Membrane compartmentalization by adherens junctions creates a spatial switch for Notch signaling and function


 Adherens junctions (AJs) create spatially and mechanically discrete microdomains at the interfaces of cells. Using a mechanogenetic platform that generates artificial AJs with controlled protein localization, clustering, and mechanical loading, we report that AJs also organize proteolytic hotspots for γ-secretase with a spatially-regulated substrate selectivity that is critical in the processing of Notch and other transmembrane proteins. Membrane microdomains outside of AJs exclusively organize Notch ligand-receptor engagement (LRE-µdomain) to initialize receptor activation. Conversely, membrane microdomains within AJs exclusively serve to coordinate regulated intramembrane proteolysis (RIP-µdomain). They do so by concentrating γ-secretase and primed receptors while excluding full-length Notch. AJs induce these functionally distinct microdomains by means of cholesterol-dependent γ-secretase recruitment and size-dependent protein segregation. By excluding full-length Notch from RIP-µdomains, AJs prevents inappropriate enzyme-substrate interactions and suppresses spurious Notch activation. Ligand-induced ectodomain shedding eliminates size-dependent segregation, releasing Notch to translocate into AJs for processing by γ-secretase. This mechanism directs radial differentiative expansion of ventricular zone-neural progenitor cells in vivo and more broadly regulates the proteolysis of large cell-surface receptors like amyloid precursor protein. These findings suggest an unprecedented role of AJs in creating size-selective spatial switches that choreograph γ-secretase processing of multiple transmembrane proteins regulating development, homeostasis, and disease.

5 cadherin types, cell types, and cell polarization states (Extended Data Fig. 1k-m), supporting the generality of AJ-mediated microdomain formation. Moreover, we observed Notch translocation into AJs after S2-cleavage, consistent with Notch relocalization from LRE-to RIP-µdomains (Extended Data Fig.  2a-e; Supplementary Note 1). These observations suggest two mechanisms by which AJs might define the compartmentalized microdomains: first, AJs recruit g-secretase that forms RIP-µdomain; second, AJs segregate Notch ligands and receptors from RIP-µdomains, limiting ligand-receptor engagement outside of AJs (i.e., LRE-µdomain).
AJs form RIP-µdomains by recruiting g g-secretase through cholesterol-rich lipid assemblies We then investigated how AJs form RIP-µdomains. Several reports have suggested possible engagement of AJs with spatially discrete lipid membrane phases 5,[40][41][42] . Similarly, g-secretase proteolytic activity is closely linked to detergent-resistant membranes [43][44][45][46][47][48][49][50] . Both of these membrane features preferentially associate with membrane proteins such as Flotillin-1 (Flot1) 5,48-51 . We therefore visualized localization of Flot1 across the cell membrane. Strong Flot1 and g-secretase fluorescence signal was seen at AJs (Fig. 2a,b and Extended Data Fig. 2f,g). These observations support the notion that both AJs and g-secretase are associated with common and long-lived lipid membrane phases enriched with Flot1, otherwise known to be short-lived and transient when alone 5 . Clustering of E-cadherin triggers rapid F-actin polymerization at the cytoplasmic leaflet of the plasma membrane. Given the established interaction between F-actin and membrane constituents like phosphatidylserine that nucleate and stabilize Flot1-containing lipid microdomains, we reasoned that AJ components may anchor phosphatidylserine leading to formation of the membrane microdomains (Raghupathy et al., 2015;Yap et al., 2015). To test this notion, we performed a coarse-grained molecular dynamic (MD) simulation of a lipid membrane comprising of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DIPC; outer leaflet), 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC; outer leaflet), N-palmitoyl-O-phosphocholineserine (PPCS; outer leaflet), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS; inner leaflet), and cholesterol (Chol; inner and outer leaflet, a key component of the discrete lipid membrane phases). We immobilized a portion (30%, red colored in Fig. 2c) of DPPS in the inner leaflet to reflect its interaction with AJ components (e.g., F-actin) and compared the results to another simulation set without DPPS immobilization. While both simulations showed lipid segregation within both the inner and outer leaflets, DPPS immobilization resulted in a microdomain having a strong transbilayer coupling (Fig. 2c) 52 . We also observed a significant decrease in lipid diffusion, indicating the microdomain stabilization (Fig. 2d,e).
To further confirm the role of discrete lipid membrane phases in recruiting g-secretase to AJs, we tested whether cholesterol-depletion disrupts g-secretase localization within AJs. Because cholesterol depletion also destabilizes native AJs 50 , we instead employed a mechanogenetic approach to maintain E-cadherin clustering while simultaneously depleting cholesterol 38, 53,54 (Fig. 2f,g). We treated cells with chloroalkane-functionalized magnetofluorescent nanoparticles (MFNs) to selectively label Halo-tagged E-cadherin in the presence of methyl-β-cyclodextrin (MβCD) that depletes cholesterol in the cell membrane. We then triggered E-cadherin clustering by applying a focused magnetic field to a certain subcellular location, generating artificial AJs (Fig. 2f,g). Similar to native AJs, vivid Flot1 and PS1 signals were seen at the artificial AJ without MβCD treatment ( Fig. 2h; Extended Data Fig. 2h,i), indicating that artificial AJs recapitulate the functional and signaling roles of native cell-cell AJs including g-secretase recruitment. In contrast, when cells were treated with MβCD, neither Flot1 nor PS1 signals were detected at the artificial AJ (Fig. 2h), suggesting that γ-secretase recruitment into AJs requires cholesterol-rich lipid assemblies. From these observations, we concluded that AJs form RIP-µdomains by recruiting and stabilizing γ-secretase through a common spatially discrete lipid assembly.

