September 24, 2015;
are hair-like cellular protrusions important in many aspects of eukaryotic biology. For instance, motile cilia
enable fluid movement over epithelial surfaces, while primary (sensory) cilia
play roles in cellular signalling. The molecular events underlying cilia
dynamics, and particularly their disassembly, are not well understood. Phosphatase and tensin
) is an extensively studied tumour suppressor, thought to primarily act by antagonizing PI3-kinase signalling. Here we demonstrate that PTEN
plays an important role in multicilia formation and cilia
disassembly by controlling the phosphorylation of Dishevelled
), another ciliogenesis regulator. DVL
is a central component of WNT signalling that plays a role during convergent extension movements, which we show here are also regulated by PTEN
. Our studies identify a novel protein substrate for PTEN
that couples PTEN
to regulation of cilia
dynamics and WNT signalling, thus advancing our understanding of potential underlying molecular etiologies of PTEN
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References [+] :
Figure 2. PTEN is required for ciliogenesis in mouse trachea and ependyma.(a) Loss of Pten causes ciliation defects in trachea. Tracheal multicilia of control littermates (top) or Pten conditional knockout (cKO) embryos (lower panel) were stained with acetylated tubulin (green). To confirm Pten loss, cells were stained for Pten (red) and F-actin (Phalloidin, blue). Note that the cell with prominent cilia in Pten cKO embryos (upper left) retains residual Pten protein. Images are representative from 15 cKO and 10 control embryos analysed from three independent experiments. Scale bar, 7 μm. (b) Loss of Pten results in cilia formation defects in ependymal cells (brain localization in schematics on the right). Control or cKO pups, induced with tamoxifen at P0–P2 were analysed for defects in ependymal cilia formation at day 9 of development as in a. Images represent 10 cKO and 14 control pups from three independent experiments. Scale bar, 7 μm. (c) Basal body (BB) polarization defects in ependymal multiciliated cells (EMCCs) at day 10 of postnatal development. Left panel shows tissue polarization in wild-type mouse of the same genetic background. Middle panel displays BB polarization in EMCC of an animal missing one allele of Pten, while the panel on the right displays effect of Pten knockout. Bottom panels illustrate the algorithm for quantification of translational polarity defects, based on Boutin et al.58 and described in Methods. Scale bar, 10 μm. (d) Quantification of Pten loss effect on basal bodies translational polarity. The angles for basal body orientation vectors (BBOVs, see Methods) were calculated and angular histogram plots are shown (on average 20 cells per field of view were analysed). Before plotting, angles within each image were normalized to the average angle calculated for each field of view and then underwent 90° rotation. Circular standard deviation (CSD) was calculated for each field of view to assess variation in BBOVs. CSD was estimated for every image plane analysed and compared between genotypes. The difference in BB polarization between control WT and Pten cKO was found significant with P<0.0001 in an unpaired Mann–Whitney test.
