FIGURE 1:

Principle of the nanoscale pulldown (NanoSPD). (A) Domain representation of full-length EGFP-tagged MYO10^FL with the ATPase motor domain, IQ motifs that bind light chains, a coiled-coil and tail domains that bind cargo. Heavy meromyosin MYO10^HMM molecules are identical but truncated after the coiled-coil. Query bait proteins are fused to the C-terminus of MYO10^HMM. (B) EGFP-tagged MYO10^FL (top panel) and truncated MYO10^HMM (bottom panel) traffic to filopodial tips in HeLa cells. Inset white boxes are shown magnified. F-actin is labeled with rhodamine phalloidin (red), and nuclei are stained with Hoechst (blue). Scale bars: 20 µm; 5 µm (inset). F-actin and MYO10 images have been uniformly inverted. (C) Using NanoSPD to test the interaction between A^BAIT and B^PREY proteins. A^BAIT is forcibly trafficked to filopodial tips by MYO10 as part of the EGFP-MYO10-A^BAIT fusion protein (green). In the presence of an interaction, RFP-B^PREY molecules (red) are actively cotransported along filopodia, resulting in concentration of bait and prey molecules at filopodial tips. (D) In the control, EGFP-MYO10^{NO BAIT} molecules (green) lacking a bait still accumulate at filopodial tips but no longer traffic RFP-B^PREY (red). Identical results are expected if RFP-B^PREY does not interact with EGFP-MYO10-A^BAIT.