XB-ART-51731J Cell Biol December 7, 2015; 211 (5): 963-73.
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The polarity protein Inturned links NPHP4 to Daam1 to control the subapical actin network in multiciliated cells.
Motile cilia polarization requires intracellular anchorage to the cytoskeleton; however, the molecular machinery that supports this process remains elusive. We report that Inturned plays a central role in coordinating the interaction between cilia-associated proteins and actin-nucleation factors. We observed that knockdown of nphp4 in multiciliated cells of the Xenopus laevis epidermis compromised ciliogenesis and directional fluid flow. Depletion of nphp4 disrupted the subapical actin layer. Comparison to the structural defects caused by inturned depletion revealed striking similarities. Furthermore, coimmunoprecipitation assays demonstrated that the two proteins interact with each other and that Inturned mediates the formation of ternary protein complexes between NPHP4 and DAAM1. Knockdown of daam1, but not formin-2, resulted in similar disruption of the subapical actin web, whereas nphp4 depletion prevented the association of Inturned with the basal bodies. Thus, Inturned appears to function as an adaptor protein that couples cilia-associated molecules to actin-modifying proteins to rearrange the local actin cytoskeleton.
PubMed ID: 26644512
PMC ID: PMC4674276
Article link: J Cell Biol
Species referenced: Xenopus laevis
Genes referenced: daam1 fmn1 fmn2 intu tub
Morpholinos: daam1 MO1 fmn1 MO1 fmn2 MO1 fmn2 MO2 intu MO1 nphp4.2 MO1 nphp4.2 MO2
Phenotypes: Xla Wt + daam1 MO (Fig. 4. B)
Article Images: [+] show captions
|Figure 1. Localization of full-length and truncated NPHP4 in multiciliated cells of the Xenopus epidermis. At the four-cell stage, Xenopus embryos were injected with mRNA for GFP-NPHP4 (green in merge), together with mRNAs for RFP-Centrin or RFP-Clamp (red in merge) to label basal bodies and ciliary rootlets, respectively. At stage 32, confocal microscopy was performed to study the localization of full-length and truncated NPHP4 in the cells with multiple motile cilia of epidermal skin. Maximum intensity projection of obtained confocal datasets is shown, unless noted otherwise. (A) Colocalization of GFP-NPHP4 with RFP-Centrin. Area enclosed by the white box is magnified in the right panel (5.3× magnification; see Fig. S1 A for separate GFP and RFP channels). (B) 3D reconstruction of confocal datasets is shown. Injection of mRNA encoding for GFP-NPHP4 (200 pg) revealed the broad localization of GFP-NPHP4 extending from the basal body to the presumptive transition zone distal to the basal body, whereas lower-dose injection of mRNA (70 pg) revealed a more confined localization of GFP-NPHP4 to the presumptive transition zone. The insets are magnified on the right panels. (C–F) Localization of NPHP4 truncations is studied. Numbers on the left of the panels indicate the first and last amino acid of the NPHP4 truncations. Expression of all constructs was confirmed by Western blot. (C) The C-terminal half of NPHP4 (amino acids 863–1,426) is sufficient for localization to the basal body. (D–F) The C-terminal half of NPHP4 contains two distinct subdomains targeting NPHP4 to the basal bodies: amino acids 863–1,250 and amino acids 1,251–1,426. (E) The first subdomain (amino acids 863–1,250) showed additional localization to the ciliary rootlet, colocalizing with RFP-Clamp. The inset is magnified by 2.4× on the right panels. (F) The C-terminal fragment spanning amino acids 1,225–1,426 showed enhanced localization to the plasma membrane (arrowhead). Area enclosed by the white box is magnified in the right panel. Bars: (main) 5 µm; (B, magnified insets) 500 nm.|
