Figure 4. Repression of shootin1... | Rockefeller University Press

Figure 4.

$Figure 4. Repression of shootin1 expression by RNAi weakens the actin flow–L1 linkage. (A, B, and D) DIC micrographs showing retrograde movement of L1-Fc–coated beads on axonal growth cones (two days in vitro) expressing a control shRNA (A), the shRNA designated against shootin1 (B), or the shRNA designated against shootin1 together with the mutant myc-shootin1 (D), which is refractory to the shRNA (left), and time series of the indicated areas at 30-s intervals (right). See Videos 6 and 7 (available). As previously described (Toriyama et al., 2006), about half of the RNAi-induced neurons polarized after 48 h in vitro. Therefore, we selected such neurons and performed the analysis on their axonal growth cones. (C) Hippocampal neurons transfected with shootin1 shRNA (left), or shootin1 shRNA together with the refractory myc-shootin1 (right) were cultured for 48 h. The vector to express the shRNAs is designed to coexpress EGFP (green). Shootin1 and myc-shootin1 were immunostained by anti-shootin1 (left) and anti-myc (right) antibodies, respectively (red). Arrows denote axonal growth cones. (E) The percentage of beads coated with L1-Fc that showed retrograde flow on growth cones expressing a control shRNA (n = 86), shootin1 shRNA (n = 71), or shootin1 shRNA and the refractory myc-shootin1 (n = 44); mean velocity of moving beads as means ± SEM (***, P < 0.0005 compared with control and RNAi + refractory shootin1); and the percentage of moving beads with indicated velocities. Bars: (B) 5 μm; (C) 50 μm.$

Repression of shootin1 expression by RNAi weakens the actin flow–L1 linkage. (A, B, and D) DIC micrographs showing retrograde movement of L1-Fc–coated beads on axonal growth cones (two days in vitro) expressing a control shRNA (A), the shRNA designated against shootin1 (B), or the shRNA designated against shootin1 together with the mutant myc-shootin1 (D), which is refractory to the shRNA (left), and time series of the indicated areas at 30-s intervals (right). See Videos 6 and 7 . As previously described (Toriyama et al., 2006), about half of the RNAi-induced neurons polarized after 48 h in vitro. Therefore, we selected such neurons and performed the analysis on their axonal growth cones. (C) Hippocampal neurons transfected with shootin1 shRNA (left), or shootin1 shRNA together with the refractory myc-shootin1 (right) were cultured for 48 h. The vector to express the shRNAs is designed to coexpress EGFP (green). Shootin1 and myc-shootin1 were immunostained by anti-shootin1 (left) and anti-myc (right) antibodies, respectively (red). Arrows denote axonal growth cones. (E) The percentage of beads coated with L1-Fc that showed retrograde flow on growth cones expressing a control shRNA (n = 86), shootin1 shRNA (n = 71), or shootin1 shRNA and the refractory myc-shootin1 (n = 44); mean velocity of moving beads as means ± SEM (***, P < 0.0005 compared with control and RNAi + refractory shootin1); and the percentage of moving beads with indicated velocities. Bars: (B) 5 μm; (C) 50 μm.