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. 2018 Jun 11;9:2279. doi: 10.1038/s41467-018-04676-w

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — Formation of a dense actin network at the centriolar patch during ependymal cell differentiation a F-actin (phalloidin, grey), centrioles (FOP, red) and cilia (GT335, green) in whole mounts of lateral ventricular walls at postnatal days 4 (P4), P6 and P15 at the apical level (colocalising with FOP staining) and subapical level; the actin network in the centriolar patch thickens as motile cilia grow; cell borders and centriolar patches are outlined with dashed white and red lines, respectively. Arrows point to long actin filaments. On the right, a sagittal view of the lateral ventricular wall and the region of interest analysed throughout the paper (red square); A: Anterior, P: Posterior, D: Dorsal, V: Ventral. b Ratio of mean phalloidin fluorescence intensities in the centriolar patch (red dashed lines in a) and on the remaining cell surface (white minus red dashed lines) as a function of ciliary length. Error bars represent the sem of n = 112 cells from three mice; P-values were determined by one-way ANOVA followed by Dunn’s multiple comparison test; ***P ≤ 0.0001. c–e High resolution microscopy (STED) of mature ependymal cells on P15 whole-mount lateral ventricular walls. c Staining of F-actin (phalloidin, grey) and centriole distal markers, Cep164 (distal appendages, red) or Centrin2-GFP (distal lumen, yellow) that colocalise with the apical actin network, or centriole proximal markers Akap450 (pericentriolar material, green) or Rootletin (rootlets, cyan) that colocalise with the subapical actin network. d Zoom-in of Cep164 (red)-, Centrin2-GFP (yellow)- and F-actin (phalloidin, grey)-labelled mature ependymal cells; note a cluster of two centrioles with merged distal appendages (arrowhead) surrounded by actin. e Three-dimensional modelling of Cep164 (red)-, Akap450 (green)- and F-actin-(grey)-positive centrioles showing the links between the apical and subapical networks, and between neighbouring centrioles. f Model of centrioles with their associated actin bundles positioned according to the markers used in this study. Scale bars=5 μm in a and 1 μm in c, d