OptIC Notch reveals mechanism that regulates receptor interactions with CSL
OptIC Notch reveals mechanism that regulates receptor interactions with CSL
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DOI：
10.1101/2023.02.20.528063
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发表时间：
2023-05
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影响因子：
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通讯作者：
J. Townson;M. Gomez-Lamarca;Carmen Santa Cruz Mateos;S. Bray
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文献类型：
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作者：
J. Townson;M. Gomez-Lamarca;Carmen Santa Cruz Mateos;S. Bray
通过受体-配体相互作用引发活性Notch信号传导，所述受体-配体相互作用导致Notch胞内结构域（NICD）的释放，所述NICD易位到细胞核中。NICD激活靶基因的转录，与DNA结合转录因子CSL（CBF 1/Su（H）/Lag-1）和辅激活因子Mastermind形成复合物。尽管如此，CSL缺乏自己的核定位序列，目前仍不清楚三方复合体的形成位置。为了探索相关机制，我们设计了一种光遗传学方法来控制NICD释放（OptIC-Notch），并监测复合物形成和靶基因激活的后果。引人注目的是，我们观察到，当未切割时，OptIC-Notch将CSL隔离在细胞质中。假设暴露的膜ΦWΦP基序是隔离的关键，我们用OptIC-Notch{ω}中的第二光敏结构域掩蔽了该基序，这足以防止CSL隔离。此外，通过OptIC-Notch或OptIC-Notch{ω}陪伴CSL的光诱导切割产生的NICD进入细胞核并诱导靶基因表达，显示出有效的光控制激活。我们的研究结果表明，暴露的ΦWΦP基序导致CSL的招聘，并建议这可能发生在细胞质之前，核进入。Notch ΦWΦP基序的光控制暴露导致CSL螯合，并表明这种相互作用可能在正常信号传导期间的核进入之前发生。
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. Despite this, CSL lacks its own nuclear localisation sequence, and it remains unclear where the tripartite complex is formed. To probe mechanisms involved, we designed an optogenetic approach to control NICD release (OptIC-Notch) and monitored consequences on 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 in 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. Summary statement Light controlled exposure of the Notch ΦWΦP motif leads to CSL sequestration and suggest this interaction could occur prior to nuclear entry during normal signalling.