Figure 5.
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.

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.

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