Dokument: Protein interactions in living cells studied by multiparameter fluorescence imaging spectroscopy (MFIS)
| Titel: | Protein interactions in living cells studied by multiparameter fluorescence imaging spectroscopy (MFIS) | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=37191 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20160216-104854-7 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Ma, Qijun [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Seidel, Claus A. M. [Betreuer/Doktorvater] Prof. Dr. Simon, Rüdiger [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 540 Chemie | |||||||
| Beschreibung: | Förster resonance energy transfer (FRET) is widely applied as a spectroscopic ruler to investigate the structures and interactions of labelled biomolecules exploiting its sensitivity to the distance. Multiparameter fluorescence image spectroscopy (MFIS) provides particular advantages to FRET imaging, because multiple fluorescence parameters can be monitored simultaneously with picosecond accuracy. In cellular studies, researchers performing image spectroscopy traditionally face the dilemma of either performing single point measurements or scanning a region of interest. Point measurements provide satisfactory photon statistics but at the expense of entirely relinquishing the image information; whereas imaging measurements contain the spatial information but with poor photon statistics in each pixel. With insufficient photon counts in FRET imaging, for example, the reason for a reduction in average donor fluorescence lifetime can hardly be assigned. The reduction can be caused by changes in FRET efficiency due to distinct protein-complex conformations, and/or changes in fraction of FRET-active species. To provide a solution to this long-standing dilemma, a novel workflow of generating population-specific pixel-integrated data for noise reduction and global analysis methods for a quantitative recovery of FRET parameters are introduced in this study. The newly developed MFIS-FRET analysis tools allow one to directly visualize and quantitatively analyze the fraction of FRET-active species and FRET efficiency. Using the determined fraction of FRET-active species and utilizing the intrinsic variations of protein concentration in each experiment, stoichiometry and dissociation constant of protein complexes can be characterized in living cells. Characterization of the FRET efficiency enables detection of even subtle FRET variations and thus provides crucial information about the structural properties of molecular complexes. As also revealed in this study, fluorescent proteins in living cells have static majorly random distance distributions, thereby allowing for the distance estimation. Hence, the MFIS-FRET data can reach the quality of traditional in vitro cuvette experiments, which greatly facilitates the protein-interaction studies in living cells.
The newly developed MFIS-FRET methodology was employed in the research of membrane localized proteins: i) ligand-dependent receptor complex formation in plant cells, ii) oligomerization of guanylate binding proteins (GBPs) in murine embryonic fibroblasts (MEFs), and iii) oligomerization of G-protein coupled receptor (GPCR) in human cells. Several important and common features of proteins, such as homo- and heteromerization (i, ii and iii), changes in interaction dynamics triggered by outside stimuli, including peptide (i), pathogen (ii) and bile acid (iii), and clustering and aggregation (i and ii) are studied in detail. i) Ligand-dependent receptor complex formation MFIS-FRET was performed in individual living plant cells over time to study the initial interaction-events occurring at the receptor level following ligand perception for the two signaling pathways of the CLAVATA3 (CLV3) and flagellin (flg) peptides. The plant peptide CLV3 regulates stem cell homeostasis, whereas the bacterial flg22 peptide elicits defense responses. It shows that the CLV and the flg pathways represent two different principles of signal transduction: flg22 first triggered receptor-like kinase (RLK) heterodimerization, and later assembly into larger complexes through homomerization. In contrast, CLV receptor complexes were preformed, and ligand binding stimulated their clustering. ii) Oligomerization of murine GBPs (mGBPs) at membrane for pathogen defense MFIS-FRET experiments are performed in uninfected and Toxoplasma gondii (T. gondii) infected MEFs, respectively, to determine the subcellular locations and concentrations of mGBPs, identify the interaction partners between the family members (mGBP1/2/3/5/6), and characterize their interaction affinities. The study shows that mGBPs can undergo concentration-dependent and species-specific oligomerization from monomeric to dimeric and oligomeric species. After the T. gondii invasion, mGBPs are recruited and thus highly enriched at the parasitophorous vacuole membrane, forming large, densely packed multimers comprising up to several thousand monomers. iii) Oligomerization pattern of the membrane-localized G-protein coupled bile acid receptor TGR5 MFIS-FRET is performed on TGR5 wild type (wt) and its two variants, TGR5 Y111F and Y111A, to investigate TGR5 assembly, structure, and multimerization affinities in living cells. The study shows that all three variants form homodimers, however, only the TGR5 wt and Y111F variant are able to also form higher-order oligomers. The TGR5 Y111A variant dimerizes at an interface between transmembrane helix 1 (TM1) and helix 8, but is barred from oligomerization, likely because a clinically relevant mutation in TM5 markedly hinders its higher-order oligomerization. In summary, this work demonstrates the unique advantages of the MFIS-FRET and new quantitative FRET analysis. The methodology introduced in this work allows one to investigate the spatiotemporally regulated protein interactions at a molecular resolution level. Researchers now can efficiently study protein localization, dynamics, concentration, aggregation, protein-complex formation, stoichiometry and binding affinity, and generate a panorama of proteins of interest in living cells. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Chemie | |||||||
| Dokument erstellt am: | 16.02.2016 | |||||||
| Dateien geändert am: | 16.02.2016 | |||||||
| Promotionsantrag am: | 18.12.2015 | |||||||
| Datum der Promotion: | 03.02.2016 |
