RT Journal Article SR Electronic T1 A Minimal Model of CD95 Signal Initiation Revealed by Advanced Super-resolution and Multiparametric Fluorescence Microscopy JF bioRxiv FD Cold Spring Harbor Laboratory SP 2022.11.29.518370 DO 10.1101/2022.11.29.518370 A1 Bartels, Nina A1 van der Voort, Nicolaas T M A1 Greife, Annemarie A1 Bister, Arthur A1 Wiek, Constanze A1 Seidel, Claus A M A1 Monzel, Cornelia YR 2022 UL http://biorxiv.org/content/early/2022/11/30/2022.11.29.518370.abstract AB Unraveling the spatiotemporal organization and dynamical interactions of receptors in the plasma membrane remains a key challenge for our mechanistic understanding of cell signal initiation. A paradigm of such process is the oligomerization of TNF receptor CD95 during apoptosis signaling, where molecular configurations are yet to be defined. Here, we scrutinize proposed oligomerization models in live cells, establishing a molecular sensitive imaging toolkit including time-resolved FRET spectroscopy, quantitative STED microscopy, confocal Photobleaching Step Analysis and FCS. CD95 interactions were probed over molecular concentrations of few to ∼ 1000 molecules/µm2, over ns to hours, and molecular to cellular scales. We further established high-fidelity monomer and dimer controls for quantitative benchmarking. Efficient apoptosis was already observed when ∼ 8 to 17% monomeric CD95 oligomerize to dimers/trimers after ligand binding. Our multiscale study highlights the importance of molecular concentrations, of the native environment, and reveals a minimal oligomerization model of CD95 signal initiation.Competing Interest StatementThe authors have declared no competing interest.