In vitro reconstitution of an mRNA-transport complex reveals mechanisms of assembly and motor activation

Single-particle motility of a SHE complex consisting of ASH1-3’+GFP-She2p(ΔhE)+She3p+Myo4p. In this montage, SHE complexes containing the RNA-binding mutant GFP-She2p(ΔhE) (right panel) are compared to complexes containing wild-type GFP-She2p and no RNA (left panel). To ensure comparability between both experiments, image sequences start at the exact same time with respect to the time point when each complex has been applied to the flow cell (i.e., 3 min). GFP is shown in green and TMR-labeled actin filaments in red. TIRFM images were taken at room temperature with 4 frames/s and are displayed at 16 frames/s. Scale bar indicates 5 µm. The time stamp indicates the total elapsed time since the beginning of the acquisition. Movie is related to Fig. 4F. This is a full-length, higher-quality version of Movie S4.