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. 2017 Feb 1;28(3):463–475. doi: 10.1091/mbc.E16-08-0583

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© 2017 Bird, Barzik, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 3: — Quantifying PPIs by NanoSPD. NanoSPD concentrates bait–prey complexes into a nanoscopic volume at filopodial tips, providing noise-free fluorescence enhancement and a defined location for imaging away from the cell body. (A) In fixed HeLa cells expressing MYO10-MYO7A(TAIL)^BAIT and MYRIP^PREY, both bait and prey coaccumulate at filopodial tips. MYO10^{NO BAIT} does not traffic MYRIP^PREY in the control, confirming the interaction and that MYRIP^PREY cannot traffic autonomously. Scale bars: 20 µm; 5 µm (magnified boxed regions). (B) Using SCA to detect molecular interactions. Pearson’s r coefficient is calculated along the filopodial shaft (dotted line, A) to measure bait and prey fluorescence correlation. The likelihood that the observed correlation can occur by random chance is estimated by bootstrapping (see Materials and Methods). (C) SCA reveals a significantly higher interaction index (t test) when MYRIP^PREY is coexpressed with MYO10-MYO7A(TAIL)^BAIT (n = 292 filopodia total) than when MYRIP^PREY is coexpressed with MYO10^{NO BAIT} (n = 264 filopodia total). Each data point is the average interaction index from a single experiment (three independent determinations). Data are mean ± SD. (D) NanoSPD can be used in nonmammalian Sf9 insect cells that produce large numbers of filopodia. In fixed Sf9 cells, MYRIP^PREY accumulates with MYO10-MYO7A(TAIL)^BAIT at filopodial tips but not when coexpressed with MYO10^{NO BAIT}. Scale bar: 10 µm. (E) Using ICA to detect PPIs in Sf9 cells. Scatter plot of bait (x-axis) and prey (y-axis) fluorescence at individual filopodia tips (from three independent determinations). MYRIP^PREY fluorescence is linearly correlated with MYO10-MYO7A(TAIL)^BAIT fluorescence, unlike cells expressing MYO10^{NO BAIT}. (F) Bar graph of MYRIP^PREY fluorescence intensities from E. Coexpression with MYO10-MYO7A(TAIL)^BAIT significantly increases the average prey fluorescence intensity at filopodial tips, compared with MYO10^{NO BAIT} (Mann-Whitney U-test). Data are mean ± SD. ****, p < 0.0001.