SPEECH AND HEARING BIOSCIENCES AND TECHNOLOGY AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY ARCHIVES JUNE 2009 02009 Anthony Wei Peng. All rights reserved. The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic copies o this the is document in whole or in art in any medium nov know or ereafter created. Signature of Author: I Mr=--. r IT Division of Health Sciences and Technology May 1,2009 Certified by: I/ - I / o v Stefan Heller, PhD Associate Professor of Otolaryngology Head and Neck Surgery Stanford University ---- .. Thesis Supervisor Accepted by: -- Ram Sasisekharan, PhD Director, Harvard-MIT Division of Health Sciences and Technology Edward Hood Taplin Professor of Health Sciences & Technology and Biological Engineering. ---- q- ~ r This page is intentionally left blank. I~~riU~...I~ ~-- -pyyur~. _ A Hair Bundle Proteomics Approach to Discovering Actin Regulatory Proteins in Inner Ear Stereocilia by Anthony Wei Peng B.S. Electrical and Computer Engineering, Cornell University, 1999 Submitted to the Harvard-MIT Division of Health Science and Technology on May 7, 2009 in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Speech and Hearing Bioscience and Technology Abstract Because there is little knowledge in the areas of stereocilia development, maintenance, and function in the hearing system, I decided to pursue a proteomics-based approach to discover proteins that play a role in stereocilia function. I employed a modified "twist-off" technique to isolate hair bundle proteins, and I developed a method to purify proteins and to process them for analysis using multi-dimensional protein identification technology (MudPIT). The MudPIT analysis yielded a substantial list of proteins. I verified the presence of 21 out of 34 (62%) existing proteins known to be present in stereocilia. This provided strong evidence that my proteomics approach was efficient in identifying hair bundle proteins. Next, I selected three proteins and localized them to murine cochlear stereocilia. StarD10, a putative phospholipid binding protein, was detectable along the shaft of stereocilia. Nebulin, a putative F-actin regulator, was located toward the base of stereocilia. Finally, twinfilin 2, a putative modulator of actin polymerization, was found at the tips of stereocilia. In order to determine the function of twinfilin 2, I localized the protein predominately to the tips of shorter stereocilia where it is up-regulated during the final phase of elongation. When overexpressed, I found that twinfilin 2 causes a shortening of microvilli in LLC-PK1/CL4 cells and in native cochlear stereocilia. The main result of this thesis was determining the sub-cellular localization of three interesting proteins and functionally characterizing one protein. My thesis also confirmed the proteomics screen I developed as an efficient method for identifying proteins in stereocilia. Thesis Supervisor: Stefan Heller Title: Associate Professor, Department of Otolaryngology - Head and Neck Surgery, Stanford University School of Medicine, Stanford, CA ~g, --- -_~.,._L -- I~---- I--- I I_ I I I_ This page is intentionally left blank. ITF~riF~'~'4%1LEP~""~"~l----Clb-~L" Acknowledgements I am very blessed to have many people in my life to support me through completing my thesis and to teach me different skills and techniques. My mentor, Dr. Stefan Heller, has been instrumental in shaping my research plan and constantly providing me support. He always had faith in me, and he continued to push me and challenge me to do better. His never-ending ideas always pushed me forward when I doubted. Dr. Tony Ricci has also provided me with microscopy knowledge and given me invaluable advice. Drs. Eduardo Corrales and Kazuo Oshima patiently taught me how to dissect the inner ear. Sabine Mann taught me how to work with Sf9 cells and helped with protein expression and antibody production. Zhigang Xu provided insight on cloning and helped with experiments. Patrick Hsu spearheaded the work with StarD10. The Heller lab has provided me with years of feedback and comments. My thesis committee (Dr. M. Charles Liberman, Dr. William Sewell, and Dr. Clifford Woolf) guided me along, put me back on the path when I started to stray, and provided helpful feedback. Drs. Inna Belyantseva and Thomas Friedman welcomed me to visit their lab and perform some of my experiments with them. My parents, Thomas, Sophia, and the Chang's have supported me through all my schooling and my research. Without their love and support I would not be where I am today. Finally, my fiance Amanda Graves for her support and love. I'm truly lucky to have her, and I am lucky to join her wonderful family who has also supported me to the end. - 1 'P+i' This page is intentionally left blank. i~i-. i -- Table of contents C hapter 1: Introd uctio n ................................................................................................. 11 1.1 Hearing O verview .......................................... ................................................. 11 1.2 Stereocilia structure importance for MET ....................................... ........ 12 1.3 Actin filament structure.....................................................13 1.4 S tereocilia S tructure ............................................................................................. 15 1.5 Stereocilia Developm ent ...................................................................................... 16 1.6 Actin Dynamics of Stereocilia.............................................. 18 1.7 Introduction to this thesis ..................................................................................... 20 Chapter 2: Development of the Proteomics Screen ..................................... .... 31 2.1 Introduction to Proteom ics ................................................ ................................. 31 2.2 Development of the sample preparation method....................................33 2.2.1 Bundle isolation and protein solubilization ..................................... .... 33 2.2.2 Protein separation and detergent removal ..................................... .... 36 2.2.3 Protein cleavage........................................................39 2.2.4 Large-scale proteomics method ..................................... ......... 40 2 .3 C o nclusio ns....................................................... ............................................. 4 1 Chapter 3: Results of the Proteomics Screen.................................................................. 55 3.1 Overview of known hair bundle proteins ............................................................... 55 3.1.1 Structural / Cytoskeletal Proteins............................. ............. 55 3.1.2 Ca2+ regulation proteins................................................ 56 3.1.3 pH regulation .............................................................................................. .. 57 3.1.4 Myosin Motors in stereocilia ..................................... ............ 57 3.1.5 Energy metabolism proteins ..................................... ........... 58 3.1.6 Other scaffolding proteins............................................... 58 3 .2 Me th o d s ............................................................................................................... 5 9 3.3 Analysis of identified proteins from the MudPIT and validation of approach........61 3.3.1. Results of proteomics screen .......................................................................... 61 3.3.2 Comparison to results from Dr. Gillespie's laboratory........................... 62 -- -- I 3.3.3 Identification of known stereocilia proteins ................................................. 63 3.4 Conclusions ....................................................................... .............................. 64 Chapter 4: Sub-cellular localization of three new stereocilia proteins identified in the proteom ic screen .......................................................................................................... 77 4.1 Introduction to the three newly identified proteins ..................................... .. 77 4.2 Methods ...................................... .............................. .................................... 79 4.2.1 Antibodies ..................................................................... ............................ 79 4.2.2 Sf9 cell protein expression and purification ................................................ 80 4.2.3 Antibody generation and affinity purification .................................... ... 81 4.2.4 HEK 293 cell transfection and im m unostaining ............................................. 82 4.2.5 W hole-mount im m uno-fluorescence .............................................................. 83 4.3 Sub-cellular localization of proteins in cochlear hair cells ..................................... 84 4.3.1 StarD10 ........................................................................ ................................. 84 4.3.2 Nebulin .............................................. ..........