AJs exclude Notch receptors from RIP-µdomains due to their large size
To interrogate how AJ drives receptor exclusion, we generated artificial AJs using mechanogenetics, which allows quantitative control over the location and mechanical loading of targeted receptors while monitoring spatial distribution of Notch signaling components (Fig. 3a) 38,53,54 . Importantly, artificial AJs are free of membrane juxtaposition. Therefore, the influence of membrane juxtaposition on protein localization can be investigated by comparing receptor distributions in native AJs to artificial AJs. To image full-length Notch explicitly, we prevented proteolytic processing by treating cells with TAPI2. Surprisingly, we observed intense Notch localization at artificial AJ ( Fig. 3b and Extended Data Fig. 3ac), which contrasted starkly with the exclusion of Notch from native AJs (Fig. 1g-i). The extraordinarily large size of the Notch extracellular domain (NECD) 55 suggested a potential explanation for these contradicting observations. Specifically, the narrow intermembrane cleft created by native AJs (20 nm) 1, 3 . In contrast, artificial AJs generated by MFNs lack a narrow intermembrane cleft, thus permitting access of Notch to the concentrated g-secretase and other components of the AJs 56 . These observations fit a model wherein the size-based physical constraint induced by AJ formation at cell-cell interface segregates Notch from the RIP-µdomain, preventing interactions that would otherwise serve to colocalize and concentrate the enzyme-substrate pair 15 .
To test a size-dependent model for Notch exclusion from AJ-induced RIP µdomains, we generated a series of U2OS cell lines stably expressing Notch variants with different truncation lengths: a partial deletion of the EGF repeats (NΔEGF1-25, approximate height: 48 nm), complete deletion of the EGF repeats but retention of negative regulatory region (NΔEGF, approximate height: 10 nm), and a complete removal of NECD (NEXT, approximate height: 4 nm) ( Fig. 3c and Extended Data Fig. 3d). We fused these Notch variants with SNAP-and mCherry-tags at their N-and C-termini, to differentially image the extracellular and intracellular domains. All cells were treated with TAPI2 and DAPT to prevent any potential proteolysis of the variants. To quantify the spatial distribution of each Notch variant relative to the AJ, we measured the average mCherry fluorescence signal inside (IIN) and outside (IOUT) of the AJ and estimated an enrichment ratio (IIN/IOUT, See METHODS for the details) where a value of 1 indicates homogenous distribution (Extended Data Fig. 3e,f). Consistent with predictions based on sizedependent protein segregation, NΔEGF1-25, the Notch variant with an ECD taller than the height of the intermembrane AJ cleft, was excluded from AJs (IIN/IOUT = 0.57) (Fig. 3d,e). NEXT with an ECD smaller than the junctional height was enriched at AJs (IIN/IOUT = 2.39) (Fig. 3d,e). Interestingly, we observed a mixed binary localization pattern of NΔEGF (intermediate height) relative to AJs, with a mean IIN/IOUT value of 1.32 (Fig. 3d,e). Some AJs displayed NΔEGF enrichment (Fig. 3d(bottom left), e(right)), consistent with the size-based prediction. Meanwhile, other AJs excluded NΔEGF (Fig. 3d(bottom right), and e(right)). These results suggest a role for the size-dependent protein segregation as a spatial switch that regulates the distribution of Notch intermediates relative to RIP-µdomain, thereby choreographing the sequential steps in Notch proteolysis. Initially, the large size of NECD presents a physical constraint preventing entry of Notch to the narrow space between membranes in the AJ cleft (i.e., RIP-µdomain), limiting ligand-receptor interactions to the LRE-µdomain. Following S2 cleavage, removal of NECD relieves the physical constraint, allowing Notch to enter into the AJ cleft. This facilitates a productive Notch-g-secretase interaction, S3 cleavage, within the RIP-µdomain and then downstream signaling.

Notch localization into RIP-µdomain triggers receptor activation regardless of S2 cleavage
We next interrogated the functional consequences of the differential localization of Notch variants with different truncation lengths into AJs (i.e., RIP-µdomains). We cultured cells with TAPI2 to decouple gsecretase processing from S2 cleavage, and measured cleaved NICD levels by immunoblotting with Notch antibodies that detect N-terminal V1744. Cells expressing N FL or NΔEGF1-25 resulted in no or minimal NICD, respectively (Fig. 3f,g and Extended Data Fig. 3d), whereas cells expressing NΔEGF produced a significant amount of NICD (Fig. 3f,g). We validated these findings using time-lapse confocal microscopic tracing of the intracellular mCherry-tag signal within AJs after removing DAPT (Extended Data Fig. 3g-l, Supplementary Note 2). Importantly, we observed a dramatic decrease in the mCherry signal within AJs but not outside AJs, confirming that g-secretase activity was localized within the RIPµdomains (Extended Data Fig. 3i). Cells expressing NEXT exhibited the highest NICD production, about a four-fold increase compared with that of NΔEGF (Fig. 3f,g and Extended Data Fig. 3d). The observed NICD production was proportional to the enrichment ratio (IIN/IOUT) of the Notch variants at AJs, suggesting the essential role of size-dependent protein segregation as a spatial switch to direct Notch activation. The substantial NICD production from the cells expressing NΔEGF indicates that, when localized together, g-secretase can process Notch, bypassing S2 cleavage. Size-dependent but ligandindependent activation of Notch receptors with an intact S2 site was observed previously in Notch variants 7 and synNotch constructs [57][58][59][60][61] , but the mechanism of this activation has been unclear. Our observations support the notion that colocalization of these Notch variants with g-secretase is sufficient to trigger S3 proteolysis and signaling.

