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Spatio-temporal protease activity and cell death induced by Natural Killer cells and soluble CD95L

Liesche, Clarissa

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Abstract

In the CD95 signaling cascade molecular processes take place, which rely on protein multimerization for activity. In this work, I aimed at measuring the initial events in CD95-mediated apoptosis, in particular the kinetics of the molecular events ligand binding, receptor oligomerization, FADD recruitment and caspase-8 activation to understand which of those processes involve critical kinetic steps. For this, I used the model cell HeLa. I have succeeded in showing and comparing the kinetics of ligand-binding, FADD recruitment and caspase-8 activation by using confocal microscopy. I have found that a critical step before caspase- 8 activation is the binding of FADD to the activated receptor. Furthermore, I found that the activation of the receptor, presumably by its oligomerization, is a limiting step in CD95-mediated apoptosis. Moreover, I showed that the internalization and activation of the receptor CD95 at the plasma membrane are competing processes. To arrive at these findings, two forms of soluble CD95L were compared, one naturally occurring form with weak activity (sCD95L) and one recombinant form with high activity (IZsCD95L). Next to biochemical methods, fluorescence-microscopy based techniques were particularly applied and I have been able to confirm that both forms are trimeric. Notably, I developed a new method to evaluate single-molecule photobleaching data suitable to infer the subunit stoichiometry of molecules at picomolar concentration. Secondly, I aimed at understanding how natural killer cells (NK cells) mediate cell death in target cells. In addition to CD95L, NK cells use own proteases to induce the death of certain cells. In this context, I have been able to distinguish the activity of caspase-8 and granzymes in individual HeLa target cells based on the method of Beaudouin et al. 2013 and by using the NK cell line NK92. While granzyme B activity was measured in cells that died early after NK-cell contact, caspase-8 activity was measured in those which died late upon NK-cell contact. Noteworthy was the observation that granzyme B was mainly active near the plasma membrane, and in lesser amounts inside the nucleus. This was consistent with my results showing that the granzyme B inhibitor serpinB9 is localized at the plasma membrane. In contrast, granzyme A was probably not active in the cytosol, but in the nucleus of the cell.

Item Type: Dissertation
Supervisor: Eils, Prof. Dr. Roland
Date of thesis defense: 24 July 2014
Date Deposited: 01 Sep 2014 08:26
Date: 2014
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
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