The plasma membrane protein “cluster of differentiation 95” (CD95) plays a prominent role in mammalian cell signalling for apoptosis, but recent studies demonstrate its additional role in proliferation1. Here, we aim to decipher how different CD95 activity states at the level of the plasma membrane affect signal transduction and the overall cell response. To this end, we interrogate CD95 interaction and complex formation after passive or active stimulation in live cells using super-resolution microscopy (FRET, STED) and an active magnetic manipulation (Magnetogenetics)2.
CD95 is a widely expressed plasma membrane protein of predominant interest in cancer therapy, whose role in the antithetic signalling for cell death and proliferation is yet to be understood. Recently, it was suggested that different activation states and the multimeric organization of CD95 at the level of the plasma membrane yield particular cell responses resulting in one or the other cell fate. In this project, we aim to decipher how CD95 receptor activation via CD95 ligand binding and its supramolecular structure formation trigger particular downstream mechanism in cancer cells. In particular, we will
- characterize the CD95 membrane complex as signalling platform for different cell responses
- investigate the oligomeric complex formation with superresolution microscopy
- actively stimulate CD95 oligomerization using a magnetic manipulation approach
To understand the relation between ligand-receptor multimerization and receptor activation, we follow an interdisciplinary approach combining experimental input with structure prediction: first, we will use live cell imaging of human cancer cells to correlate CD95 receptor dynamics with the overall cell response. Second, we will monitor CD95 active-nonactive conformational states by Förster Resonance Energy Transfer and Stimulated Emission Depletion (FRET and STED) to identify CD95 complexes and their structural hallmarks. Third, we will use a magnetogenetic manipulation approach to actively manipulate CD95 and induce different degrees of receptor accumulation. We will correlate the kinetics of receptor activation, oligomerization and downstream signalling in a multiparametric approach to quantify how complex CD95 receptor states determine the cell fate decision. Finally, using receptor mutants which block particular interactions and downstream signalling, we will confirm our hypotheses.
Our results will uncover how signalling activation is modulated by the capacity of its receptor to form characteristic membrane complexes.
1. G. Gülcüler Balta, et al., "3D cellular architecture modulates tyrosine activity thereby switching CD95 mediated apoptosis to survival". Cell Reports (2019), 29, 2295-2306
2. C. Monzel, et al. “Magnetic Control of Cellular Processes Using Biofunctional Nanoparticles”. Chemical Science (2017), 8, 7330-8, https://doi.org/10.1039/C7SC01462G
3. R. M.L. Berger et al., .... Cornelia Monzel*, and Amelie Heuer-Jungemann*,
"Nanoscale FasL Organization on DNA Origami to Decipher Apoptosis Signal Activation in Cells"
Small (2021), 2101678 https://doi.org/10.1002/smll.202101678 *corresponding authors
4. N. Bartels, N. T. M. van der Voort, A. Greife, A. Bister, C. Wiek, C. A. M. Seidel, C. Monzel, "A Minimal Model of CD95 Signal Initiation Revealed by Advanced Super-resolution and Multiparametric Fluorescence Microscopy" bioRxiv (2022), https://doi.org/10.1101/2022.11.29.518370
Project leader: Prof. Dr. Cornelia Monzel,
Researcher: Nina Bartels,
Research area: Biophysics, Cell biology, nanotechnology