Spatial mutations alter Notch signaling
Prevailing models of Notch proteolysis by g-secretase are based on the notion that S2-cleavage of Notch serves to potentiate the cleavage by modifying the molecular interface at the enzyme-substrate pair 57,61,62 . For example, it has been suggested that g-secretase selectively recognizes S2-cleaved Notch (i.e., NEXT) through hydrogen bonding between a glutamate residue in nicastrin and the new N-terminus of NEXT 62 . Another model proposes that S2 cleavage serves to reduce steric repulsion between nicastrin and NECD, strengthening their interaction 61 . However, another key feature of S2 cleavage is that it generates a smaller molecular intermediate that can uniquely access AJs, thereby colocalizing Notch with g-secretase and significantly increasing its concentration near the enzyme active site. This suggests a third model, wherein g-secretase activity on full length Notch and its intermediates is blocked by maintaining concentrations of Notch below the KM for g-secretase due to their compartmentalization within LRE-and RIP-µdomains, respectively.
To explicitly test the consequences of membrane compartmentalization on g-secretase processing and signaling, we designed three experiments that induce spatial mutations of Notch. First, we employed a DNA-mediated crosslinking strategy to enhance NΔEGF -a Notch variant that exhibited a binary localization relative to AJs and relatively low Notch activation -enrichment at the RIP-µdomain (i.e., AJ) 63 . We generated cells co-expressing SNAP-NΔEGF-mCherry and Halo-Ecad-GFP and promoted colocalization of these two molecules by treating the cells with complementary benzylguanine (BG)-and chloroalkane-modified oligonucleotides in the presence of TAPI2 and DAPT (Fig. 4a). Notch-E-cadherin heterodimers were formed efficiently as evidenced by the appearance of a higher molecular weight band corresponding to the DNA-linked complex on western blots (Extended Data Fig. 4a,b). Compared to untreated cells (IIN/IOUT = 1.32 ± 1.06), we observed further enrichment of NΔEGF at AJs in the presence of DNA crosslinking (IIN/IOUT = 1.89 ± 0.91) (Fig. 4b). We then maintained cells in TAPI2 but removed DAPT to allow S3 cleavage. We observed a decrease in mCherry signal at AJs after DAPT removal, indicating efficient S3 cleavage without S2 cleavage (Extended Data Fig. 4c-e). Accordingly, in western blots, we observed increased V1744-terminated NICD levels from the cells treated with DNA crosslinkers, compared with the untreated control (Fig. 4c). These results suggest that the new molecular interfaces produced by mechanical activation leading to S2 cleavage are not necessary when g-secretase is concentrated with its substrate. Considering that DNA crosslinking (molecular weight = 21.4 kD) increases the ECD size of NΔEGF, the observed increase in NICD production cannot be explained by the nicastrin-induced steric repulsion model. Rather, this result favors a model wherein the increased concentration of the NΔEGF at RIP-µdomains facilitates its interaction with g-secretase and thus promotes S3 cleavage.
To further test the importance of size-dependent spatial segregation, we induced spatial mutation of NEXT (i.e., the Notch variant that showed the strongest enrichment at the AJ and activation) by chemically conjugating it with macromolecules of increasing hydrodynamic size: polyethylene glycol with an average molecular weight of 3.4 kD (PEG3.4k, 2.5 nm), branched PEG20k (bPEG20k, 4.0 nm), linear PEG20k (ℓPEG20k, 8.0 nm), DNA-streptavidin conjugates (DNA-stv, 9.5 nm), and human immunoglobulin G (hIgG, 12 nm) ( Fig. 4d; Extended Data Fig. 4f). Specifically, we conjugated BG-modified polymers and proteins to the extracellular SNAP tag (4.0 nm) of the variant via BG-SNAP chemistry (Fig.  4d). Grafting of these macromolecular pendants onto NEXT increases the size of the Notch construct but does not modify the N-terminal amine for hydrogen bonding with nicastrin. In the presence of DAPT, we observed a size-dependent distribution of NEXT at the AJ, where the larger pendants resulted in a greater decrease in mCherry signal at the AJ. With pendants smaller than 5 nm (i.e., PEG3.4k and bPEG20k), NEXT remained enriched at the AJ with IIN/IOUT of 2.21 and 2.01, respectively (Fig. 4d,e). When ℓPEG20k, DNA-stv, or hIgG were added, we observed a binary localization pattern of NEXT (i.e., enriched at or excluded from the AJs) with mean IIN/IOUT values of 1.06, 0.82, or 0.98, respectively (Fig. 4d,e). These observations were similar to the mixed spatial behavior of NΔEGF having a comparable ECD size, where 8 only a subset of AJs allowed Notch colocalization. We then examined the signaling consequences for each spatial mutation of NEXT. Following S3 cleavage, NICD traffics to the nucleus, allowing us to measure nuclear the mCherry signal as a proxy for Notch pathway activation. The PEG3.4k or bPEG20k addition did not significantly alter nuclear mCherry signal of NEXT, compared with cells with no pendant addition (Fig. 4f,g and Extended Data Fig. 4g). Conjugation of ℓPEG20k and DNA-stv resulted in a substantial decrease in nuclear mCherry signal to 0.39 and 0.37 fractional intensity, respectively (Fig.  4f,g and Extended Data Fig. 4g). hIgG addition suppressed nuclear mCherry signal further to 0.27 (Fig.  4f,g and Fig. 4g). We summarized the NICD production for all Notch variants as a function of the Notch enrichment factor, IIN/IOUT, in Fig. 4h, clearly visualizing the spatial dependence of S3 cleavage.
Finally, we investigated whether g-secretase can process full-length Notch when specifically directed to AJs by mechanogenetics. In this case, the biophysical constraints to Notch localization caused by membrane juxtaposition are eliminated and hence no exclusion of Notch receptors from RIP-µdomains is expected (Fig. 3a). We generated artificial AJs on the cells expressing SNAP-N FL -mCherry using mechanogenetics, in the presence of TAPI2 (to prevent S2 cleavage) but without DAPT (to allow gsecretase activity). Contrary to the experiment with DAPT (Fig. 3b), we observed no enrichment of mCherry signal at the artificial AJ, presumably due to NICD release (Fig. 4i,j and Extended Data Fig.  4h,i). To confirm that the loss of mCherry signal corresponded to bona fide signaling from Notch, we employed a UAS-Gal4 reporter system that detects Notch activation with the nuclear mCherry fluorescence 37,38,64,65 . To a cell recombinantly expressing SNAP-Notch-Gal4 (SNAP-N FL -Gal4) and Halo-Ecad-GFP, we again generated artificial AJs via mechanogenetics and measured the nuclear mCherry fluorescence every two hours. Note that no source of S2 cleavage (e.g., no ligand-immobilized substrate) was added. We observed strong nuclear mCherry signal from the cells with artificial AJs, but no signal from neighboring cells (Fig. 4k,l and Extended Data Fig. 4j). Together, these results suggest that the AJ-mediated membrane microcompartmentalization at cellular interface serves as a critical substrateselection mechanism for g-secretase.

The AJ-mediated RIP-µdomain is indispensable for Notch signaling
Given the significant role of AJs creating LRE-and RIP-µdomains, we next interrogated Notch signal activation in cells lacking AJs. In a first experimental approach we minimized physical contact between cells, and hence AJ formation, by sparsely plating UAS-Gal4 reporter cells expressing SNAP-N FL -Gal4 on a Dll4-coated substrate -effectively decoupling cell-cell contact from Notch-Dll4 interactions by allowing ligand presentation from the glass substrate rather than neighboring cells. After 16 hours from cell seeding, we measured the mCherry fluorescence in cells having no prior contact with other cells. For comparison, we also analyzed the mCherry signal in cells with robust AJs within high-density culture. While cells with physical contacts with adjacent cells exhibited a robust increase in nuclear mCherry fluorescence signal, those without cell-cell contact elicited no increase in signal (Fig. 5a-c and Supplementary Video S1). Reestablishing AJs by plating cells on a substrate coated with Ecad-Fc and Dll4-Fc rescued the Notch signaling of the solitary cells (Fig. 5a,d and Supplementary Video S2). We further confirmed AJ-dependent Notch activation in cells cultured with heterogeneous densities across a Dll4-coated substrate (Fig. 5b). These results support a model wherein AJs (or some other means of enriching g-secretase to form RIP-µdomain) are required for Notch processing at the cell surface and downstream signaling. Critically, E-cadherin seems to function in this capacity in a manner that is independent of its role in mediating cell-cell contact. In a second experimental approach, we knocked out the gene encoding E-and N-cadherin (CDH1/2) in the reporter cell line via CRISPR-Cas9 (Extended Data Fig. 5a-d), then plated the cells at high density on Dll4-Fc coated plates. Strikingly, E-cadherin knockout (Ecad-KO) resulted in abrogation of Notch activation even with robust cell-cell contact (Fig. 5e,f and Extended Data Fig. 5e-g). Reintroduction of plasmids encoding E-cadherin or N-cadherin into Ecad-KO cells recovered Notch activation with substantial nuclear mCherry signal (Fig. 5e,f and Extended Data Fig. 5e-g). Single cell analysis of the nuclear fluorescence signal exhibited a clear positive correlation with E-cadherin expression in the respective cells, confirming AJ-dependent Notch signaling (Fig. 5g).

