OptIC Notch reveals mechanism that regulates receptor interactions with CSL

Abstract

Active Notch signalling is brought about through receptor-ligand interactions that result in the release of the Notch intracellular domain (NICD) which translocates to the nucleus. NICD activates transcription at target sites in a tripartite 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 when and how the tripartite complex is formed. To probe the mechanisms involved, we designed an optogenetic approach to control NICD release (OptIC-Notch) and monitor consequences on complex formation and target gene activation. Strikingly we observed that, when uncleaved, OptIC-Notch sequesters 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 activity, showing efficient light controlled Notch activation. Our results demonstrate that exposure of the ΦWΦP motif leads to recruitment of CSL and suggests that this is an important regulatory step in Notch signalling that can occur in the cytoplasm prior to nuclear entry.

Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind

“…One possible consequence of the prolonged CSL enrichment in Notch OFF conditions is that target loci will retain "memory" of previous Notch activation that would make them more receptive to a subsequent exposure to Notch activity. To investigate, we took advantage of the temporal control provided by optogenetic release of NICD using OptIC-Notch{ω} 69 . We compared the response to Notch activation in cells that had been 'preactivated' with blue light for 4 hours and subsequently kept in the dark for 24 hours, and 'naïve' cells, which had been kept solely in the dark and hence had no prior Notch activity (Figure 5D-F).…”

“…For photomanipulation, transgenes UAS-Cry2-TevC and UAS-OptICNotch{ω} were recombined onto one chromosome and the conditions used were as described 69 and see below.…”

“…The OptIC-Notch{ω} system was used 69 in which blue light conditions induce association of two components, CRY2-TEVc and CIBN-TEVn-mCherry-NICD, which leads to the cleavage and release of NICD, mimicking Notch activation. Larvae were maintained under strict dark conditions and for imaging were dissected under an amber light to maintain OFF conditions.…”

“…Larvae were maintained under strict dark conditions and for imaging were dissected under an amber light to maintain OFF conditions. Activation was achieved by transferring larvae to a blue light incubator for the indicated times or by exposure to a blue laser (458 nm) during live-imaging, as described previously 69 .…”