|Title:||Development and application of methods for spatio-temporal perturbation and analysis of protein interactions via intracellular protein microarrays|
|Abstract:||Many processes in mammalian cells, including adhesion and migration, require dynamic rearrangements of specialized filament structures, which are collectively called the cytoskeleton. Those dynamic rearrangements are controlled in space and time by signaling proteins, including the Rho family of small GTPases. In this thesis, the development and application of techniques to measure and perturb the activity of RhoGTPases in space and time is presented. First, a miniaturized, intracellular protein interaction array was developed to study multiple protein interactions inside an individual living cell. Using this technology, two distinct protein interactions were simultaneously monitored in individual cells to uncover cell-to-cell variance in their dynamic response to acute pharmacological perturbation. To generate those intracellular interaction arrays, bio-orthogonal artificial receptors were used that do not perturb normal cellular function. To optimize the processing of those receptors on the secretory pathway, their design was systematically improved by deletion analysis and insertion of linkers and glycosylation motifs. Those optimizations lead to a significant improvement of the plasma membrane targeting of artificial receptors. To perturb protein activities in living cells, a new class of optimized artificial receptors, termed ‘ActivatorPARCs’ was generated, that are based on subcellular targeting via chemically induced dimerization. Recruitment of ActivatorPARCs to subcellular regions in the plasma membrane enabled perturbation of RhoGTPase activity in space and time. To increase the speed of this perturbation and to enable more flexible control of spatial perturbations, ActivatorPARCs that are based on photo chemically induced dimerization were generated. Lateral diffusion of the uncaged photodimerizer was eliminated by covalent linkage on immobilized receptors. This enabled 'Molecular activity painting', a novel technique, in which rapid and stable perturbations can be directly “painted” with light inside individual living cells. The application of those tools to directly study spatio-temporal signal propagation in cellular reaction networks is discussed.|
|Subject Headings:||Cell and systems biology|
|Appears in Collections:||Chemische Biologie|
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