2026-09-19T15:57:56Zhttps://dspace.mit.edu/server/oai/requestoai:dspace.mit.edu:1721.1/545882022-01-13T07:53:45Zcom_1721.1_7582com_1721.1_7581col_1721.1_131022
Stefan Heller.
Peng, Anthony Wei
Harvard University--MIT Division of Health Sciences and Technology.
Harvard University--MIT Division of Health Sciences and Technology
2010-04-28T17:05:46Z
2010-04-28T17:05:46Z
2009
2009
http://hdl.handle.net/1721.1/54588
569417655
Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 137-154).
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.
by Anthony Wei Peng.
Ph.D.
154 p.
eng
Massachusetts Institute of Technology
M.I.T. theses are protected by
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http://dspace.mit.edu/handle/1721.1/7582
Harvard University--MIT Division of Health Sciences and Technology.
A hair bundle proteomics approach to discovering actin regulatory proteins in inner ear stereocilia
Thesis
application/pdf
Publication
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