Studium vlastností a interakcí nukleoproteinů pomocí pokročilých metod časově rozlišené fluorescenční mikroskopie

Vacková, Anežka

The main aim of this thesis was implementation and testing of an additional imaging method for investigation of protein-protein interactions in our laboratory and to extend already established heteroFRET (Förster resonance energy transfer) method used with the fluorescence lifetime imaging (FLIM). We performed homoFRET experiments with both the steady-state and time-resolved anisotropy-imaging methods. The presence of homoFRET between fluorophores was done by partial fluorophore bleaching and by the "red edge" analysis. We compared the methods, especially their sensitivity, experimental requirements, complexity, and informational value. We effectively applied steady-state and time-resolved anisotropy imaging to six proteins of interest; both proved comparably effective and reliable as heteroFRET-FLIM for detection of the protein interactions. The red edge method appeared problematic due to background contribution, which could not be suppressed with the current setup and instrumentation. We validated steady-state and time-resolved anisotropy imaging by studying well- described oligomerizing nucleophosmin variants (NPMwt and NPMmut) and noninter- acting mutant NPMcut, expressed in living HEK-293T cells. Then, we also probed nu- cleolin (NCL) and free fluorescent tags NowGFP and mVenus, aiming to find...

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