Myosin VI interacts with SGs in a Ca²⁺-dependent manner. (A) Bovine adrenal medulla fractionation was assessed by SDS-PAGE and Western blotting with the indicated antibodies (Golgi: GM130; early endosomes: Rab5; endoplasmic reticulum: protein disulfide-isomerase (PDI); plasma membrane: SNAP25; and SG: Synaptotagmin-I and VAMP2). Purified SGs were obtained by pooling fractions 11 and 12. (B) Schematic representation of the experimental approach used to identify cytosolic proteins interacting with SGs in a Ca²⁺-dependent manner. (C, top) Purified SGs and cytosol were incubated in the presence of increasing free [Ca²⁺]. Myosin VI recruitment to SGs was analyzed by Western blotting. Synaptotagmin-I was used as a loading control. (bottom) Quantification of myosin VI recruitment to SGs (n = 3). (D, left) PC12 cells immunolabeled with anti–myosin VI (green) and anti–Synaptotagmin-I (red) antibodies. Arrows mark colabeled SGs. The enlarged images (insets) show the association between endogenous myosin VI and Synaptotagmin-I. Bars: (main images) 5 µm; (insets) 1 µm. (right) Analysis of the colocalization between Synaptotagmin-I (Syt-I) and myosin VI, EEA1, or VAMP2. (E) Paraformaldehyde-fixed PC12 cells were immunolabeled with the anti–myosin VI antibody and labeled with 5-nm protein A–gold for EM. Arrowheads show myosin VI–positive SGs. Error bars are means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.