Department of Physiology Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK.
Development. 2023 Jun 1;150(11). doi: 10.1242/dev.201785. Epub 2023 Jun 8.
Active Notch signalling is elicited through receptor-ligand interactions that result in release of the Notch intracellular domain (NICD), which translocates into the nucleus. NICD activates transcription at target genes, forming a complex with the DNA-binding transcription factor CSL [CBF1/Su(H)/LAG-1] and co-activator Mastermind. However, CSL lacks its own nuclear localisation sequence, and it remains unclear where the tripartite complex is formed. To probe the mechanisms involved, we designed an optogenetic approach to control NICD release (OptIC-Notch) and monitored the subsequent complex formation and target gene activation. Strikingly, we observed that, when uncleaved, OptIC-Notch sequestered CSL in the cytoplasm. Hypothesising that exposure of a juxta membrane ΦWΦP motif is key to sequestration, we masked this motif with a second light-sensitive domain (OptIC-Notch{ω}), which was sufficient to prevent CSL sequestration. Furthermore, NICD produced by light-induced cleavage of OptIC-Notch or OptIC-Notch{ω} chaperoned CSL into the nucleus and induced target gene expression, showing efficient light-controlled activation. Our results demonstrate that exposure of the ΦWΦP motif leads to CSL recruitment and suggest this can occur in the cytoplasm prior to nuclear entry.
活性 Notch 信号通过受体-配体相互作用引发，导致 Notch 细胞内结构域（NICD）释放，NICD 易位到细胞核内。NICD 与 DNA 结合转录因子 CSL [CBF1/Su(H)/LAG-1]和共激活因子 Mastermind 形成复合物，在靶基因上激活转录。然而，CSL 缺乏自身的核定位序列，复合物形成的确切位置仍不清楚。为了探究涉及的机制，我们设计了一种光遗传学方法来控制 NICD 的释放（OptIC-Notch），并监测随后的复合物形成和靶基因激活。引人注目的是，我们观察到未切割的 OptIC-Notch 将 CSL 隔离在细胞质中。假设接近膜的 ΦWΦP 基序的暴露对于隔离至关重要，我们用第二个光敏感结构域（OptIC-Notch{ω}）掩盖了这个基序，这足以防止 CSL 隔离。此外，由 OptIC-Notch 或 OptIC-Notch{ω}的光诱导切割产生的 NICD 引导 CSL 进入细胞核，并诱导靶基因表达，显示出高效的光控激活。我们的结果表明，ΦWΦP 基序的暴露导致 CSL 的招募，并表明这可以在核内进入之前在细胞质中发生。
