Figure 2. Vinculin accumulates at apical... | Rockefeller University Press

Figure 2.

$Vinculin accumulates at apical pole of NSCs with the AJs. (A) Image of a chicken NT transverse section at stage HH18 showing the expression of vinculin mRNA by in situ hybridization. Scale bar = 50 μm. (B) High magnification image spanning the entire apicobasal length showing a NT at stage HH18 24 hpe with VCL-T12·mCherry (gray scale). Scale bar = 10 μm. (C) Apical region of an NT slice as in B, stained with anti-N-cadherin (green) and VCL-T12·mCherry (magenta), channels are shown separately at the right-hand panels (gray scale). Scale bar = 10 μm. (D) ST-tagged β-catenin was electroporated for 24 h into stage HH12 chicken NTs. At HH18, β-catenin·ST and the endogenous associated proteins were purified on Streptactin columns. The presence of ST (β-catenin) and vinculin in the elution fractions was assessed by Western blot. L, lysate; FT, flow through; W, wash; E, elution. (E) Drawing of the procedure followed to obtain “en face” pictures of the apical pole of neural precursors. A low magnification image of a test electroporation is shown. Centrioles, primary cilia, actin, and nucleus are labeled with PACT, Arl13b, phalloidin, and DAPI, respectively. Scale bar = 10 μm. (F) “En face” pictures of chicken NTs transfected at stage HH12 for 24 h with Cep152-GFP (yellow), VCL·mCherry (magenta), and β-Catenin·flag (green). Different combinations of channels are shown for clarity. Arrows indicate the centrioles position. The images are Z projections of seven confocal planes spaced 0.15 µm. Scale bar = 1 μm. (F′) X-axis projections of the fluorescence intensity of a rectangular area (cyan dotted rectangle) that contained the centrioles. (F″) The mean fluorescence intensity of the mentioned area was calculated in 37 consecutive confocal planes spaced 0.15 µm. Scale bar = 1 μm. (G) “En face” images of chicken NTs transfected at stage HH12 for 24 h with Cep152-GFP (green), β-Catenin·Flag (magenta), and Arl13b-RFP (cyan). Nuclei were stained with DAPI. The encircled area in the left-handed panel is enlarged in the right-handed panels, where different channel combinations are shown for clarity. Scale bar = 1 μm. Source data are available for this figure: SourceData F2.$

Vinculin accumulates at apical pole of NSCs with the AJs. (A) Image of a chicken NT transverse section at stage HH18 showing the expression of vinculin mRNA by in situ hybridization. Scale bar = 50 μm. (B) High magnification image spanning the entire apicobasal length showing a NT at stage HH18 24 hpe with VCL-T12·mCherry (gray scale). Scale bar = 10 μm. (C) Apical region of an NT slice as in B, stained with anti-N-cadherin (green) and VCL-T12·mCherry (magenta), channels are shown separately at the right-hand panels (gray scale). Scale bar = 10 μm. (D) ST-tagged β-catenin was electroporated for 24 h into stage HH12 chicken NTs. At HH18, β-catenin·ST and the endogenous associated proteins were purified on Streptactin columns. The presence of ST (β-catenin) and vinculin in the elution fractions was assessed by Western blot. L, lysate; FT, flow through; W, wash; E, elution. (E) Drawing of the procedure followed to obtain “en face” pictures of the apical pole of neural precursors. A low magnification image of a test electroporation is shown. Centrioles, primary cilia, actin, and nucleus are labeled with PACT, Arl13b, phalloidin, and DAPI, respectively. Scale bar = 10 μm. (F) “En face” pictures of chicken NTs transfected at stage HH12 for 24 h with Cep152-GFP (yellow), VCL·mCherry (magenta), and β-Catenin·flag (green). Different combinations of channels are shown for clarity. Arrows indicate the centrioles position. The images are Z projections of seven confocal planes spaced 0.15 µm. Scale bar = 1 μm. (F′) X-axis projections of the fluorescence intensity of a rectangular area (cyan dotted rectangle) that contained the centrioles. (F″) The mean fluorescence intensity of the mentioned area was calculated in 37 consecutive confocal planes spaced 0.15 µm. Scale bar = 1 μm. (G) “En face” images of chicken NTs transfected at stage HH12 for 24 h with Cep152-GFP (green), β-Catenin·Flag (magenta), and Arl13b-RFP (cyan). Nuclei were stained with DAPI. The encircled area in the left-handed panel is enlarged in the right-handed panels, where different channel combinations are shown for clarity. Scale bar = 1 μm. Source data are available for this figure: SourceData F2.