AJ-mediated membrane compartmentalization regulates neuronal progenitor cell differentiation in vivo
Notch signaling is essential for the maintenance of stemness, self-renewal, and differentiation of neural progenitor cells (NPCs) 66,67 . In the mammalian cerebral cortex, Notch signaling orchestrates developmental neurogenesis, where it modulates a balance between tangential proliferative (i.e., symmetric division) and radial differentiative (i.e., asymmetric division) expansion of the apical ventricular-zone NPCs (VZ-NPCs) to establish a stratified neuronal organization 68 . Interestingly, only radial expansion of VZ-NPCs accompanies its differentiation, suggesting that Notch signaling in VZ-NPCs may be coupled with cells' spatial cues. Several reports also emphasize the critical role of apical-endfoot AJs in Notch signaling and the decision-making process of VZ-NPC development (i.e., proliferation vs. differentiation) [69][70][71] .
Given the essential role of the AJ-mediated microdomain formation for Notch signaling in cell line models, we reasoned that apical-endfoot AJs may also organize proteolytic hotspots for Notch activation. To test this hypothesis, we mapped the spatial distribution of Notch and g-secretase relative to Ncadherin-based AJs in VZ-NPCs of the developing mouse brain (E13.5) ( Fig. 6a-g). Consistent with observations in cell lines, Notch and PS1 exhibited exclusion (MOC = 0.14 ± 0.05, n = 9) from and enrichment (MOC = 0.69 ± 0.07, n = 9) within AJs, respectively, confirming compartmentalization between LRE-and RIP-µdomains ( Fig. 6c-g). We also captured the spatial distribution of the Notch activation intermediate by intracerebroventricular injection of DAPT into postnatal mice (P3). The immunostaining showed inclusion of Notch signal within AJs, presumably resulting from NEXT accumulation (Extended Data Fig. 6a) as observed in cell lines (Extended Data Fig. 2a,b). These results support the notion that AJs also drive compartmentalized microdomains and serve as a spatial switch regulating Notch signaling in vivo.
To understand the function of the AJs on VZ-NPC development, we disrupted AJs via dominantnegative cadherin expression preventing the RIP-µdomain formation. We retrovirally transfected a plasmid encoding a dominant-negative form of E-cadherin with the extracellular domain truncation (DNcad) 71 and a C-terminal GFP tag to VZ-NPCs of developing mice (P3) via intracerebroventricular injection (Fig. 6h). 48 hours after transfection, we analyzed NPC differentiation via TuJ1 immunostaining, a neuronal marker. While mice transfected with a control plasmid (n = 3) showed negligible TuJ1 signal, those with DN-cad plasmid transfection (n = 5) exhibited robust TuJ1 expression, presumably through downregulation of Notch signaling ( Fig. 6i-k and Extended Data Fig. 6b,c). These results support that AJ-mediated membrane microdomain compartmentalization modulates NPC maintenance and differentiation via Notch signaling.

Size-dependent spatial dynamics and proteolysis of amyloid precursor proteins
To test whether AJs serve as proteolytic hotspots with size dependent substrate selectivity for other large cell surface proteins, we investigated the processing of amyloid precursor protein (APP). APP plays a central role in amyloid beta (Aβ) pathology, which can cause failures in many organs such as brain, heart, kidney, and vasculature [72][73][74][75] . Interestingly, APP has a strikingly similar topology and proteolytic cleavage sequence to that of Notch. Like Notch, upon activation, APP is processed by two rounds of proteolysis: first a-or β-secretase and then g-secretase releasing its extracellular and intracellular domains, respectively 72,73,75 . We generated U2OS cells co-expressing APP-GFP and SNAP-N-cadherin (SNAP-Ncad) and monitored the cell surface spatial dynamics of APP intermediates relative to N-cadherin-based AJs (NAJs) in the presence of protease inhibitors. Having an intermediate ECD size (80 kD), full-length APP showed binary localization (i.e., excluded or enriched) relative to AJs in the presence of inhibitors (Fig. 7a,b and Extended Data Fig. 7a,b), similar to the Notch variant with EGF repeat truncation (i.e., NΔEGF). APP diffused into the NAJs after ECD shedding by a-or β-secretase, and then was processed by g-secretase within it (Fig. 7a,b and Extended Data Fig. 7a,b).
APP proteolysis by g-secretase produces more soluble p3 and Aβ40 predominately, along with less soluble and pathogenic Aβ42 and longer isoforms 72,73,75 . It has been previously shown that local acidic pH environment (e.g., pH 5.5) leads to a gain in the proportion of pathogenic Aβ species 76 . Additionally, N-cadherin expression in cells stabilizes an open conformation of PS1 that favors Aβ40 production over Aβ42 77 . Given our previous observation that loss of AJs leads to a decrease in cell surface g-secretase, we hypothesized that APP processing would be biased under these conditions towards Aβ42. We tested this hypothesis by constructing U2OS cell lines lacking both E-and N-cadherins (CDH1/2-KO cells) using CRISPR-Cas9 (Extended Data Fig. 5a-d). We then transfected plain U2OS cells or CDH1/2-KO cells with a plasmid encoding APP and measured APP fragment production by ELISA. While no significant changes were observed in total Aβ(40+42) and soluble APPa (sAPPa) (Fig. 7c,d), CDH1/2-KO cells produced higher relative levels of Aβ42, the isoform prone to severe fibril aggregation, compared to cells with endogenous cadherin expression (Fig. 7e).

