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Klufas Dissertation FINAL.pdf (3.73 MB)

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Resolving Membrane Receptor Multimerization in Live Cells using Time Resolved Fluorescence Methods

Klufas, Megan J
http://orcid.org/0000-0001-9913-695X
http://rave.ohiolink.edu/etdc/view?acc_num=akron151017994353956

Abstract Details

2017, Doctor of Philosophy, University of Akron, Chemistry.
The cell membrane is a complex environment made up of thousands of molecular components. The dynamic assembly of these components regulates a myriad of cellular functions, but it is difficult to measure in a biologically relevant context. Pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) is a time resolved fluorescence technique that was used to obtain concentration, mobility and co-diffusion (fc) of membrane proteins in live cells. Several lines of evidence support the hypothesis that homo-dimerization (or even oligomerization) facilitates the function of membrane proteins. The goal of this research was to elucidate the dynamic organization and relative affinity of membrane protein-protein interactions. In order to accomplish this goal, a mathematical model was developed to interpret the cross-correlation value obtained from PIE-FCCS and to quantify the dynamic interactions of membrane receptors in a more rigorous way. This thesis describes three main projects. The first project focused on determining the homo-dimerization of the neuronal membrane protein PlexinA4 before and after ligand stimulation. PIE-FCCS measurements of protein controls with varying degrees of homo-multimerization were used to determine that PlexinA4 receptors assemble into preformed dimers. In the next project, a mathematical model was developed to interpret the PIE-FCCS figure of merit, fc. Several controls systems with varying dimerization affinity and degrees of oligomerization were measured and analyzed to verify the accuracy of the model. Lastly, the organization of two class A G protein-coupled receptors (GPCRs) was investigated using PIE-FCCS and a new labeling strategy. The organization of these membrane proteins showed a high degree of cell to cell variability. A simple monomer-dimer equilibrium model failed to describe the range of single cell data, so the mathematical model was altered to describe a cluster model that agreed with the experimental data. This thesis lays the groundwork for application of the mathematical model to quantify the organization of other membrane receptors using PIE-FCCS.
Adam Smith, Ph.D. (Advisor)
Chrys Wesdemiotis, Ph.D. (Committee Member)
David Perry, Ph.D. (Committee Member)
Michael Konopka, Ph.D. (Committee Member)
Mesfin Tsige, Ph.D. (Committee Member)
155 p.
Biochemistry; Biophysics; Chemistry
fluorescence correlation spectroscopy; fluorescence cross-correlation spectroscopy; pulsed interleaved excitation; homodimerization; homo-oligomerization; membrane organization; protein-protein interactions

Recommended Citations

Citations

  • Klufas, M. J. (2017). Resolving Membrane Receptor Multimerization in Live Cells using Time Resolved Fluorescence Methods [Doctoral dissertation, University of Akron]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=akron151017994353956

    APA Style (7th edition)

  • Klufas, Megan. Resolving Membrane Receptor Multimerization in Live Cells using Time Resolved Fluorescence Methods. 2017. University of Akron, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=akron151017994353956.

    MLA Style (8th edition)

  • Klufas, Megan. "Resolving Membrane Receptor Multimerization in Live Cells using Time Resolved Fluorescence Methods." Doctoral dissertation, University of Akron, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=akron151017994353956

    Chicago Manual of Style (17th edition)

akron151017994353956
189
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This open access ETD is published by University of Akron and OhioLINK.