|Figure 2. Xenopus Nphp4 is essential for building functional cilia. (A and B) Confocal microscopy of multiciliated cells of Xenopus epidermal skin. Cilia and apical cell surface are labeled with anti–acetylated tubulin (Ac-tub; red in merge) and membrane-targeted GFP (mGFP; green in merge). (A) Maximum intensity projection (top), and 3D reconstruction projected in the x-z plane (lateral) for the confocal datasets are shown. The apical surface is indicated by a solid white line. Embryos injected with 20 ng control morpholino (ctl MO) showed normal ciliogenesis, whereas 20 ng nphp4 ATG MO–injected embryos showed ciliogenesis defects. (B) Single optical sections comparing the signals for anti–acetylated tubulin at levels 1 and 2 in ctl MO– and nphp4 ATG MO–treated Xenopus embryos (see dashed white lines in the lateral views of A) revealed that ciliary microtubules are nucleated below the apical cell surface in nphp4-deficient, but not ctl MO–treated, Xenopus embryos. (C) Scanning EM of the Xenopus epidermis confirmed the ciliogenesis defect in 16 ng nphp4 ATG MO– and 8 ng nphp4 SB MO–injected Xenopus embryos. (D) Labeling of basal bodies and the apical cell surface with RFP-Centrin and mGFP revealed that basal bodies are aligned along the apical cell surface and distributed uniformly across the cell surface in ctl MO–injected Xenopus embryos. In contrast, basal bodies in nphp4 MO–injected Xenopus embryos failed to migrate to the apical cell surface and remained within the cytoplasm. (Top) Maximum intensity projection of confocal datasets. (Bottom) Serial confocal images projected in the x-z plane with the apical cell surface indicated by a dashed white line. (E) The multiciliated cells of the Xenopus epidermis formed a dense actin cytoskeleton (phalloidin, green) at the apical cortex. Apically localized basal bodies (RFP-Centrin, red) were embedded in this actin web (left). In nphp4-deficient Xenopus embryos, basal bodies remained within the cytoplasm below the apical actin web, which is also thinner than in control cells (right). Confocal 3D datasets were processed with Imaris software, and the lateral (top), top (middle), and cytoplasmic (bottom) views are shown. (F) Depletion of nphp4 resulted in a decreased rate of cilia-driven fluid flow as revealed by the movement of polypropylene beads across the epidermis. Shown is a summary of four independent experiments (n ≥ 25). Error bars, SEM; t test; *, P < 0.002. (G) nphp4-deficient Xenopus embryos showed a mild perturbation of basal body polarization, revealed by the relative position of the basal bodies (RFP-Centrin) and the ciliary rootlets (GFP-Clamp). A moderate dose of 6.8 ng nphp4 SB MO was injected to allow apical docking of basal bodies, a prerequisite for polarization. The right panels show the magnified images for the regions enclosed by white boxes. Only a small number of basal bodies were incorrectly polarized (asterisk) in nphp4-deficient cells. The polarization was quantified by angular measurements of Clamp/Centrin pairs. Depletion of nphp4 resulted in a moderate increase of circular standard deviation as compared with the control. Error bars, SEM. (H) Transmission electron microscopy detected occasional ciliary axonemes (white arrowhead, box 1), and a large number of basal bodies (black arrowheads, box 2) in the cytoplasm of nphp4-depleted Xenopus multiciliated cells. Bars: (A–E) 5 µm; (G) 1 µm; (H) 200 nm.|
|Figure 3. Nphp4 is essential for the organization of the subapical actin layer. (A) Serial confocal images for actin (phalloidin) and ciliary rootlets (GFP-Clamp) of cells with multiple motile cilia revealed that the subapical actin layer became irregular in nphp4-deficient Xenopus embryos, whereas apical actin pool remained unaffected. The depth of each optical section from the first section is shown on the left. A magnified image of the inset is shown on the right side (4× magnification). (B) Triple staining for actin (phalloidin, red in merge), ciliary rootlets (GFP-Clamp, green in merge), and basal bodies (γ-tubulin, blue in merge) revealed that actin filaments in the subapical actin layer connect a basal body with the ciliary rootlet of the neighboring basal body. In nphp4-deficient cells, the subapical actin layer was poorly nucleated and failed to provide the connection between basal bodies. Top panels show the maximum intensity projection of serial confocal images. Single optical sections at the level of the subapical actin pool (boxed area) are magnified and shown in the bottom panels (4.5× magnification). Right panels are higher magnification images of the single optical sections depicting the subapical actin organization (10.5× magnification). act, actin; bb, basal body; rtl, ciliary rootlet. Bars, 5 µm.|
