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dc.contributor.advisorIngber, Donald Elliott
dc.contributor.advisorStultz, Collin Melveton
dc.contributor.authorGee, Elaine Pei-San
dc.date.accessioned2013-02-20T14:10:22Z
dash.embargo.terms2014-06-21en_US
dc.date.issued2013-02-20
dc.date.submitted2012
dc.identifier.citationGee, Elaine Pei-San. 2012. Biophysical and Molecular Determinants in Cell Tension-Mediated Fibronectin Unfolding that Drive Fibrillogenesis. Doctoral dissertation, Harvard University.en_US
dc.identifier.otherhttp://dissertations.umi.com/gsas.harvard:10402en
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:10318179
dc.description.abstractAssembly of the extracellular matrix (ECM) protein fibronectin (FN) is a mechanical process that involves cell binding to FN through cell surface integrin receptors and application of tensional forces generated in the cell's contractile actin cytoskeleton. Deformation-induced exposure of cryptic sites, defined as buried molecular recognition sites, in FN has been proposed as a mechanism by which cell tension drives FN fibrillogenesis. The primary integrin attachment site on FN is the RGD loop in the 10FNIII domain. In this thesis, I set out to define the molecular biophysical mechanism by which cell tension application at the RGD site promotes unfolding and thereby induces FN-FN self-assembly leading to matrix fibril formation. Chapter 1 of this dissertation provides an overview of the current knowledge behind the biophysical and molecular basis of FN assembly in the ECM and its key role in development and disease. In Chapter 2, steered molecular dynamic simulations show that the 10FNIII domain under force applied through its N-terminus and RGD loop (N-to-RGD) unfolds to a preferred kinetic intermediate with solvent-exposed N-terminal hydrophobic residues in a manner different from past analyses in the literature where force through the N- and C- termini leads to multiple unfolding pathways. Use of single-molecule atomic force spectroscopy in Chapter 3 experimentally reveals that a mechanically stable intermediate of 10FNIII exposed by N-to-RGD pulling shows a length extension that agrees with the predicted kinetic intermediate. Results of biochemical and cellular studies using synthetic peptides with sequences from the 10FNIII intermediate show in Chapter 4 that the twenty-three amino acid sequence that spans the unraveled N-terminus of the predicted intermediate mediates FN multimerization and contains a minimal seven amino acid sequence we call the multimerization motif that is sufficient to induce FN-FN multimer assembly. Finally, Chapter 5 summarizes the new insights supported by this work regarding the role that mechanical forces applied at the cell binding site in 10FNIII plays in the physiological unfolding of FN with respect to FN fibrillogenesis and ECM assembly.en_US
dc.language.isoen_USen_US
dash.licenseMETA_ONLY
dc.subject10FNIIIen_US
dc.subjectcell tensionen_US
dc.subjectcryptic sitesen_US
dc.subjectfibronectin fibrillogenesisen_US
dc.subjectmechanical unfoldingen_US
dc.subjectRGD loopen_US
dc.subjectbiophysicsen_US
dc.titleBiophysical and Molecular Determinants in Cell Tension-Mediated Fibronectin Unfolding that Drive Fibrillogenesisen_US
dc.typeThesis or Dissertationen_US
dash.depositing.authorGee, Elaine Pei-San
dash.embargo.until10000-01-01
thesis.degree.date2012en_US
thesis.degree.disciplineBiophysicsen_US
thesis.degree.grantorHarvard Universityen_US
thesis.degree.leveldoctoralen_US
thesis.degree.namePh.D.en_US
dc.contributor.committeeMemberBlacklow, Stephenen_US
dc.contributor.committeeMemberShih, Williamen_US
dc.contributor.committeeMemberSmith, Michaelen_US
dc.contributor.committeeMemberHogle, Jamesen_US
dash.contributor.affiliatedGee, Elaine Pei-San


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