Experimental Medical Physics, Heinrich-Heine University, Düsseldorf, Germany.
Molecular Physical Chemistry, Heinrich-Heine University, Düsseldorf, Germany.
Sci Adv. 2024 Aug 30;10(35):eadn3238. doi: 10.1126/sciadv.adn3238.
Unraveling the concentration-dependent spatiotemporal organization of receptors in the plasma membrane is crucial to understand cell signal initiation. A paradigm of this process is the oligomerization of CD95 during apoptosis signaling, with different oligomerization models being discussed. Here, we establish the molecular-sensitive approach cell lifetime Förster resonance energy transfer image spectroscopy to determine CD95 configurations in live cells. These data are corroborated by stimulated emission depletion microscopy, confocal photobleaching step analysis, and fluorescence correlation spectroscopy. We probed CD95 interactions for concentrations of 10 to 1000 molecules per square micrometer, over nanoseconds to hours, and molecular to cellular scales. Quantitative benchmarking was achieved establishing high-fidelity monomer and dimer controls. While CD95 alone is primarily monomeric (96%) and dimeric (4%), the addition of ligand induces oligomerization to dimers/trimers (~15%) leading to cell death. This study highlights molecular concentration effects and oligomerization dynamics. It reveals a minimal model, where small CD95 oligomers suffice to efficiently initiate signaling.
揭示质膜中受体的浓度依赖性时空组织对于理解细胞信号起始至关重要。细胞凋亡信号中 CD95 寡聚化就是一个很好的例子，目前有不同的寡聚化模型在讨论。在这里，我们建立了分子敏感的细胞寿命Förster 共振能量转移成像光谱法来确定活细胞中 CD95 的构象。这些数据得到了受激发射损耗显微镜、共聚焦光漂白分步分析和荧光相关光谱法的证实。我们在 10 到 1000 个分子/平方微米的浓度范围内，在纳秒到小时，以及分子到细胞的尺度上，探测了 CD95 的相互作用。通过建立高保真的单体和二聚体对照，实现了定量基准测试。虽然单独的 CD95 主要是单体 (96%)和二聚体 (4%)，但配体的加入诱导寡聚化为二聚体/三聚体 (~15%)，从而导致细胞死亡。这项研究强调了分子浓度效应和寡聚化动力学。它揭示了一个最小的模型，其中少量的 CD95 寡聚体就足以有效地启动信号。