|Figure 4. daam1-deficient multiciliated cells recapitulate the ciliary phenotypes of nphp4-deficient cells. (A) GFP-tagged Daam1 (green in merge) preferentially localized to a region distal to the basal body (RFP-Centrin, red in merge) at the apical surface. Maximum intensity projection (top) or 3D reconstruction (bottom) of confocal images is shown. (B) Confocal imaging of cilia (Ac-tub, red). As compared with the control, reduced number of cilia formed above the apical cell surface (membrane-associated GFP [mGFP], green) after injection of 12 ng daam1 MO. The apical cell surface is depicted by dashed white lines on the right panels. (C) The ciliogenesis defect was confirmed by scanning electron microscopy. (D) daam1-deficient Xenopus embryos show a decreased rate of cilia-driven fluid flow across the epidermis (four independent experiments with n > 45; error bars, SEM; t test; *, P = 0.0014). Depletion of daam1 was also associated with a polarity defect and an increase in circular SD (t test; *, P < 0.001). (E) Basal bodies in daam1-deficient multiciliated cells failed to migrate to the apical cell surface and remained in the cytoplasm. Basal bodies and ciliary rootlets were labeled with RFP-Centrin and GFP-Clamp. Maximum intensity projection (top) or projection in the x-z plane (bottom) of serial confocal images are shown. The apical cell surface is indicated by dashed white line. (F) The subapical actin layer displayed irregularities in daam1–deficient multiciliated cells. Ciliary rootlets were labeled with GFP-Clamp (green in merge) and actin stained with phalloidin (red in merge). (Top) Maximum intensity projection of serial confocal images. Single optical sections at the level of apical and subapical actin layer of the boxed area are shown magnified in the middle and bottom panels (4× magnification). Bars, 5 µm.|
|Figure 5. Inturned plays a role similar to Nphp4 for normal ciliogenesis, and basal body localization of Inturned requires Nphp4. (A) GFP-tagged INTURNED (green in merge) preferentially localized to a region distal to the basal body labeled by RFP-Centrin (red in merge). Maximum intensity projection (top) and 3D reconstruction (bottom) of confocal images are shown. The representative localization pattern is shown magnified in the inset (4.3× magnification). (B) Confocal imaging of cilia stained with anti–acetylated tubulin (Ac-tub). Reduced number of cilia formed above the apical cell surface after injection of 17 ng inturned MO. Maximum intensity projection (top) and the projection in the x-z plane (bottom) of confocal images are shown. Dashed line indicates the apical surface of cells as revealed by membrane-associated GFP (mGFP; green). (C) 8 ng inturned MO–injected embryos presented with a decreased rate of ciliary fluid flow across the epidermis. Shown is the summary of four independent experiments (n ≥ 39; t test; *, P = 0.0014). (D) Injection of 8 ng inturned MO resulted in a reduced nucleation of the subapical actin pool as well as fragmentation of apical actin web. Actin and ciliary rootlet were stained with phalloidin (red in merge) and GFP-Clamp (green in merge), respectively. (Top) Maximum intensity projection of serial confocal images. The area enclosed by white boxes was magnified (5.6×); single optical sections at the level of apical actin pool (middle) and subapical actin pool (bottom) are shown. Bars, 5 µm. (E) Localization of GFP-NPHP4 (green in merge) to the basal body (RFP-Centrin, red in merge) was only slightly affected by the depletion of inturned (7 ng MO). Areas enclosed by the white boxes are magnified in the insets (2.5×). Only a small subset of basal bodies in the inturned-deficient cells revealed a reduced accumulation of NPHP4 (asterisks). (F) Depletion of nphp4 (8 ng MO) resulted in a reduced colocalization of GFP-INTURNED (green in merge) with the basal bodies, and GFP-INTURNED revealed a more homogenous distribution in nphp4-deficient cells. (G) Whereas GFP-Daam1 was closely associated with the actin filaments in Xenopus epidermal cells microinjected with a control MO (ctl MO; Fig. S3 F), the depletion of nphp4 or inturned in Xenopus epidermal skin cells decreased both actin and GFP-Daam1 in the subapical region. The image depicts three z sections at a −600 nm in cells as indicated. Bars, 1 µm.|
References [+] :
Adler, Inturned localizes to the proximal side of wing cells under the instruction of upstream planar polarity proteins. 2004, Pubmed