Figure 4. PTEN regulates cilia disassembly and phosphorylation of DVL2.(a) Top: time-course scheme for cilia disassembly in hTERT-RPE1 cells transfected (Trx.) with siControl (siCTL) or siPTEN. Bottom: representative images of cells starved and after serum addition, stained for acetylated tubulin (cilia axoneme, green), pericentrin (centrioles, red), 4′,6-diamidino-2-phenylindole (DAPI, cell nuclei, blue); scale bar, 20 μm. (b) PTEN loss promotes cilia disassembly. The percentage of ciliated cells from a was quantified by automated imaging (Supplementary Fig. 4 and Methods). Graph shows mean with error bars (s.e.m.) from n=7 (n=4 for a 7-h time point), with ∼1,000 cells for each condition (***P<0.001 by a t-test). (c) DVL2 peptide sequence containing the serine-143 phosphorylation site (in red), recognized by the antibody. (d) PTEN knockdown enhances DVL2 phosphorylation on serine 143 during cilia formation. hTERT-RPE1 cells were transfected with siCTL or siPTEN and lysates were analysed at 24, 48 and 96 h post transfection. Cells were starved for the last 48 h before lysis. (e) Phospho-S143-DVL2 levels during cilia disassembly. The experiment was performed as in a with siCTL- or siPTEN-transfected hTERT-RPE1 cells. Cell lysates were analysed by immunoblotting using the indicated antibodies. Samples in d and e are representative from at least four independent experiments. (f) siRNA-resistant PTEN rescues accelerated cilia disassembly caused by PTEN knockdown. Cilia disassembly was evaluated in hTERT-RPE1 lines expressing siRNA-resistant Flag-tagged PTEN (3F-PTENr) or empty vector (pCAGIP), upon transfection with siCTL or siPTEN. Cilia disassembly was quantified as in b. Results are plotted as mean with error bars showing s.e.m.; n=4, **P<0.01 by a t-test. (g) Expression of DVL2 S143A rescues the effect of PTEN knockdown on cilia disassembly rates. Cilia disassembly was performed in hTERT-RPE1 stable cell lines expressing either 3F-DVL2 or its S143A mutant in the presence of siDVL2 (targeting its endogenous 3′-untranslated region) upon siPTEN or siCTL transfection. Cilia disassembly was quantified as in b and is plotted as percentage of ciliated cells (mean with error bars showing s.e.m. of n=4; **P=0.01 by a t-test) with an average of 170 cells analysed per condition in each experiment.
Figure 5. PTEN affects ciliogenesis by targeting DVL2.(a) PTEN-dependent cilia disassembly is blocked by CK1δ-ɛ inhibitor but not by PI3-kinase inhibitor. hTERT-RPE1 cells transfected as in Fig. 4a were treated with 10 μM of LY294002 (PI3K inhibitor) or 40 μM of IC261 (CK1δ-ɛ inhibitor) for a total of 4 h (2 h before plus 2 h post serum addition). Ciliation was quantified at 0 h (starved) and 2 h of disassembly. Graph represents three independent experiments; data are mean with error bars showing s.e.m.; ***P<0.001 by a t-test, with 300 cells or more per condition. (b) Representative images from a stained for acetylated tubulin (axonemes, green) and pericentrin (centrioles, red). Scale bar, 20 μm. (c) DVL2 binds PTEN in vitro. hTERT-RPE1 cells overexpressing 3Flag-DVL2 wild-type (WT) or S143A mutant were lysed and precipitated using glutathione-sepharose beads coupled to either His-GST-PTEN(1–353) WT or DACS mutant and analysed by immunoblotting. (d,e) PTEN dephosphorylates DVL2 pSerine-143 peptide. (d) Dephosphorylation of pS143-peptide (Fig. 4c) was analysed in an enzyme-linked immunosorbent-type assay, after 10 min incubation with varied concentrations of PTEN WT, purified using baculoviral system. The percentage of remaining phospho-serine-143 was quantified by normalizing to a condition containing the peptide without PTEN. Experiments were performed in triplicates. Results are plotted as the mean with error bars indicating s.d. (n=3). (e) pS143 dephosphorylation efficiency by PTEN variants (10 μM) was compared with PTEN WT as in d. Experiments were carried out in triplicate, n=3. (f) PTEN dephosphorylates DVL2 at serine 143. Immunoprecipitated 3Flag-DVL2 from HEK293T cells was incubated with PTEN variants for 1 h. Serine-143 phosphorylation was analysed by immunoblotting. Figure represents three independent experiments. (g) A protein phosphatase dead PTEN mutant (Y138L) fails to rescue multicilia defects in Xenopus. Effects on multicilia were quantified by visual analysis of acetylated tubulin (axoneme) staining of embryos' epidermis. Embryos were injected with indicated morpholinos, hPTEN constructs and Centrin-RFP as a lineage tracer. The percentage of normal multiciliated cells was plotted as mean with error bars in s.e.m. from four independent experiments; **P<0.01 by a t-test, with more than 500 cells per condition. (h) Model for PTEN function during cilia formation and stability.
Stages of ciliogenesis and regulation of ciliary length.