Dynamics of p53 tetramers in live single cells

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Dynamics of p53 tetramers in live single cells

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dc.contributor.advisor Lahav, Galit
dc.contributor.author Gaglia, Giorgio
dc.date.accessioned 2014-06-06T14:50:47Z
dc.date.issued 2014-06-06
dc.date.submitted 2014
dc.identifier.citation Gaglia, Giorgio. 2014. Dynamics of p53 tetramers in live single cells. Doctoral dissertation, Harvard University. en_US
dc.identifier.other http://dissertations.umi.com/gsas.harvard:11450 en
dc.identifier.uri http://nrs.harvard.edu/urn-3:HUL.InstRepos:12269874
dc.description.abstract Protein homo-oligomerization is the process through which identical peptides bind together to form higher order complexes. Self-interactions in many cases are constitutive and stable, used as building blocks for biological structures, such as rings, filaments and membranes. Further, homo-oligomerization can also be a regulatory process that influences the proteins' function such as change in transcriptional activities for transcription factors. Innovative methods to measure oligomerization in live cells are needed in order to understand regulation and function of homooligomerization in the native cellular context. This thesis examines the case of the tumor suppressor p53, whose homo-tetramerization greatly influences its activity as a transcription factor. We develop methods to quantify p53's self-interaction in individual living cells and follow it in time after DNA damage. The two methods we developed have complementary qualities and different applications. We first use fluorescent correlation spectroscopy to study the molecular events occurring in the first three hours of the p53 in response to double strand breaks. We find that in the absence of stress p53 is present in a mixture of, monomers, dimers and tetramers. When damage is sensed, oligomerization is rapidly induced and nearly all p53 is found bound in tetramers. We combine our data with a mathematical framework to propose the existence of a dedicated mechanism triggering p53 oligomerization independently of protein stabilization. Next, we use bimolecular fluorescent complementation to probe for tetramerization in the longer timescales of p53's response to ultraviolet radiation. In this context we find that even though the rate of p53 accumulation increases with the dose of radiation, p53 tetramers are formed at a steady rate. We hence propose the existence of an inhibitory mechanism that prevents the oligomerization reaction from following a linear input-output relation. We identify ARC, a known cofactor of p53, as part of this inhibitory mechanism. Downregulation of ARC restore the linear relation between to total and tetrameric p53. Finally, in both experimental setups higher oligomerization lead to an increase in p53 activity, underscoring the connection between regulation of oligomerization and the transcriptional activity of p53 in cancer cells. Collectively, this work emphasizes the importance of precise measurements to investigate the regulation and function of higher order complexes and provides generally applicable methods to quantify homo-oligomerization in live single cells. en_US
dc.language.iso en_US en_US
dash.license LAA
dc.subject Systematic biology en_US
dc.subject DNA damage en_US
dc.subject mathematical modeling en_US
dc.subject p53 en_US
dc.subject protein dynamics en_US
dc.subject tetramerization en_US
dc.title Dynamics of p53 tetramers in live single cells en_US
dc.type Thesis or Dissertation en_US
dash.depositing.author Gaglia, Giorgio
dc.date.available 2014-06-06T14:50:47Z
thesis.degree.date 2014 en_US
thesis.degree.discipline Systems Biology en_US
thesis.degree.grantor Harvard University en_US
thesis.degree.level doctoral en_US
thesis.degree.name Ph.D. en_US
dc.contributor.committeeMember Shah, Jagesh en_US
dc.contributor.committeeMember Mitchison, Timothy en_US
dc.contributor.committeeMember Cluzel, Philippe en_US
dc.contributor.committeeMember Lee, Sam en_US

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