PT - JOURNAL ARTICLE AU - Bartels, Nina AU - van der Voort, Nicolaas T M AU - Greife, Annemarie AU - Bister, Arthur AU - Wiek, Constanze AU - Seidel, Claus A M AU - Monzel, Cornelia TI - A Minimal Model of CD95 Signal Initiation Revealed by Advanced Super-resolution and Multiparametric Fluorescence Microscopy AID - 10.1101/2022.11.29.518370 DP - 2022 Jan 01 TA - bioRxiv PG - 2022.11.29.518370 4099 - http://biorxiv.org/content/early/2022/11/30/2022.11.29.518370.short 4100 - http://biorxiv.org/content/early/2022/11/30/2022.11.29.518370.full 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.