FIGURE 7:

Simplified NanoSPD 2.0 assays using the GFP-nanotrap. NanoSPD 2.0 uses MYO10^HMM fused to the GFP-binding nanotrap (MYO10^NANOTRAP) to capture EGFP-tagged bait proteins and transport them along filopodia. (A) Image of fixed HeLa cells expressing nonfluorescent MYO10^NANOTRAP, EGFP-MYO7A(TAIL)^BAIT, and mCherry-MYRIP^PREY. Both bait and prey robustly accumulate at filopodial tips (magnified panels shown below). (B) mCherry-MYRIP^PREY does not accumulate at filopodial tips (magnified panels shown below) when coexpressed with mCherry-MYO10^NANOTRAP and EGFP (EGFP^{NO BAIT}). This confirms that the GFP-nanotrap does not bind mCherry-MYRIP^PREY directly. Scale bars: 25 μm; 5 μm (magnified regions). (C) SCA of data from A and B shows a significantly higher interaction index (t test) in the presence of EGFP-MYO7A(TAIL)^BAIT vs. EGFP^{NO BAIT}. Data are mean ± SD with each data point representing the average interaction index from a single experiment. (D) ICA of data from A and B reveals the significant increase (Mann-Whitney U-test) of mCherry-MYRIP^PREY fluorescence at filopodial tips in the presence of EGFP-MYO7A(TAIL)^BAIT vs. EGFP^{NO BAIT}. Data are mean ± SD with each data point representing a single filopodium. (E) Bait (x-axis) vs. prey (y-axis) scatter plot of fluorescence at filopodial tips from D. Each data point represents a single filopodium. ****, p < 0.0001; all data are from three independent experimental determinations (n = 285–295 filopodia total).