Skip to Main Content
Frequently Asked Questions
Submit an ETD
Global Search Box
Need Help?
Keyword Search
Participating Institutions
Advanced Search
School Logo
Files
File List
M Sc Thesis v27-final-final-last final-last last fina tomerl - v4.pdf (14.72 MB)
ETD Abstract Container
Abstract Header
Sas4 N terminal as a potential binding probe for tubulin-GDP
Author Info
Yuan, Wenjue
Permalink:
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1399559850
Abstract Details
Year and Degree
2014, Master of Science, University of Toledo, Biology (Cell-Molecular Biology).
Abstract
As a conserved organelle, the centrosome and its regulated biogenesis is required for cilium formation, accurate cell division, and genome integrity (Nigg and Raff, 2009). Centrosomes consist of a centriole pair surrounded by an amorphous protein network known as pericentriolar material or PCM (Nigg and Raff, 2009). PCM assembly is a tightly regulated step during centrosome biogenesis that determines the size and capability of centrosomes (Conduit et al., 2010; Kirkham et al., 2003; Piehl et al., 2004). During PCM assembly, Sas4, one of the PCM proteins activated by tubulin in GDP bound conformation, provides a scaffold for preassembled cytoplasmic complexes to be tethered to the centrosome. Tubulin directly binds to Sas4; together, they are components of cytoplasmic complexes of centrosomal proteins (Gopalakrishnan et al., 2011; Hung et al., 2004). On the basis of the guanine nucleotide bound state, tubulin can act as a molecular switch in PCM recruitment. While tubulin-GTP prevents Sas4 from forming protein complexes, tubulin-GDP promotes it (Gopalakrishnan et al., 2012). Since tubulin-GDP, instead of tubulin-GTP, promotes formation of centrosomal protein complex recruited by Sas4, it is interesting to note that in a normal, healthy cell, the proportion of GTP: GDP in cytoplasm is near 100:1 (Gamberucci et al., 1994). Thus, in this study, we hypothesize that in order to guarantee proper PCM assembly, Sas4 specifically binds to tubulin-GDP. In other words, Sas4 could be utilized as a binding probe specifically for tubulin-GDP conformation. Our research focuses on in vitro experiments that identify Sas4-tubulin interaction and the specificity under different guanine nucleotides. A number of in vitro biochemistry assays were carried out in order to test Sas4-Tubulin interaction in different guanine nucleotide statuses. Co-Immunoprecipitation, Pulldown assay, Native PAGE-Far Western and Far Western Dot-Blot methods were utilized to test our hypothesis. The results are consistent with our hypothesis that Sas4 specifically recognizes tubulin-GDP, and it is further confirmed by the fact that tandem-Sas4, a recombinant protein derived from our newly built plasmid, specifically increased the binding strength to tubulin-GDP by at least 50%. Our results further confirmed that Sas4N specifically binds tubulin-GDP and function as a core in scaffolding centrosomal protein in the maturation of centrosome. Meanwhile , it also give us a potential tool to utilize Sas4, which specifically binds to tubulin-GDP, to gain more details of insights on the function of free tubulin-GDP as molecule switch instead of a building block in biological process like microtubule dynamic instability
Committee
Tomer Reiss (Committee Chair)
John Plenefisch (Committee Member)
Song-Tao Liu (Committee Member)
William Taylor (Committee Member)
Pages
80 p.
Subject Headings
Biochemistry
;
Biology
;
Biomedical Engineering
;
Biomedical Research
;
Cellular Biology
;
Developmental Biology
;
Health Sciences
;
Molecular Biology
;
Molecular Chemistry
;
Molecules
Keywords
centrosome
;
Sas4
;
tubulin
;
GTP
;
GDP
;
GMPCPP
Recommended Citations
Refworks
EndNote
RIS
Mendeley
Citations
Yuan, W. (2014).
Sas4 N terminal as a potential binding probe for tubulin-GDP
[Master's thesis, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1399559850
APA Style (7th edition)
Yuan, Wenjue.
Sas4 N terminal as a potential binding probe for tubulin-GDP.
2014. University of Toledo, Master's thesis.
OhioLINK Electronic Theses and Dissertations Center
, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1399559850.
MLA Style (8th edition)
Yuan, Wenjue. "Sas4 N terminal as a potential binding probe for tubulin-GDP." Master's thesis, University of Toledo, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1399559850
Chicago Manual of Style (17th edition)
Abstract Footer
Document number:
toledo1399559850
Download Count:
543
Copyright Info
© 2014, all rights reserved.
This open access ETD is published by University of Toledo and OhioLINK.