The aim of my PhD research was to develop and establish techniques for surface functionalization of nanoparticles, which can be employed to study the dynamics, function and activity of recombinantly expressed as well as endogenous proteins inside living cells. A prerequisite to achieve this goal was the ability to bio-functionalize nanoparticles with proteins in the cytoplasm of living cells. The HaloTag technology was utilized for generic site-specific targeting of nanoparticles to proteins. Fast and efficient targeting of nanoparticles to proteins was then achieved by using an engineered clickHTL exhibiting fast reactivity towards the HaloTag-enzyme. Application of this approach to track individual proteins in the outer membrane of mitochondria revealed that the physicochemical properties of the nanoparticles biased the mobility of the targeted proteins. To circumvent this, a model nanoparticle was systematically engineered in order to identify physicochemical properties that are important for tracking intracellular membrane proteins without affecting their diffusion dynamics. Nanoparticles exhibiting stealth properties were finally obtained upon densely coating the nanoparticle surface with PEG2k. These particles were mono-functionalization with clickHTL, to ensure labeling in a 1:1 stoichiometry, and could be successfully used for unbiased tracking of individual membrane proteins. Beyond the observation of proteins, generic approaches that allow intracellular manipulation and probing of protein activities are desired. To this end, 500 nm superparamagnetic nanoparticles were used as mobile nanoscopic hotspots self-assebled into active signaling platforms. Inside living cells, precise and accurate manipulation of endogeneous Rac1 activity was possible at different subcellular locations and over extended time periods. These experiments demonstrated that Rac1 signaling is dependent on the subcellular-context by spatial isolation of distinct signaling pathways. Furthermore, these MNPs provided well defined platforms for selective spectroscopy in order to quantify bait-prey protein interactions in the cytoplasm as was demonstrated by the interaction of cdc42 and N-WASP.
| Identifer | oai:union.ndltd.org:uni-osnabrueck.de/oai:repositorium.ub.uni-osnabrueck.de:urn:nbn:de:gbv:700-2014011612234 |
| Date | 16 January 2014 |
| Creators | Liße, Domenik |
| Contributors | Prof. Dr. Jacob Piehler, Prof. Dr. Andreas Herrmann |
| Source Sets | Universität Osnabrück |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis |
| Format | application/pdf, application/zip |
| Rights | http://rightsstatements.org/vocab/InC/1.0/ |
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