Discussion
Unlike most other juxtacrine signaling systems, the Notch ligand-receptor interaction (a binding switch) is converted into intracellular signals only following multiple additional regulatory steps gated by mechanical, enzymatic, and spatial events. These include unfolding of the negative regulatory region (a mechanical switch), S2-and S3-cleavage (proteolytic switches), and finally translocation of the NICD from the cell membrane to the nucleus (a spatial switch) [24][25][26] . Our study reveals that Notch integrates an additional spatial switch via AJ-driven interfacial membrane compartmentalization to tightly choreograph the critical and irreversible enzymatic cleavage sequence prior to NICD release. Previously, it was thought that this enzymatic sequence was regulated by modification of the molecular interface between Notch and nicastrin after S2-cleavage 61,62 . Our model is not incompatible with a contribution of the nicastrin-Notch chemical interface on g-secretase activity. However, it strongly suggests that the AJdriven membrane compartmentalization is the major regulator of Notch-g-secretase interaction and signaling, functioning by increasing the concentration of the g-secretase substrate to the point that it exceeds the KM and is efficiently processed by the enzyme. Particularly, mechanogenetic experiments shown in Fig. 3a,b and Fig. 4i-l, respectively, supports the notion that Notch with an intact S2 site is effectively engaged and then processed so long as the spatial constraint of juxtaposed cell membranes at AJs is removed.
The operating principle of this new spatial switch is closely related to another unique feature of Notch receptor: its unusually tall extracellular domain. The functional residues responsible for ligand binding are located near the N-terminus, which protrudes above the crowded cell surface, where they are poised to engage ligands on neighboring cells. Surprisingly, however, it has also been shown that replacing the EGF-like domain repeats with a smaller ligand binding domain (e.g., synNotch) maintains the receptor function 78,79 . Why then does Notch receptor bear such a massive ECD? Our study provides insight into this question, where the large ECD is crucial for its spatial segregation from g-secretase thereby minimizing nonspecific ligand-independent activation. Low level NICD production was observed even for Notch variants with partial EGF truncation (NΔEGF1-25) and levels gradually increased upon successive truncations. NΔEGF1-25 has comparable size to smaller Notch family homologs, including C. elegans LIN-12/Notch and GLP-1/Notch (13 and 10 EGF repeats, respectively), suggesting the relevance of a spatial switch across the Notch family and metazoans. We also observed mixed distribution of NΔEGF variant that is smaller than AJ cleft heights. We interpreted that this unanticipated exclusion might result from a lateral crowding effect in high-density AJs, because density of cadherin clusters within AJs varies with the size, type, and degree of junction maturation 80,81 and the glycosylated negative regulatory region domain 82 can be susceptible to steric crowding. Our model also explains previous observations where synNotch with a relatively small ECD exhibited significant ligand-independent activation (10-50% of ligand-induced activation) 59 .
We also show that size-dependent spatial segregation regulates APP cleavage and Aβ production. It has been previously shown that g-secretase presenting in different subcellular compartments cleaves APP into diverse Aβ isoforms 73,75,83 . Our study shows that, after the ECD cleavage, AJ potentiates cell surface processing of APPs within the junction, yielding Aβ40 predominantly, while removal of AJ produces more Aβ42. To establish the relevance of this observation to APP processing will require further investigation in a neuronal system, but our results in model cell lines are consistent with the predominant secretion of Aβ from the synapse, where N-cadherin junctions localize 84 . More importantly, these finding suggest that AJ may represent proteolytic hotspots with size-dependent substrate selectivity across a more diverse range of cell-surface proteins.
Our study also suggests a critical role of the AJ-mediated membrane compartmentalization in VZ-NPC maintenance and differentiation during development. It has been previously proposed that apicalendfoot AJs promote Notch signaling in NPCs 69-71 , but the mechanism underlying precise Notch signal regulation was unclear. Our findings suggests that Notch signaling is maintained by creating RIPµdomains within AJs, and disruption of the AJs downregulates Notch signaling and hence promotes NPC differentiation. This result also provides important insights on the molecular mechanism of how environmental spatial and physical changes of cells (i.e., VZ-NPC detachment and radial migration) direct cell signaling (i.e., Notch signaling) and differentiation, finally enabling spatiotemporally coordinated tissue development.
The size-dependent segregation of Notch from the RIP-µdomain has important analogies to the kinetic segregation model of T cell activation, where the large CD45 phosphatase is excluded from T cell receptor (TCR) immunological synaptic clefts 18,19,85 . However, there are several distinct features of the Notch spatial switch compared to the kinetic segregation model. First, unlike the immunological synapse where TCR and CD45 remain constant in size throughout activation, Notch undergoes a dramatic size change during the course of cell surface activation, enabling its dynamic spatial redistribution and sequential proteolysis. Second, the role of AJs in Notch signaling is not limited to creating a physical barrier, but also plays the critical role of recruiting and concentrating g-secretase to facilitate processing of S2-cleaved Notch at the cell surface. Third and finally, the consequences of size-dependent segregation on signaling are reversed in comparison to the immunological synapse. While spatial segregation of CD45 enables sustained TCR phosphorylation and downstream signaling, Notch segregation from AJs inhibits signal activation. Our result extends the relevance of size-dependent spatial segregation models beyond immune cells 18,19,85,86 , supporting the notion that size-dependent protein segregation can serve as a general mechanism for regulating a broad range of receptor signaling at the cell-cell interface, including Notch and APPs. It is also important to note that our model may not be limited to the AJs, but may be extended to other cell-cell junctions that provides an environment for sizedependent protein segregation while effectively concentrating proteases.
Overall, AJ-mediated interfacial membrane compartmentalization not only sheds light on the mechanism underlying the sequential proteolysis of Notch and APPs, but also may extend to other receptors processed by g-secretase. Finally, we anticipate further implications of our work in other areas of research such as providing new design principles for synthetic receptors like synNotch, as well as new therapeutic approaches that target Notch and APP signaling by spatial mutation in cancer and neurodegenerative diseases.
S Artavanis-Tsakonas, M. R. R. L. Notch signaling: cell fate control and signal integration in development. Science 284, 770-776 (1999 . (c, f, and i) Overlays are box and whisker plots; ****P < 0.0001, ns: non-significant; oneway ordinary ANOVA followed by Tukey's multiple comparison testing.  Fig. 2c. (f) A schematic showing mechanogenetic interrogation of g-secretase and cholesterol-rich ordered lipid microassemblies relative to the artificial AJs. Artificial AJs were formed by clustering Ecad-GFP labeled with magnetofluorescent nanoparticles (MFNs) by application of an external micromagnetic tweezer (µMT). MβCD was used for cholesterol depletion in the cell membrane before artificial AJ formation. (g) Epifluorescence images showing the formation of an artificial AJ by mechanogenetics. After stimulation by µMT, vivid MFN and E-cadherin signals at the magnetic focus were seen, indicating formation of AJs. Scale bar, 5 µm. (h) Confocal fluorescence images of E-cadherin, presenilin1, and Flotillin-1 (Flot1) at the artificial AJ with and without MβCD treatment. After cholesterol depletion, no PS1 recruitment was seen at the AJ, suggesting that g-secretase recruitment to the AJ requires lipid microdomain formation at the AJ. Scale bar, 2 µm.  . (f,g) Western blot analyses of cleaved NICD levels in the cells stably expressing N FL , NΔEGF1-25, NΔEGF, and NEXT. All cells were transfected with Ecad-GFP and incubated with TAPI2 for 24 hr. β-actin levels represent the loading control. A representative image of immunoblotting (f), and quantification (g) of cleaved NICD levels. The average intensity of each NICD band relative to respective β-actin band was quantified and then normalized to that of NEXT (mean ± s.d.; n = 4 biological replicates). (e-g) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: non-significant; one-way ANOVA followed by Tukey's multiple comparison test. The average intensity of NICD band was normalized to that of β-actin band in each sample. (mean ± s.d.; **P < 0.01; n = 5 biological replicates; ordinary one-way ANOVA). (d) Spatial mutation of NEXT via chemical ligation of macromolecular pendants (denoted as P). BG-modified polymers or proteins were conjugated to the extracellular SNAP tag of NEXT. Cartoons depicting shape and hydrodynamic size of different pendants are shown. Confocal images showing size-dependent spatial mutation of NEXT (red) at the AJs (green). The top row shows maximum projection images of the cells treated with the indicated pendants. Scale bar, 5 µm. The middle row shows confocal z resliced images along the white dashed lines in the maximum projection images. Yellow and green arrowheads indicate the AJs enriches with and those that excludes Notch, respectively. Scale bar, 3 µm. The bottom row shows line profiles quantifying fluorescence signals from NEXT (red) and E-cadherin (green) along the white lines in the zresliced images. Images and line profiles are representative of n ≥ 15 biological replicates. (e) Quantification in IIN/IOUT of NEXT with macromolecular pendants (n ≥ 15). (F and G) Confocal fluorescence images (f) and quantification (n ≥ 180) (g) of nuclear mCherry signal for the NEXTexpressing cells treated with macromolecular pendants. DAPI signal (blue) indicate cell nucleus. Scale bar, 5 µm. Cells expressing N FL were used as a negative control. (h) A plot representing the NICD level of various Notch variants as a function of the enrichment factor (IIN/IOUT, mean ± s.e.m.; n ≥ 15 biological replicates). All Notch variants with different truncation length, DNA crosslinking, or pendant addition used in Fig. 2 and 3 were included. median ± s.e.m.; n ≥ 4 for western blot for NICD levels; n ≥ 180 for nuclear mCherry fluorescence.

