Fig. 3: Processive myosin-7a complexes are predominantly dimeric.

A TIRF view of single-molecule motility assays with mCherry-tagged M7BP demonstrates that GFP-myosin-7a (green) and M7BP-mCherry (red) are colocalized in the majority of the puncta moving along actin filaments. Actin filaments were labeled with Alexa-Fluor 647 phalloidin (gray). Scale bar = 5 μm. B Kymograph analysis of box in A shows that the processive movements (diagonal lines) of GFP-myosin-7a and M7BP-mCherry are overlaid. C The histograms of fluorophore numbers (GFP for myosin-7a and mCherry for M7BP) determined by fluorescence intensity analysis. N = 661 quantified complexes. D Schematic of the three-color labeling experiments to test the hypothesis that the motile complex is composed of dimerized myosin-7a. Halo-tagged myosin-7a was labeled with TMR (red), Alexa-Fluor 488 (green), or Alexa-Fluor 660 (blue) and equal quantities of all three labeled myosins were simultaneously introduced into the flow chamber. E Movie frames show that myosin-7a labeled with three different colors moves processively along actin with low motor unit numbers (as most puncta exhibit the original or a mixture of two colors). Actin is labeled with Alexa-Fluor 405 phalloidin and displayed in gray). Unlabeled M7BP is present at the concentration equal to that of total myosin-7a. Images 1, 2, and 3 correspond to the regions in box 1, 2, and 3 at particular time points. Scale bar = 5 μm (left panel); Scale bar = 1 μm in images of 1, 2, and 3. F Kymograph analysis of the three-color single-molecule assays showing examples of different-color fluorophores moving together. G Frequency distribution histogram of the numbers of HaloTag fluorophores in each complex indicating that the motile complexes predominantly contain two myosin molecules. N = 1485 quantified complexes.