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

Ahimou F, Mok LP, Bardot B, Wesley C - J. Cell Biol. (2004)

Bottom Line: Notch signaling is repeatedly used during animal development to specify cell fates.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, The University of Vermont, VT 05405, USA.

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.

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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.
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fig3: 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.

Mentions: O-fucosyl transferase Ofut1 is known to modify N, and its RNA interference (RNAi) knock-out in S2 cells has been shown to significantly reduce N binding to Dl (Okajima and Irvine, 2002; Okajima et al., 2003). To confirm that we are indeed measuring Dl binding ability, we knocked down Ofut1 expression in S2-N cells by RNAi. Ofut1 protein levels were significantly reduced on the third and fourth day after dsOfut1 RNA treatment, when compared with S2-N control cells (Fig. 3 A). Cell surface biotinylation and streptavidin immunoprecipitation showed that although the levels of N at the cell surface were lower than control levels on d 3, it was higher on d 4 (Fig. 3 B). AFM measurements on the same population of cells showed that detachment forces with S2-Dl cantilever was almost zero on both days (Fig. 3 C). To confirm by an independent method that S2-N (Ofut1−) cells have lost their ability to bind S2-Dl cells, we performed cell aggregation assays using the same batch of d 4 cells used in AFM and expression assays. Results showed that although the control S2-N cells formed aggregates as usual, S2-N (Ofut1−) cells did not (Fig. 3 D). Thus, all our experiments indicated that the detachment forces we measured with the different Notch receptors were due to the differences in their Dl binding strengths.


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

Ahimou F, Mok LP, Bardot B, Wesley C - J. Cell Biol. (2004)

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.
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Related In: Results  -  Collection

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fig3: 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.
Mentions: O-fucosyl transferase Ofut1 is known to modify N, and its RNA interference (RNAi) knock-out in S2 cells has been shown to significantly reduce N binding to Dl (Okajima and Irvine, 2002; Okajima et al., 2003). To confirm that we are indeed measuring Dl binding ability, we knocked down Ofut1 expression in S2-N cells by RNAi. Ofut1 protein levels were significantly reduced on the third and fourth day after dsOfut1 RNA treatment, when compared with S2-N control cells (Fig. 3 A). Cell surface biotinylation and streptavidin immunoprecipitation showed that although the levels of N at the cell surface were lower than control levels on d 3, it was higher on d 4 (Fig. 3 B). AFM measurements on the same population of cells showed that detachment forces with S2-Dl cantilever was almost zero on both days (Fig. 3 C). To confirm by an independent method that S2-N (Ofut1−) cells have lost their ability to bind S2-Dl cells, we performed cell aggregation assays using the same batch of d 4 cells used in AFM and expression assays. Results showed that although the control S2-N cells formed aggregates as usual, S2-N (Ofut1−) cells did not (Fig. 3 D). Thus, all our experiments indicated that the detachment forces we measured with the different Notch receptors were due to the differences in their Dl binding strengths.

Bottom Line: Notch signaling is repeatedly used during animal development to specify cell fates.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, The University of Vermont, VT 05405, USA.

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.

Show MeSH
Related in: MedlinePlus