Plasmid construction
Plasmid constructs used in this study are listed in Supplementary Table 1. All constructs used in this paper were assembled using standard restriction enzyme-based cloning, in-fusion cloning, and/or Gibson isothermal assembly. The maps, sequences, and construction details of all plasmids are available upon request. All constructs were sequenced to confirm mutation. Complete details of all cloning procedures are available upon request.
Flag-human Notch1 (N FL )-Gal4 and pGF1-UAS-H2B-mCherry were gifts from S. Blacklow (Harvard University). Flag-human N FL -Gal4 was provided in a Tet-ON Flp-IN vector (pcDNA5). SNAP-N FL -mCherry and SNAP-N FL -Gal4 were constructed as previously reported 38 . All Notch1 variants with partial or full extracellular domain truncation were constructed by linearizing and amplifying SNAP-hN1-mCherry vector via inverse PCR while omitting the sequence corresponding the ECD truncation. Notch ectodomain sequences of amino acid 23-981, 23-1426, 23-1709 were deleted for SNAP-ΔEGF1-25-mCherry. SNAP-ΔEGF-mCherry. and SNAP-NEXT-mCherry, respectively. Note that similar Notch variants with partial ECD truncation were reported previously 78 where the structural integrity and function of Notch negative regulatory region (NRR) domain were preserved. Ecad-GFP was purchased from Addgene (Addgene plasmid # 28009; http://n2t.net/addgene:28009). SNAP-Ecad-GFP and Halo-Ecad-GFP were constructed first by linearizing and amplifying Ecad-EGFP vector via inverse PCR. SNAP-and Halo-tags were then inserted in frame with E-cadherin, downstream of the E-cadherin pro-peptide (amino acid #155) and upstream of the extracellular domain sequence, using Gibson assembly (NEB). pCMV6-Flotillin-1-Halo (Flot1-Halo) was constructed by replacing the myc-tag within the pCMV6-Flotillin1-myc (purchased from Origene (MR206823)) with the Halo-Tag sequence in frame with Flot1 using In-Fusion cloning (Clontech). Human amyloid precursor protein1-EGFP (APP-EGFP) was created by cloning human APP695 (a gift from C. Miller of King's College London) into a pEGFP-N1 plasmid (Clontech). To facilitate membrane distribution mapping, we used APP constructs used in confocal imaging lack YENPTY motifs (Fig. 7a,b), and compared the result with full-length APP (Fig. 7a,b). APP ΔYENPTY -EGFP was constructed by first linearizing the vector via inverse PCR, and deleting the sequence of final 15 amino acids upstream of C-terminus of APP (amino acid 681-695), which includes YENPTY motif (amino acid 682-687) using Gibson assembly.

Transfection and cell line generation
All cell lines expressing recombinant proteins used in this study are listed in Supplementary Table 1. U2OS stable cell lines were constructed from parental U2OS T-rex cell lines (Flp-IN, Tet-ON engineered cell line, gift from S. Blacklow). Constructs were inserted into the engineered Flp-IN site by co-transfection with a plasmid containing the Flp-recombinase (pOG44) via electroporation with the Neon Transfection System (ThermoFisher) according to manufacturer's protocol (Shock Conditions: 1230V, 10ms, 4 pulses, number of cells 5 x 10 6 ). The amount of total DNA used was 10 µg/well: 1 µg of DNA containing the desired construct and 9 µg pOG44. Cells transfected with desired plasmids were incubated in a selection medium containing 400 µg/mL hygromycin (Invitrogen) for at least 10 days. All cells with Notch truncation and reporter were further sorted for inducible expression of Notch variants via fluorescence-activated cell sorting (FACS) on a FacsAria2 (BD) by staining for the appropriate tag (SNAP or Halo) with fluorescently tagged antibody. For single-cell monoclonal population establishment, fluorescently-positive bulk-sorted populations were plated into 96 well plates at 0.2 cells/well by serial dilution and grown in selection medium. Each clonal cell population was tested and selected based on the levels of Notch reporter activity or Notch membrane expression. U2OS cells expressing recombinant proteins transiently were generated by transfecting plasmids encoding desired proteins using Neon-based electroporation. Cells were allowed to settle in a 6-well cell culture dish post electroporation for 6-8 hr. To remove dead cells, cells were lifted and re-plated on a fibronectin-coated glass bottom dish with 1 x 10 5 cells per well density. MDCK cells were plated at 70% density then transfected with N FL -mCherry utilizing Lipofectamine 3000 (ThermoFisher) or Neon electroporation according to the manufacturer's protocol (Shock Conditions: 1,650V, 20ms, 1 pulse, number of cells 5 x 10 6 ). HUVEC cells were transfected via electroporation with SNAP-N FL via the BioRad Gene Pulser system (250 V, 20 ms square wave, 1x10 6 cells/mL Gene Pulser Electroporation Buffer, 5 µg/mL SNAP-N FL -mC). All cells transiently expressing recombinant proteins were incubated for 24-48 hr from the transfection, and then used for further analyses.
Micro-magnetic tweezers (μMT) set up. The μMT was set up and aligned on the inverted microscope with point-scanning confocal imaging capabilities (Nikon) as previously described 38,54,87 . The needle probe -NdFeB magnet assembly was attached to the z-translation stage (Sutter Instrument, MP-325) and its location was carefully aligned with the microscopic objective lens while observing the dummy substrate filled with DPBS. The µMT tip was positioned at the center of the objective oculus with bright-field illumination using the X-Y translation stage linked to PIMikroMove (Physik Instrumente) and µManager (UCSF). Using the z translation stage, the µMT was carefully lowered to set the height of the tip to 10 µm above the focal plane while recording the X-Y coordinates and the z-position of the needle probe.

Preparation of cells expressing recombinant Flotilin-1 for mechanogenetics experiments.
U2OS cells were co-transfected with SNAP-Ecad-GFP (5 µg) and Flot1-Halo (5 µg) plasmids using Neon electroporation. 24 hr later, cells were re-plated on a #1.5 glass-bottomed dish (MatTek, d = 10 mm) coated with collagen at a density of 1 x 10 5 cells per dish. To fluorescently label Flot1-Halo, cells were treated in a complete McCoy's 5A medium containing 3.5 μM cell membrane permeable Halo-ligand 660 dye (Promega) for 30 minutes at 37°C. Cells were washed three times with DPBS, incubated with a phenol red-free complete medium, and then mechanogenetically stimulated (see below). For the cholesterol depletion experiment, we also treated cells 10 mM of methyl-β-cyclodextrine (MβCD) (Sigma-Aldrich) in serum-free McCoy's 5A medium for 30 min at 37°C and washed with complete medium three times. To label SNAP-Ecad-GFP with MFNs, cells were first treated with 5 µM of an oligonucleotide bearing benzylguanine (BG-T60ACTG10) for 45 minutes at 37°C, washed two times with 10 ml of serum free medium, and then incubated with serum-free medium containing 10 nM monovalent MFNs bearing complementary sequence (T60CAGT10) and 0.5% alkali casein for 10 min at 37°C, 5% CO2. Cells were washed with 10 ml of complete medium two times, and then incubated with phenol red-free medium for mechanogenetic experiments on a confocal or wide-field epifluorescence microscope.
Preparation of cells expressing human Notch1 receptor for the mechanogenetic experiment. Inducible U2OS cells stably integrated with SNAP-N FL -mCherry were transfected with Halo-Ecad-GFP (10 µg) using Neon electroporation. 24 h later, cells were re-plated on a collagen (or fibronectin)-coated glassbottomed dish. To induce surface expression of SNAP-N FL -mCherry. cells were incubated with complete medium containing doxycycline (Sigma, 2 µg/mL) for 18 h. To inhibit γ-secretase activity, cells were treated with DAPT (5 µM) and further incubated for 6 h. Cells were treated with 5 µM of an oligonucleotide bearing chloroalkane (Cl-T60ACTG10) for 45 minutes at 37°C and labeled with MFNs via the procedure described above.

