The adhesion force of Notch with Delta and the rate of Notch signaling

Abstract

Notch signaling is repeatedly used during animal development to specify cell fates. Using atomic force microscopy on live cells, chemical inhibitors, and conventional analyses, we show that the rate of Notch signaling is linked to the adhesion force between cells expressing Notch receptors and Delta ligand. Both the Notch extracellular and intracellular domains are required for the high adhesion force with Delta. This high adhesion force is lost within minutes, primarily due to the action of Presenilin on Notch. Reduced turnover or Delta pulling accelerate this loss. These data suggest that strong adhesion between Notch and Delta might serve as a booster for initiating Notch signaling at a high rate.

Figure 1.

Notch signaling and the various Notch receptors used in this paper. (A) The SuH/N^intra signaling pathway. (B) The structures of Notch receptors used in this paper; relative activities shown on an arbitrary scale. TM, transmembrane domain.

Figure 2.

The detachment force between Notch receptors and Delta. (A) A force–distance graph generated between an S2-Dl cantilever and an S2-N cell in 1× PBS+Ca ²⁺. (B) Detachment forces between S2-Dl or S2-N cantilevers and different cells. (C) Force–distance graphs generated when the same S2-Dl cantilever was used successively on an S2-N cell, an S2-NΔ1-18 cell, and an S2-N cell, in 1× PBS+Ca ²⁺. (D) Cell aggregates of S2-Dl or S2 cells with S2 cells expressing Notch receptors. (E) Correlation (r) between the detachment force and the average number of cells in aggregates (at 10 min). Cell number = aggregation size/S2-NΔ1-18+S2-Dl aggregate size (∼1 cell); SD: N = 20.44, N^1-2155 = 8.4, N^nd3 = 13.9, Nmf = 16.7). (F) Aggregated cells after forceful separation. White arrows = Notch cluster regions coextensive with Dl contact regions; black arrow = an N^1-2155 cluster region partially coextensive with Dl contact region. Left, Texas red images with an N antibody (C458.2H); right, Nomarski images of the same cells. Bars, 2 μm (in F).

Figure 3.

Ofut1 RNAi abolishes the detachment force between N and Dl. (A) Western blots showing the levels of Ofut1 in S2-N cells treated with Ofut1 dsRNA [S2-N(Ofut1⁻)] or not [S2-N(control)]. (B) N levels on S2-N cell surfaces after treatment with Ofut1 dsRNA. Cell surface = streptavidin bead precipitates in 40 μl; Total = 40 μl total extracts used. Same extracts were used for A and B. (C) Detachment forces between S2-Dl cantilevers and S2-N(Ofut1⁻) or S2-N(control) cells, in 1× PBS+Ca ²⁺. (D) Aggregates of S2-Dl and S2-N(Ofut1⁻) or S2-N(control) cells.

Figure 4.

Surface scans of cells expressing the different Notch receptors or Dl. (A) High-resolution AFM height images (100 × 100 nm; z range, 7 nm) of the surfaces of live heat-shocked S2 cell or S2 cells expressing the different proteins, scanned in 1× PBS+Ca²⁺. (B) Similar images of the surfaces of live S2 cells expressing Dl or N through the actin promoter. (C) Deflection images (z range, 16 nm) of the surface of an in situ “heat-shocked” S2-N cell. Higher resolution height images (100 × 100 nm; z range, 10 nm) on the right.

Figure 5.

Temporal change in detachment force and SuH/N^intra signaling. (A) Detachment forces between the S2-Dl cantilevers and the S2 cells expressing Notch receptors, in 1× PBS+Ca²⁺. (B) Western blot showing the levels of N after treatment with S2-Dl cells. (C and D) Western blots showing the levels of active Psn in the different cell mixtures. Asterisk corresponds to a cross-reacting band. (E) Western blots showing the increase in levels of S2- and S3-cleaved N fragments in S2-Dl–treated S2-N cells. (F) Northern blots showing the levels of E(spl)C m3 RNA in the different cell mixtures. (G) Semi-quantitative PCR showing relative levels of E(spl)C m3 RNA. rp49 RNA amplification shows levels of total RNA in the samples.

Figure 6.

Presenilin inhibitor blocks the loss of detachment force between Notch receptors and Dl. (A) Detachment forces between the S2-Dl cantilevers and 1% DMSO-pretreated S2 cells expressing Notch receptors, in 1× PBS+Ca²⁺. (B) Detachment force between the S2-Dl cantilevers and 1% DMSO + Psn inhibitor–pretreated S2 cells expressing Notch receptors, in 1× PBS+Ca²⁺. (C) A Western blot showing the amounts of Notch in the presence or absence of the Psn inhibitor. (D) A Western blot showing the amounts of NΔB molecules in the absence or presence of 5× Psn inhibitor.

Figure 7.

Chloroquine accelerates the loss of detachment force between Notch receptors and Dl. (A) Detachment forces between the S2-Dl cantilevers and chloroquine-pretreated S2 cells expressing Notch receptors, in 1× PBS+Ca²⁺. (B) A Western blot showing the levels of N molecules, in chloroquine-pretreated or untreated S2-N cells, in the presence of S2 or S2-Dl cells.

Figure 8.

Dl pulling promotes the loss of detachment force between N and Dl. Detachment forces at various speeds of retraction of S2-Dl cantilevers from S2-N (A and B) or S2-N^1-2155 (C and D) cells that were either not pretreated (A and C) or pretreated with 1% DMSO + Psn inhibitor (B and D), in 1× PBS+Ca²⁺. (E) Detachment forces between cantilevers carrying untreated (nt) or 1% DMSO + Psn inhibitor–pretreated (t) S2-Dl cells and untreated (nt) or 1% DMSO + Psn–inhibitor pretreated (t) S2-N cells, in 1× PBS+Ca²⁺. (F) Northern blots showing the levels of E(spl)C m3 RNA in the different cell mixtures, at different times of aggregation.