Abstract
Planar cell polarity (PCP) and intercellular junctional complexes establish tissue structure and coordinated behaviors across epithelial sheets. In multiciliated ependymal cells, rotational and translational PCP coordinate cilia beating and direct cerebrospinal fluid circulation. Thus, PCP disruption results in ciliopathies and hydrocephalus. PCP establishment depends on the polarization of cytoskeleton and requires the asymmetric localization of core and global regulatory modules, including membrane proteins like Vangl1/2 or Frizzled. We analyzed the subcellular localization of select proteins that make up these modules in ependymal cells and the effect of Trp73 loss on their localization. We identify a novel function of the Trp73 tumor suppressor gene, the TAp73 isoform in particular, as an essential regulator of PCP through the modulation of actin and microtubule cytoskeleton dynamics, demonstrating that Trp73 is a key player in the organization of ependymal ciliated epithelia. Mechanistically, we show that p73 regulates translational PCP and actin dynamics through TAp73-dependent modulation of non-musclemyosin-II activity. In addition, TAp73 is required for the asymmetric localization of PCP-core and global signaling modules and regulates polarized microtubule dynamics, which in turn set up the rotational PCP. Therefore, TAp73 modulates, directly and/or indirectly, transcriptional programs regulating actin and microtubules dynamics and Golgi organization signaling pathways. These results shed light into the mechanism of ependymal cell planar polarization and reveal p73 as an epithelial architect during development regulating the cellular cytoskeleton.
Highlights
The specific orientation of cells within the plane of the tissue, named planar cell polarity (PCP), is an essential feature of animal tissues[1]
Two independent transfection experiments were performed with two clones per genotype. quantitative reverse transcription-PCR (qRT-PCR) assays were repeated four times by duplicate
Kruskal-Wallis test together with Dunn’s multiple comparisons test was performed to evaluate statistical differences; *p < 0.05, ***p < 0.001, n.s: non-significant. b The Golgi apparatus was marked by GM130 and in the magnification panel the contour of a single cell is delineated by a white line. c–g Analysis of MT cytoskeleton changes induced upon TAp73 expression (+Dox) in TAp73-Saos-2 cells stained with specific antibodies against TAp73 (c, red), Vangl[2] (d–f, green), αtubulin (d, g, blue), acetylated α-tubulin (e, red), EB3 (g, red), and Phalloidin (g, green)
Summary
All animal experiments were carried out in accordance with European (European Council Directive 2010/63/UE) and Spanish regulations (RD 53/2013) as well as institutional animal ethical guidelines. For the quantification of number and intensity of pMLC2 membrane dots, five independent mice from each genotype were considered, counting six non-overlapping fields (1 cell/field; ±20 μm × 20 μm) of each animal. Image analysis was performed using ZEN blue software, analyzing number and intensity of p-MLC dots in membrane region. Orthogonal view of z-stack image analysis was performed with ZEN blue software, counting the number of cells with BBs aligned with the plasma membrane and cells with BBs scattered within the cytoplasm. For the quantification of Vangl[2] and Frizzled localization at the plasma membrane, confocal z-stack images were taken before and after transiently transfecting the indicated expression vectors into p73KO-iPSCs. Singleplane images were obtained from z-stacks.
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