Mechanogenetic regulation of artificial E-cadherin junctions.
To induce MFN and hence cadherin clustering, the μMT was carefully directed towards a targeted subcellular location until the tip-tomembrane distance (d) reached 10 µm. As the tip approached the target membrane, the formation of an artificial E-cadherin junctions (AJs) was monitored every 5 minutes. After 30 min of mechanogenetic stimulation, the spatial distribution of MFNs and artificial AJs was monitored using time-lapse confocal fluorescence imaging. To investigate g-secretase processing of full-length Notch, the spatial distribution of membrane mCherry (S-N FL -mC) or nuclear mCherry signals (S-N FL -Gal4) were monitored using timelapse live cell confocal imaging. To observe localization of membrane microdomains, the spatial distribution of Flot1 fluorescence signal was monitored using live cell confocal imaging. Time-lapse live cell confocal imaging was performed using a 60x Plan-Apo oil objective (NA 1.4) on a Nikon A1 laser scanning confocal microscope equipped with an environmental chamber maintaining cells at 37°C, 5% CO2. Cells were immediately fixed with 4% paraformaldehyde (Life Technologies) in DPBS for 15 minutes and washed with DPBS 3 times for 5 minutes before immunostaining. , and a temperature-and CO 2controlled stage top incubator (Okolab, Bold Line). Unless otherwise noted, confocal microscopy was performed using Plan-Apo 60x, 1.4 NA or Plan-Apo 100x, 1.4 NA oil objectives (Nikon) on a Nikon A1R laser scanning confocal microscope. Images were acquired using Galvano scanning mode and confocal zoom of 3-4x magnification.

Monitoring the dynamic spatial localization of Notch intermediates
To activate Notch, we plated cells expressing SNAP-N FL -mCherry on a substrate coated with Dll4 fused with a Fc fragment (Dll4-Fc). Briefly, a glass bottom dish (Lab-Tek II Chambered Coverglass, ThermoFisher, or 7-mm glass-bottomed dish, MatTek) was coated with fibronectin (Hamster, 5 µg/ml) and Dll4-Fc (2.5 µg/ml) for 1 hr at 37°C, and washed thoroughly with 10 ml of PBS. A negative control dish was also prepared by coating it with fibronectin only. U2OS cells co-expressing SNAP-N FL -mCherry and Ecad-GFP were plated and incubated with doxycycline (2 μg/mL), TAPI2 (100 μM) 88 , and/or DAPT (5 μM). Different combinations of inhibitors were used to capture the respective intermediates (See Fig.  1a-d and Extended Data Fig. 2a-c). After 48 hr, cells were labeled with SNAP-647 (NEB, 5 µM) and then fixed as detailed above. Inhibitor concentrations were maintained during wash and fixation steps. For DAPT washout experiments cells were plated and activated via Dll4-Fc ligand as described above. DAPT inhibitor was removed by washing in media at each time point (0, 0.5, 1.5, 3, 6, and 12 hrs). At each time point, cells were washed three times with large volumes of PBS and fixed 4% with PFA. The spatial distribution of Notch intermediates and AJs was monitored using spinning disk confocal fluorescence microscopy (Zeiss Cell Observer Z1), equipped with Yokagawa spinning disk and Evolve 512 EMCCD Camera (Photometrics). Images were obtained with Plan-Apo 63x, 1.4 NA or Plan-Apo 100x 1.46 NA oil objectives (Zeiss) with solid-state lasers of 405, 488, 561 nm, and 647 nm. The microscope was controlled with Zeiss Zen software (Zeiss).
Plasma membrane staining using DiI dyes U2OS cells expressing Ecad-GFP were plated on a fibronectin-coated glass bottom dish (MatTek, D = 7.0 mm) at a density of 1 x 10 4 cells per dish. After 48 hr, the dish was filled with a complete McCoy's 5A medium containing 5 µM of DiI plasma membrane labeling dyes (Invitrogen) and incubated for 10 min at 37°C, 5% CO2. Cells were then washed 3 times with complete medium. The spatial distribution of AJs and DiI membrane staining and Ecad-GFP was monitored using spinning disk confocal microscopy (See Extended Data Fig. 3f).

