![Figure 6. Myosin VI SI recruits SGs in an activity- and F-actin–dependent manner. (A) Bright-field, epifluorescence, and EM images of PC12 cells expressing GFP-MyoVI-SIfull processed for correlative EM. Arrows indicate dense-core SGs clustered in the region of transfected cells where the GFP-MyoVI-SIfull–positive structures are located (insets). (B and C) Live-cell confocal images of PC12 cells coexpressing NPY-mCherry with GFP-MyoVI-SIfull (B) or GFP-MyoVI-NIfull (C) before (control) and after 100 µM nicotine (nicotine) stimulation. The insets highlight the NPY-mCherry–positive SGs clustered around GFP-MyoVI–positive structures. The NPY-mCherry images are shown as pseudocolor images. A region of interest around a GFP-MyoVI–positive structure was used for quantification of NPY-mCherry fluorescence (right graphs) in the conditions described in B and C. (D and E) Quantification of NPY-mCherry fluorescence around GFP-MyoVI-SItail (D)– or GFP-MyoVI-NItail (E)–positive structures before (control, black) and after 100 µM nicotine stimulation (nicotine, red). Average NPY-mCherry fluorescence is expressed as a percentage of initial fluorescence. (F) GFP-MyoVI-SIfull and GFP-MyoVI-NIfull were expressed in wild-type (wt) or myosin VI stable knockdown (myosin VI KD) PC12 cells. After fixation, cells were imaged by confocal microscopy, and the number of cells harboring large GFP-MyoVI–positive structures (large structures) or small punctate cytosolic distribution (small puncta) were counted and expressed as a percentage of the total number of transfected cells (n = 3 independent experiments). (G, left) Confocal images of myosin VI stable knockdown PC12 cells expressing GFP-MyoVI-SIfull or GFP-MyoVI-NIfull immunolabeled with Synaptotagmin-I (red). The enlarged images show the association between expressed GFP-MyoVI and Synaptotagmin-I (Syt-I). (right) Analysis of the colocalization between Synaptotagmin-I and the different GFP-MyoVI. Bars: (B, C, and G [main images]) 5 µm; (B, C, and G [insets]) 1 µm. Error bars are means ± SEM. **, P < 0.01; ***, P < 0.001.](https://cdn.rupress.org/rup/content_public/journal/jcb/200/3/10.1083_jcb.201204092/5/jcb_201204092_fig6.jpeg?Expires=1771608263&Signature=ZqQZ~G9-KPhYSy9Tkr7NgDeWRKLpaD8VFOHN0Brsk6ZgUTyhXRrUtQI4~5sbbYTKigiNBMEfaXGp5R6HuJw2Vi1UEvypUisO9nhRYh3hFkxon5uDZDmjRvMdGwwRmMQpvHm4I0zhJ29ZHEdHptw1zKvR6kz7yG3VDAFB4cIm3Ak1qWR7IpzqBzNovJzm7dqbQ8lJp8KT0qHQ1k807W48y6zhaZjmZIuTzwOwZopwp2CGSOQ44EjnkmFav2JYkl78m5Gmxh7EKcD5mZt5l~QbCfibfgcp1LW31P16pKjPCGRcm3ctkzwAsXAGCt7-UMDssaaShtXQSYrdPAOMZlU4tA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Myosin VI SI recruits SGs in an activity- and F-actin–dependent manner. (A) Bright-field, epifluorescence, and EM images of PC12 cells expressing GFP-MyoVI-SIfull processed for correlative EM. Arrows indicate dense-core SGs clustered in the region of transfected cells where the GFP-MyoVI-SIfull–positive structures are located (insets). (B and C) Live-cell confocal images of PC12 cells coexpressing NPY-mCherry with GFP-MyoVI-SIfull (B) or GFP-MyoVI-NIfull (C) before (control) and after 100 µM nicotine (nicotine) stimulation. The insets highlight the NPY-mCherry–positive SGs clustered around GFP-MyoVI–positive structures. The NPY-mCherry images are shown as pseudocolor images. A region of interest around a GFP-MyoVI–positive structure was used for quantification of NPY-mCherry fluorescence (right graphs) in the conditions described in B and C. (D and E) Quantification of NPY-mCherry fluorescence around GFP-MyoVI-SItail (D)– or GFP-MyoVI-NItail (E)–positive structures before (control, black) and after 100 µM nicotine stimulation (nicotine, red). Average NPY-mCherry fluorescence is expressed as a percentage of initial fluorescence. (F) GFP-MyoVI-SIfull and GFP-MyoVI-NIfull were expressed in wild-type (wt) or myosin VI stable knockdown (myosin VI KD) PC12 cells. After fixation, cells were imaged by confocal microscopy, and the number of cells harboring large GFP-MyoVI–positive structures (large structures) or small punctate cytosolic distribution (small puncta) were counted and expressed as a percentage of the total number of transfected cells (n = 3 independent experiments). (G, left) Confocal images of myosin VI stable knockdown PC12 cells expressing GFP-MyoVI-SIfull or GFP-MyoVI-NIfull immunolabeled with Synaptotagmin-I (red). The enlarged images show the association between expressed GFP-MyoVI and Synaptotagmin-I (Syt-I). (right) Analysis of the colocalization between Synaptotagmin-I and the different GFP-MyoVI. Bars: (B, C, and G [main images]) 5 µm; (B, C, and G [insets]) 1 µm. Error bars are means ± SEM. **, P < 0.01; ***, P < 0.001.
