PD for scramble, N1KO, LAR-KO, and Trio-KO cells cultured under static conditions expressing TMD-mApple or mApple infection control. and confirming in mouse models, we identify that activation of the Notch1 transmembrane receptor directly regulates vascular barrier function through a non-canonical, transcription independent signaling mechanism that drives adherens junction assembly. Shear stress triggers Dll4-dependent proteolytic activation of Notch1 to reveal the Notch1 transmembrane domain C the key domain that mediates barrier establishment. Expression of the Notch1 transmembrane domain is sufficient to rescue Notch1 knockout-induced defects in barrier function, and does so by catalyzing the formation of a novel receptor complex in the plasma membrane consisting of VE-cadherin, the transmembrane protein tyrosine phosphatase LAR, and the Rac1 GEF Trio. This complex activates Rac1 to drive adherens junction assembly and establish barrier function. Canonical Notch transcriptional FLT3-IN-1 signaling is highly conserved throughout metazoans and is required for many processes in vascular development, including arterial-venous differentiation3, angiogenesis4, and remodeling5; here, we establish the existence of a previously unappreciated non-canonical cortical signaling pathway for Notch1 that regulates vascular barrier function, and thus provide a FLT3-IN-1 mechanism by which a single receptor might link transcriptional programs with adhesive and cytoskeletal remodeling. (Fig. 1l) and (Extended Data Fig. 6a), likely too rapid for a transcription-dependent response. knockout of endothelial Notch1 resulted in loss of barrier function, increasing vascular permeability in the lung vasculature, quantified by EB extravasation17 (Fig. 1nCp, Extended Data Fig. 5b). Together, these results suggested an important role for shear stress in maintaining endothelial integrity, and that the Notch1 receptor potentially regulates this effect through a non-transcriptional mechanism. Upon Notch1 activation, the extracellular domain (ECD) of Notch1 is cleaved, which allows -Secretase-mediated cleavage of the ICD to leave behind the transmembrane domain (TMD) in the plasma membrane18. Given the observed increase in ICD cleavage with flow, we generated a library of CRISPR/Cas9-mediated Notch1 truncation mutants and recombinant rescue constructs (Fig. 2a, Extended Data Fig. 6e) to determine whether these subdomains of Notch1 contribute to regulating vascular permeability. Truncation of ICD (ICD-KO) resulted in constitutively low permeability and elaborated AJs (Fig. 2bCd) in static conditions, while truncation of both ICD and TMD (ICD-TMD-KO) increased permeability under flow (Fig. 2b). These data suggested that ICD was not critical for Notch1-induced barrier function, while TMD was necessary. Indeed, expression of TMD alone, as well as TMD-ICD, in Notch1-KO cells rescued barrier function and AJ assembly (Fig. 2eCg). Interestingly, Notch1-KO cells expressing TMD-ICD harboring a point mutation that prevents cleavage of the ICD (V1754G19, Extended Data Fig. 6b) failed to rescue barrier function (Fig. 2e), and TMD-ICD FLT3-IN-1 failed to rescue in the presence of DAPT, while cells expressing TMD alone maintained barrier function irrespective of DAPT exposure (Fig. 2h). Together, these findings are consistent with a model wherein TMD is the key component of Notch1 for regulating barrier function, and the barrier forming activity of TMD requires cleavage of ICD. Open in a separate window Figure 2 The Notch1 transmembrane domain mediates barrier function through interaction with VE-cadherina, A library of endogenous Notch1 truncation mutants and over-expression rescue constructs were generated to examine the key functional domains of Notch1 that regulate barrier function. b, PD for ECs with CRISPR/Cas9-mediated endogenous truncation of Notch1 ICD (ICD-KO) or truncation of the TMD and ICD (TMD-ICD-KO) cultured statically, under flow, or in the presence of rDll4-coated collagen. c, Fluorescent micrographs of VE-cadherin and actin for ICD-KO and TMD-ICD-KO ECs under flow conditions. d, Quantification of junctional area measured from VE-cadherin immunostained micrographs. e, PD for N1-KO ECs expressing TMD-ICD-mApple, TMD-ICD V1754G-mApple, TMD-mApple, or mApple infection control cultured statically, under flow, or in the presence of rDll4-coated collagen. f, Fluorescent micrographs of VE-cadherin (magenta), actin (green), and DAPI (blue) in static N1-KO cells expressing Rabbit Polyclonal to GLU2B TMD-mApple or mApple infection control. g, Quantification of junctional area measured from VE-cadherin immunostained micrographs. h, PD for static N1-KO cells expressing TMD-ICD-mApple or ICD-mApple exposed to DAPT or DMSO load control. i, Immunofluorescent images FLT3-IN-1 of Notch1-KO cells expressing either mApple or TMD-mApple, co-stained for VE-cadherin. Co-localization of Notch1 TMD and VE-cadherin (red arrow) is lost at free edges (blue arrow). j, Immunoprecipitation of VE-cadherin and N-Cadherin from Notch1-KO cells expressing either mApple or TMD-mApple. Immunoblotting.