Extended Data Fig. 6: Spatial mutations of Notch to study the effect of membrane compartmentalization on the signaling.

A representative western blot of lysate from cells expressing NΔEGF and Halo-Ecad-GFP after 2 hr incubation with or without DNA crosslinkers. The blot was labelled with anti-SNAP (a) and anti-Ecadherin (b) antibodies. The expected mass of NΔEGF, E-cadherin monomer, and the complex with the Notch construct and E-cadherin forming a heterodimer are 90 kD, 158 kD, and 230 kD, respectively. β-actin detection was used to assess protein loading. In both blots, predicted bands representing Notch-E-cadherin heterodimers (solid black lines) and SNAP-NΔEGF-mCherry or Halo-Ecad-GFP monomers (dashed black lines) are indicated. (c) A representative maximum intensity projection of XY images (i) and a XZ-resliced image (ii) showing exclusion of full-length Notch1 (SNAP-N^FL-mCherry) from the AJs (green) after the DNA crosslinking. (d) Representative confocal maximum intensity projection images showing the distribution of NΔEGF relative to the AJs after crosslinking. Cells were treated with or without DAPT. Scale bar, 10 µm. (e) Single-cell confocal z-resliced images showing intracellular mCherry signal at the AJ under DNA and DAPT treatment (left) and after washing out DAPT (right). Removing DAPT elicited a significant reduction in mCherry signal intensity from the AJ. Scale bar, 5 µm. (f) Paired analysis of multiple cells expressing NΔEGF in enrichment factor (I_IN/I_OUT) after DAPT washout. Each dot represents I_IN/I_OUT value before and after DAPT washout from a single cell. Each line corresponds to the I_IN/I_OUT changes before and after DAPT washout in a same single cell (paired two-tailed Student’s t test; n = 6 cells examined across 2 independent experiments). (g) Larger area (1 ×1 mm²) confocal fluorescence images shown in Fig. 4f. Scale bar, 200 µm. (h) Dynamic light scattering spectra of BG-modified macromolecules used in the experiment to induce spatial mutation of NEXT in Fig. 4d-g. (i) Western blot analyses showing that spatial mutations of NEXT alter the level of Notch activation. Representative western blot from three independent experiments. The blot was probed with specific antibodies for cleaved NICD (Val¹⁷⁴⁴) and β-actin. Each lane was loaded with the lysates from NEXT-expressing cells incubated with different BG-modified polymers or proteins for 20 h. The lysate from N^FL was used as control. (j,k) Representative images of artificial AJs formed in live cells. Cells treated with both TAPI2 and DAPT (j) or with only TAPI2 but no DAPT (k). Magnified images were shown in lower panels. An intense mCherry signal was observed at the artificial AJ with TAPI2 and DAPT treatment, while no enrichment of Notch1 signal was seen from cells without DAPT. Scale bar, 5 µm (low-magnification), 2 µm (zoomed-in images). (l) Representative time-lapse images showing Notch signal activation in UAS-Gal4 reporter cells with artificial AJs (white arrows). Cells were cultured in the presence of TAPI2 and no source of S2 cleavage. Neighboring cells without magnetic stimulation were used as internal negative controls. Images were acquired using epifluorescence imaging every 2 hr for 24 hr. Scale bar, 50 µm.

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