Myosin VI SI mediates the caging of SG to the cortical actin network in myosin VI knockdown cells. (A and B) Myosin VI stable knockdown (KD) PC12 cells coexpressing NPY-mCherry with GFP-MyoVI-SIfull (A) or GFP-MyoVI-NIfull (B) were imaged by TIRF microscopy before and after nicotine stimulation. Videos were acquired at a rate of one frame per second. The insets highlight the trajectories of GFP-MyoVI–positive (arrowheads) and GFP-MyoVI–negative (arrows) SGs during 12 s after stimulation. (C) Average MSD of NPY-mCherry–positive SGs tracked in untransfected myosin VI stable knockdown cells and in myosin VI stable knockdown cells expressing GFP-MyoVI-SIfull. (D) Average MSD of NPY-mCherry–positive SGs tracked in untransfected myosin VI stable knockdown cells and in myosin VI stable knockdown cells expressing GFP-MyoVI-NIfull. (E) Time-lapse series of a single NPY-mCherry–positive SG appearing in the TIRF plane and becoming highly restricted upon association with GFP-MyoVI-SIfull. The arrow points to the beginning of the trajectory of the SG. The arrowhead shows the end of this trajectory or the position of the SG at each time point. Video 1 shows the complete time series for this panel. (F) Time-lapse series of a single SG labeled with NPY-mCherry appearing in the TIRF plane and becoming GFP-MyoVI-NIfull positive with little effect on its movement. The arrow points to the beginning of the trajectory of the SG. The arrowhead shows the end of this trajectory or the position of the SG at each time point. Video 2 shows the complete time series for this panel. Bars: (A [main images], B [main images], E, and F) 5 µm; (A [insets] and B [insets]) 1 µm. Error bars are means ± SEM. ***, P < 0.001.