Single-cell cleavage kinetics of SNAP-NΔEGF-mC
A 6-channel µ-slide flow chamber (Ibidi, VI 0.4) was coated with fibronectin (2.5 µg/mL) for 1 hr at 37°C and washed with PBS four times. U2OS cells co-expressing SNAP-N∆EGF-mCherry and Ecad-GFP were plated on the µ-slide flow chamber by applying 60 µL of single cell suspension at a density of 3 x 10 5 cells/mL. After 3 hr, the channel was filled with a complete McCoy's 5A medium containing doxycycline (2 µg/mL), TAPI2 (100 µM), and DAPT (5 µM). Cells were grown for 48 hr in normal growth medium to reach 70-80% confluency and form cadherin adherens junctions. Cells were labeled with BG-Alexa Fluor 647 (NEB) for 30 min to stain cell surface N∆EGF. Multiple cells with stable AJs were identified using large-area epi-fluorescence scanning (500 µm x 500 µm), and the spatial distribution of SNAP-NΔEGF-mCherry at AJs under TAPI2 and DAPT inhibition was imaged by confocal z-stack (step size = 0.2 µm, total range of z stacks = 10 µm) scanning from basal to apical membranes. Then, DAPT containing media was removed and replaced by flowing complete medium containing doxycycline and TAPI2 at a flow rate of 50 µl/min for 10 minutes using a syringe pump. Localization of extracellular (NECD) and intracellular (NICD) domain at the AJs before and during DAPT washout was monitored every 30 minutes in multiple color channels (NICD, mCherry; NECD, AF647; AJ, GFP) by time-lapse confocal zstack microscopy for 12 hr. Time-lapse live cell confocal imaging was performed using a 60x Plan-Apo oil objective (NA 1.4) on a Nikon A1 laser scanning confocal microscope equipped with an environmental chamber maintaining cells at 37°C, 5% CO2.
Western Blot analysis U2OS cells co-expressing Notch variants and Ecad-GFP (or Halo-Ecad-GFP) were incubated with culture media containing doxycycline (2 µg/mL) and TAPI2 (100 µM) in a 6-well plate at a density of 1x10 6 cells per well. After 24 hr, cells were washed with ice-cold DPBS twice and lysed in RIPA (Invitrogen) or 1% NP-40 (Invitrogen) supplemented with complete protease and phosphatase inhibitor cocktail (100x; Cell Signaling Technology) at 4°C while gently shaking for 30 minutes. Insoluble fractions were removed by centrifugation of the cell lysates at 13,000 r.p.m. for 10 minutes. Total protein concentrations in lysates were determined by a BCA assay (Bio-Rad). 20 µg of whole cell lysates were then mixed with 4x Laemmli sample buffer (Bio-Rad) with 10% β-mercaptoethanol (BME) and heated to 95°C for 5 minutes. For western blot analysis of DNA-crosslinked heterodimers, the cell lysates were mixed with 4x Laemmli sample buffer (Bio-Rad) without BME before boiling to denature. Samples were then loaded into a 4-15% Mini-Protein TGX precast gel (Bio-Rad) and were run at 70 V for 30 minutes and then 120 V for 45 minutes. Separated proteins were transferred to a PVDF membrane using Mini Trans-Blot Cell (100 V constant, 1 hr) or the Trans Turbo Blot system (Bio-Rad). Membranes were blocked for 1hr at room temperature in blocking solution (5% w/v nonfat dry milk in 1x TBST). The membranes were probed with anti-V1744 NICD antibody (1:1000; Cell Signaling Technology #4147), anti-SNAP (1:1000; NEB), anti-Notch1 (1:1000, Cell Signaling Technology #3447 or #4380), anti-mCherry (1:500, Abcam #167453), anti-E-cadherin (1:100, Santa Cruz Biotechnology, sc-8426), and anti-β-actin (1:5000; Cell Signaling Technology #4970) antibodies overnight at 4°C with gentle rocking. The membranes were washed in TBST three times for 5 minutes and incubated with an anti-rabbit (Cell Signaling Technology, # at 1:2000 for NICD, mCherry, SNAP detection and at 1:10000 for β-actin detection) or anti-mouse (Cell Signaling Technology, # at 1:2000 for E-cadherin detection) HRP conjugated antibody. The target proteins were visualized by chemiluminescence using an ECL detection kit and a ChemiDoc MP imaging system (Bio-Rad). Quantification of band intensities by densitometry was carried out using the Image Lab software (Bio-Rad).
Spatial mutation of SNAP-NΔEGF-mCherry via DNA crosslinking DNA-mediated crosslinking of SNAP-NΔEGF-mCherry with Halo-Ecad-GFP. DNA crosslinkers including benzylguanine (BG)-and chloroalkane (Cl) modified oligonucleotides were synthesized as previously described (Kwak et al., 2019;Liang et al., 2018). To prepare 10x crosslinking DNA stock solution, complementary BG-and Cl-modified oligonucleotides were hybridized in situ. BG-T10(ACTG)5 and Cl-T10(CAGT)5 were mixed at equimolar concentration (20 µM) in PBS, incubated at 95°C on a dry heat block for 5 min, and slowly cooled down to room temperature for 2 hr. U2OS cells co-expressing SNAP-NΔEGF-mCherry and Halo-Ecad-GFP were cultured in a 6-well plate for western blot analysis at a density of 1 x 10 6 cells per mL or in a channel of an Ibidi µ-slide for confocal imaging analysis at a density of 3 x 10 5 cells per mL. Cells were grown to 70-80% confluency for typically 24 hr, followed by overnight incubation with complete medium containing doxycycline (2 µg/mL), TAPI2 (100 µM) and DAPT (5 µM). Cells were then serum starved with 2 ml of serum-free medium with doxycycline, DAPT, and TAPI2 for 6 hrs. Before adding DNA crosslinkers, cells were washed and placed in 450 µl of serum-free media. 50 µl of prewarmed 10x DNA crosslinker stock solution was added to each well and incubated at 37°C. Western blot analysis to validate receptor crosslinking were performed after 30-minute incubation of the DNA crosslinkers as detailed above.
Live cell confocal time-lapse imaging. After overnight incubation with the DNA crosslinkers, imaging was performed on an inverted laser scanning confocal microscope (Nikon A1) equipped with an environmental chamber at 37°C and 5% CO2. Images were obtained with a Plan-Apochromat 60x, 1.4 NA oil objective (Nikon) with solid-state lasers of 405, 488, 561 nm, and 647 nm. Additionally, the microscope was equipped with Ti-E Perfect Focus System (Nikon). To examine the effect of DNA-mediated crosslinking on spatial distribution of SNAP-NΔEGF-mCherry at AJs, multiple AJs were imaged in entirety from basal to apical sides for Halo-Ecad-GFP and SNAP-NΔEGF-mCherry using a 488 nm and 561 nm laser respectively, for a 12 µm range at a z-step size of 0.25 µm. To monitor dissipation of SNAP-NΔEGF-mCherry at AJs upon removal of DAPT inhibition, fresh phenol red-free McCoy's 5A medium containing doxycycline and TAPI2 was introduced into the channel using a syringe pump for 10 min, and confocal z-stack images of the previously selected AJs were acquired every 30 minutes for 6 hr. Images were acquired using NIS-element software (Nikon), and image post-processing and analyses were done using Fiji/ImageJ and custom-built scripts.
Synthesis of BG-modified DNA-streptavidin conjugates. DNA oligonucleotides bearing biotin-and BGfunctional groups were synthesized by reacting biotin-(ACTG)5-NH2 (IDT DNA) with BG-GLH-NHS as described above 63 . Equimolar amounts of streptavidin (10 nmol) and BG-DNA-biotin (10 nmol) were dissolved in PBS (0.5 ml) for 2 hr, forming streptavidin-BG complex. The solution was concentrated to approximately 50 µl using an Amicon centrifugal filter (MWCO: 30k) and then diluted again with 0.45 ml of PBS. This concentration and reconstitution step was repeated three times to remove unconjugated DNA.
Synthesis of BG-modified human IgG. hIgG (10 mg) and BG-GLA-NHS (0.82 mg) were dissolved in 850 µl of PBS and 150 µl of anhydrous DMSO, respectively. Two solutions were mixed and reacted for 2 hr at room temperature with gentle shaking. The solution was desalted with NAP-10 and then with NAP25 pre-equilibrated with PBS. The proteins were further concentrated until the final volume is 300-500 µl using Amicon centrifugal filter (MWCO: 30k). The IgG concentration was determined by measuring the absorbance at 280 nm.
Spatial mutation of SNAP-NEXT-mCherry using the BG-modified macromolecules. U2OS cells coexpressing SNAP-NEXT-mCherry and Ecad-GFP were incubated in complete McCoy's 5A medium containing doxycycline (2 µg/ml), TAPI2 (100 µM), DAPT (5 µM), and respective BG-modified macromolecules (10 µM). After 24 h, cells were fixed and imaged by confocal microscopy to determine the enrichment factor of SNAP-NEXT-mCherry at AJs. Images were taken with a 100x objective and 3x confocal zoom. 20 stage positions per each treatment were manually selected and their coordinates were stored in the computer. In each position, confocal z stacks of DAPI, Ecad-GFP and Notch-mCherry were acquired for a 12 µm range at a Z step-size of 0.25 µm to monitor the AJs in their entirety from basal to apical sides. To assess the levels of Notch activation, a set of identical experiment but without DAPT was performed. After 24 h, cells were fixed, stained with DAPI, and imaged by confocal microscopy to determine nuclear mCherry signal. Images were taken with a 60x objective and 1x confocal zoom. 5 stage positions per each condition were selected manually. For each position, a confocal large-area scan of DAPI, Ecad-GFP, and SNAP-NEXT-mCherry was acquired for a 1 mm x 1 mm area.

Plate-bound Dll4 Notch activation in high-density grouped versus solitary cells
To activate Notch, we plated SNAP-N FL -Gal4 reporter cells on a substrate coated with Dll4-Fc as detailed above. Two different cell seeding densities were used: We plated cells with a density of 1 x 10 3 cells per 10 mm glass-bottomed dish (MatTek, No. 1.5 glass), predominantly yielding solitary cells. We also plated cells with a density of 1 x 10 4 cells per dish, predominantly yielding high-density grouped cells.