Dokument: Advanced Molecular-Sensitive Imaging to Unravel Spatio-Temporal Hallmarks of CD95 Receptor Activation

Titel:	Advanced Molecular-Sensitive Imaging to Unravel Spatio-Temporal Hallmarks of CD95 Receptor Activation
URL für Lesezeichen:	https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=63571
URN (NBN):	urn:nbn:de:hbz:061-20240919-091951-0
Kollektion:	Dissertationen
Sprache:	Englisch
Dokumententyp:	Wissenschaftliche Abschlussarbeiten » Dissertation
Medientyp:	Text
Autor:	Bartels, Nina Maria [Autor]
Dateien:	[Dateien anzeigen] Adobe PDF [Details] 41,52 MB in einer Datei [ZIP-Datei erzeugen] Dateien vom 03.09.2023 / geändert 03.09.2023
Beitragende:	Prof. Dr. Monzel, Cornelia [Gutachter] Prof. Dr. Seidel, Claus [Gutachter]
Dewey Dezimal-Klassifikation:	500 Naturwissenschaften und Mathematik » 530 Physik
Beschreibung:	Unraveling the spatio-temporal organization and dynamical interactions of receptors in the plasma membrane remains a key challenge for our mechanistic understanding of cell signal initiation. To study membrane receptors in their natural environment, integrated in a plasma membrane of human cells, requires minimally invasive and highly sensitive techniques. In biological sciences, fluorescence microscopy and spectroscopy techniques are powerful tools to study intact cells. However, each of these techniques exhibits particular spatio-temporal regimes of superior performance and few techniques are hitherto capable of relating measured intensities to molecular numbers and concentration. To solve these issues, the present work advances molecular-sensitive imaging techniques and establishes a generic concept to uncover molecular mechanisms of signal initiation. A paradigm of a ligand-induced cell signal initiation process is the oligomerization of TNF (tumor necrosis factor) receptor CD95 in the signaling pathway for apoptosis (i.e. a coordinated natural form of cell death). A deregulation of the CD95 signaling mechanism was shown to be characteristic in some types of cancer leading to proliferation rather than cell death [1]. As the switch from signaling for death to life is hypothesized to occur via different CD95 activity states, the mechanism of molecular organization of CD95 on the cell membrane is of high interest, where the correct molecular configuration is yet to be defined. Here, the proposed oligomerization models are scrutinized in live cells by establishing high-fidelity monomer and dimer controls as well as molecular sensitive imaging techniques such as confocal Photobleaching Step Analysis (cPBSA), a quantitative STED analysis, an advanced FRET approach, and using FCS. Thus, CD95 interactions are probed over the whole dynamic range from μs to hours, molecular to cellular scales and with particular focus on molecular concentrations. The obtained results reveal a minimal model of signal initiation, where signaling dynamics scale with molecular concentrations. Suggested high oligomerization states are not observed. Instead, ligand coupling induces a switch of 6 − 15% CD95 monomers to dimers/trimers evenly distributed in the plasma membrane. This suffices to trigger apoptosis efficiently. As a result of the CD95 oligomerization study, the methods of choice were successfully advanced to tap their full potential for quantitative molecular imaging studies. In this course, • STED images were subjected to a quantitative object and brightness analysis, • a novel approach for robust PBSA is presented using a confocal setup and ubiquitous fluorescent proteins, and • FRET is advanced towards a quantitative interpretation and high precision of oligomer studies. Since a cell signal initiation is a transient and potentially localized process, this study highlights the importance of combining complementary techniques for a full understanding of this process. Additionally, an excursion into (pre-)clinical research highlights the benefits of microscopy in this field with a microscopic study of therapeutic membrane receptors. In cancer immunotherapy, chimeric antigen receptor (CAR) T-cell therapy represents a major advancement in personalized treatment strategies, which has led to an exponential growth within this research field. CARs are genetically engineered to bind a specific tumor-associated antigen and start the T-cell activation pathway in order to fight the malignancies. In this study, multi-channel, time-lapse widefield microscopy is utilized in combination with bioimage data analysis to visualize and characterize the killing of cancer cells induced by novel CAR designs. A time-lapse study of a novel 2nd generation CAR generated against CD44v6 shows concentration dependent effect in the killing efficiency using varying effector-to-target ratios. In further studies, the agglomeration of CD19 CARs expressed in T- and NK cells into immunological synapses with the antigen-carrying target cells could be visualized. The specificity of this interaction was shown in comparison to non-specific effector and target cell combinations. Overall, advanced fluorescence microscopy could contribute towards a more differentiated understanding of the CAR-induced killing mechanism and complement the clinical standard approaches. Altogether, this thesis contributes towards a greater understanding of receptors in the cellular plasma membrane. To do so, it provides technical developments for quantitative molecular imaging. Based on these advancements, a general concept/approach to understand cell signal initiation mechanism is presented including the workflow and necessary tools along with statistical analysis. In particular an understanding of cell apoptosis signal initiation, which is essential for a multitude of vital processes during development, homeostasis, elimination of malignant cells is achieved.
Lizenz:	Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz
Fachbereich / Einrichtung:	Mathematisch- Naturwissenschaftliche Fakultät » WE Physik
Dokument erstellt am:	19.09.2024
Dateien geändert am:	19.09.2024
Promotionsantrag am:	21.11.2018
Datum der Promotion:	30.06.2023