In Vivo Investigation of Protein Interactions in Caenorhabditis Elegans by Forster Resonance Energy Transfer Microscopy

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biophysics and Computational Biology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Breusegem, Sophia Yannick

Contributors dc:contributor

• Clegg, Robert M.

• Biology, Animal Physiology

Language dc:language

eng

Identifier

(MiAaPQ)AAI3069977

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/85425