Figure 5.

Role of ligand tether and lateral mobility on Notch activation levels. A reporter cell line engineered by Sprinzak et al. containing full length Notch1 and a YFP reporter controlled by a synthetic 12XCSL promoter was used to measure Notch activation levels. (A) Mean fluorescence intensity of reporter cells was measured after 48 h for the following surface types (left to right): supported membrane with histidine-tagged variants of DLL4, supported membrane with laterally mobile DLL4-mCherry-biotin (fluid), supported membrane with hindered DLL4-mCherry-biotin (hindered), functionalized glass with nonfluid DLL4-mCherry-biotin (nonfluid), DLL4-mCherry physisorbed with fibronectin, and DLL4/Fc physisorbed with fibronectin, ^∗P < 0.001. Error bars represent SE for experiments performed in triplicate. Orange rectangles represent streptavidin and purple circles represent biotin moieties. Abbreviations for mCherry = mC; biotin = bio. Representative images of Notch reporter cells on supported membranes with (B) 165 molecules/μm² of nonfluid DLL4-mCherry-biotin and (C) 5150 molecules/μm² of nonfluid DLL4-mCherry-biotin. Scale bar = 10 μm. (D) Calibration of reporter cell line response to different surface densities of nonfluid DLL4-mCherry-biotin. The data were fit to a Hill function, which is indicated by the dashed line. To see this figure in color, go online.