Advancing Fluorescence Fluctuation Microscopy in Living Cells: From Non-stationary Signals to Ternary Protein Interactions
2015-10
Loading...
View/Download File
Persistent link to this item
Statistics
View StatisticsJournal Title
Journal ISSN
Volume Title
Title
Advancing Fluorescence Fluctuation Microscopy in Living Cells: From Non-stationary Signals to Ternary Protein Interactions
Authors
Published Date
2015-10
Publisher
Type
Thesis or Dissertation
Abstract
Fluorescence fluctuation spectroscopy (FFS) is a powerful method for quantifying protein interactions. By exploiting the brightness of fluorescence intensity fluctuations we are able to measure the stoichiometry of protein complexes. FFS is particularly valuable because it allows real-time measurements within living cells, where protein complex formation plays a crucial role in the regulation of cellular processes. However, intensity fluctuations are frequently altered by the cell environment in subtle and unanticipated ways, which can lead to failure of the available FFS analysis methods. This thesis demonstrates that measuring in very small volumes, such as yeast and E. coli cells, can introduce a significant bias into the measured brightness as a result of cumulative sample loss, or photodepletion. This loss leads to a non-stationary signal, which is incompatible with the implicit assumption of a stationary process in conventional FFS theory. We addressed this issue by introducing a new analysis approach that serves as a foundation for extending FFS to non-stationary signals. FFS measurements in cells are also currently limited to the study of binary interactions involving two different proteins. However, most cellular processes are mediated by protein complexes consisting of more than two different proteins. Observation of pairwise interactions is not sufficient to unequivocally determine the binding interactions involving three or more proteins. To address this issue, we extended FFS beyond binary interactions by developing tricolor heterospecies partition analysis to characterize ternary protein systems. The method is based on brightness analysis of fluorescence fluctuations from three fluorescent proteins that serve as protein labels. We verified tricolor heterospecies partition analysis by experiments on well-characterized protein systems and introduced a graphical representation to visualize interactions in ternary protein systems.
Description
University of Minnesota Ph.D. dissertation. October 2015. Major: Physics. Advisor: Joachim Mueller. 1 computer file (PDF); xi, 196 pages.
Related to
Replaces
License
Collections
Series/Report Number
Funding information
Isbn identifier
Doi identifier
Previously Published Citation
Suggested citation
Hur, Kwang Ho. (2015). Advancing Fluorescence Fluctuation Microscopy in Living Cells: From Non-stationary Signals to Ternary Protein Interactions. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/177159.
Content distributed via the University Digital Conservancy may be subject to additional license and use restrictions applied by the depositor. By using these files, users agree to the Terms of Use. Materials in the UDC may contain content that is disturbing and/or harmful. For more information, please see our statement on harmful content in digital repositories.