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. 2011 May 24;17(1):71–84. doi: 10.1038/mp.2011.57

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Copyright © 2012 Macmillan Publishers Limited

This work is licensed under the Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

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Effects of Shank3^WT and mutants on growth cone dynamics. (a) Shank3 is found in the growth cone of neonatal rat-cultured hippocampal neurons on DIV2. Neurons are labeled for Shank3 (green), F-actin (phalloidin; blue) and either tubulin (anti-Tuj1; red) or cortactin (anti-cortactin, red). Scale bars represent 20 μm. Insets represent high magnifications of the axonal growth cone. Shank3 colocalized with F-actin (blue) and with cortactin (red) in axonal growth cones. (b) Localization of Shank3 overexpressed in young neurons. Green fluorescent protein (GFP)–Shank3^WT or Shank3^STOP or GFP–C3 (green) was transfected in young neurons (DIV0). Immunostaining for MAP2 (red) and actin (phalloidin; blue) revealed that Shank3^WT accumulates in growth cones, whereas Shank3^STOP is restricted to the cell body. (c) Time-lapse video microscopy of growth cones from rat hippocampal neurons co-transfected with the control (GFP–C3) and Shank3^WT or mutated forms. Merge represents three frames with different colors (0 min in green, 5 min in red and 10 min in blue). Scale bars represent 5 μm. (d) Quantification of growth cone motility with the motility index.²⁸ ^***P<0.001 and ^*P<0.05 compared with the control; °°°P<0.001 compared with Shank3^WT.

Effects of Shank3^WT and mutants on growth cone dynamics. (a) Shank3 is found in the growth cone of neonatal rat-cultured hippocampal neurons on DIV2. Neurons are labeled for Shank3 (green), F-actin (phalloidin; blue) and either tubulin (anti-Tuj1; red) or cortactin (anti-cortactin, red). Scale bars represent 20 μm. Insets represent high magnifications of the axonal growth cone. Shank3 colocalized with F-actin (blue) and with cortactin (red) in axonal growth cones. (b) Localization of Shank3 overexpressed in young neurons. Green fluorescent protein (GFP)–Shank3^WT or Shank3^STOP or GFP–C3 (green) was transfected in young neurons (DIV0). Immunostaining for MAP2 (red) and actin (phalloidin; blue) revealed that Shank3^WT accumulates in growth cones, whereas Shank3^STOP is restricted to the cell body. (c) Time-lapse video microscopy of growth cones from rat hippocampal neurons co-transfected with the control (GFP–C3) and Shank3^WT or mutated forms. Merge represents three frames with different colors (0 min in green, 5 min in red and 10 min in blue). Scale bars represent 5 μm. (d) Quantification of growth cone motility with the motility index.²⁸ ^***P<0.001 and ^*P<0.05 compared with the control; °°°P<0.001 compared with Shank3^WT.