In Vivo Investigation of Protein Interactions in Caenorhabditis Elegans by Forster Resonance Energy Transfer Microscopy
Breusegem, Sophia Yannick
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https://hdl.handle.net/2142/85425
Description
Title
In Vivo Investigation of Protein Interactions in Caenorhabditis Elegans by Forster Resonance Energy Transfer Microscopy
Author(s)
Breusegem, Sophia Yannick
Issue Date
2002
Doctoral Committee Chair(s)
Clegg, Robert M.
Department of Study
Biophysics and Computational Biology
Discipline
Biophysics and Computational Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Animal Physiology
Language
eng
Abstract
In the second part of the thesis interactions of PAT-4, the C. elegans homologue of integrin-linked kinase (ILK), were investigated by FRET microscopy in living transgenic animals expressing CFP- and YFP-fusion proteins. Quantitative FRET microscopy in living C. elegans was most complicated by large autofluorescence contributions to the measured signals. Heterogeneous absolute and relative fusion protein expression levels were observed, and photobleaching of CFP during the measurements had to be accounted for. The CFP photobleaching kinetics were also measured to quantify the FRET efficiency. At high signal-to-noise, different FRET microscopy methods were reliable. The measurements indicate inter-PAT-4/ILK interactions in the muscle attachment structures in vivo, as well as interactions between PAT-4/ILK and PAT-6/actopaxin and between PAT-4/ILK and betaPAT-3 integrin. The thesis discusses advantages and disadvantages of the different measurement methods, and future strategies for the elucidation of the three-dimensional organization of the nematode's muscle attachment structures by in vivo FRET microscopy.
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