Cortactin mediates the linkage between F-actin and shootin1 as a clutch molecule. (A) Cosedimentation of shootin1 with F-actin in the presence of cortactin. Polymerized actin (5 µM) was incubated with purified cortactin (1.5 µM; C), purified shootin1-myc (1.5 µM; S), or both (C+S). After centrifugation, the pellets were immunoblotted with anti-cortactin or anti-myc antibody. F-actin was detected by Coomassie brilliant blue (CBB) staining. (B) Hippocampal neurons were transfected with miRNA against cortactin (#1 or #2) or control miRNA, and cultured on polylysine-coated coverslips for 48 h. They were then fixed and immunostained with anti-shootin1 antibody. The vectors for the miRNAs are designed to coexpress EGFP. Arrowheads indicate an axonal growth cone. Relative fluorescence intensities of shootin1 in growth cones are shown on the right (n = 289 neurons). (C) DIC micrographs showing retrograde movement of L1-CAM-Fc–coated beads on axonal growth cones (3 d in vitro) expressing a control miRNA or cortactin miRNA, and a time series of the indicated areas at 30-s intervals (right). See Videos 5 and 6. The bottom panel illustrates the percentage of beads that showed retrograde flow on growth cones expressing control miRNA (n = 38) or cortactin miRNA (n = 38; left), the mean velocity of moving beads (middle), and the percentage of moving beads with the indicated velocities (right). Data represent means ± SEM (error bars); *, P < 0.05; ***, P < 0.001. Bars: (B) 50 µm; (C) 10 µm.