bioimagesUnknownTakayuki YasunagaThe Journal of Cell Biologyhttps://www.ebi.ac.uk/biostudies/studies/S-JCBD-201502043JCB

Confocal microscopy revealing the reducued accumulation of GFP-INTURNED to the basal bodies (RFP-centrin) in nphp4 MO-treated multi-ciliated cell.

Confocal microscopy of the ctl MO-treated Xenopus multi-ciliated cell showing the dense actin cytoskeleton (Alexa Fluor 488-phalloidin) at the apical cell surface. This actin cytoskeleton is comprised of two distinct layers: the apical actin layer which forms a meshwork-like structure immediately below the apical plasma membrane, and the subapical actin layer which forms an evenly distributed filament-like structure. The ciliary rootlets were labeled by GFP-clamp.

Amino acids 863-1065 of NPHP4 are not sufficient for the localization to the basal bodies (RFP-centrin).

Confocal microscopy revealed reduced nucleation of the subapical actin network in inturned MO-treated multi-ciliated cell. Inturned depletion also affected the apical actin network. Ciliary rootlets and actin cytoskeleton were labeled by GFP-clamp and Alexa Fluor 568-phalloidin, respectively.

Confocal microscopy showing the normal localization of GFP-INTURNED at the basal bodies(RFP-centrin) in ctl MO-treated multi-ciliated cell.

N-terminal region of NPHP4 (amino acids 1-515) fails to localize to the basal body (RFP-centrin).

Confocal microscopy for multi-ciliated cell revealing the ciliogenesis defects in Daam1 MO-treated cells. Cilia and apical cell surface were labeled using an anti-acetylated tubulin antibody (red) and mGFP, respectively.

Confocal microscopy of the nphp4 ATG MO-treated Xenopus multi-ciliated cell revealed that basal bodies (RFP-centrin) remained within the cytoplasm below the apical actin cytoskeleton (Alexa Fluor 488-phalloidin). The apical actin cytoskeleton was also slightly thinner than in control cells.

Confocal microscopy showing the basal body localization of GFP-NPHP4 in ctl MO-treated multi-ciliated cell. Basal bodies were labeled by RFP-centrin.

Amino acids 1050-1426 of NPHP4 localize to the basal bodies (RFP-centrin).

Amino acids 863-1250 of NPHP4 is not confined to the basal body, but showed additional localization to the ciliary rootlets, co-localizing with RFP-clamp

Confocal microscopy for multi-ciliated cell showing normal ciliogenesis in control MO-treated cell. Cilia and apical cell surface were labeled using an anti-acetylated tubulin antibody (red) and mGFP, respectively.

Confocal microscopy revealed that subapical actin layer become irregular in nphp4 MO-treated multi-ciliated cell, while the apical actin layer remained unaffected. The actin cytoskeleton and ciliary rootlets were labeled by Alexa Fluor 568-phalloidin and GFP-clamp, respectively.

Confocal microscopy showing the apical localization of basal bodies in ctl MO-treated multi-ciliated cell. Basal bodies and ciliary rootlets were labeled by RFP-centrin and GFP-clamp, respectively.

Confocal microscopy showing normal ciliogenesis in ctl MO-treated multi-ciliated cell. Cilia and apical cell surface were labeled by an anti-acetylated tubulin antibody (red) and mGFP, respectively.

Scanning electron microscopy of the Xenopus epidermis showing the normal ciliogenesis in ctl MO-treated embryos.

The apical surface of the control multi-ciliated cells of the Xenopus epidermis is decorated with more than one hundred cilia (ac-tub). The apical cell surface is labeled by membrane-targeted GFP (mGFP).

Confocal microscopy revealed ciliogenesis defects in inturned MO-treated multi-ciliated cell. Cilia and apical cell surface were labeled by an anti-acetylated tubulin antibody (red) and mGFP, respectively.

After co-transfection of Flag-tagged capu and V5-tagged full length (FL) or truncations of NPHP4 (N4), IP with an anti-Flag antibody was performed. Both C-terminal and N-terminal domains of NPHP4 were co-precipitated with capu. Numbers indicate the first and last amino acid of the NPHP4 truncations.

C-terminal region of NPHP4 (amino acids 511-1426) is sufficient for the localization to the basal bodies (RFP-centrin).

Confocal microscopy showing that many of the basal bodies are remaining within the cytoplasm in Daam1 MO-treated multi-ciliated cell. Basal bodies and ciliary rootlets were labeled by RFP-centrin and GFP-clamp, respectively.

Confocal microscopy showing the association of GFP-NPHP4 to the apically migrating basal bodies (RFP-centrin).

Confocal microscopy of the nphp4 MO-treated Xenopus multi-ciliated cell showing the minor perturbation of basal body polarization, as revealed by the relative position of the basal bodies (RFP-centrin) and the ciliary rootlets (GFP-clamp).

V5-tagged human NPHP4 was co-transfected with Flag-tagged CD2AP (negative control), fly cappuccino (capu), human SPIRE, or human INTURNED (INTU) in HEK 293T cells. After immunoprecipitation (IP) with an anti-Flag antibody, NPHP4 co-precipitated with capu and INTU (WB, Western blot).

Flag-tagged INTU was co-expressed with V5-tagged NPHP4 and DAAM1. Both NPHP4 and DAAM1 co-precipitated with INTU.

High speed confocal microscopy showing the fast and coordinated beating of cilia (mGFP) in ctl MO-treated multi-ciliated cell.

C-terminal half of NPHP4 (amino acids 863-1426) is sufficient for the localization to the basal bodies (RFP-centrin).

Confocal microscopy showing the co-localization of GFP-fused Lifeact (Lifeact-GFP) with the Alexa Fluor 568-phalloidin-labeled F-actin structure in multi-ciliated cell of the fixed Xenopus embryo.

Flag-tagged DAAM1 was co-expressed with V5-tagged NPHP4 and INTU. After IP with anti-Flag, INTU co-precipitated with DAAM1. However, NPHP4 co-precipitated only in the presence of INTU with DAAM1.

Amino acids 1225-1426 of NPHP4 localize to the basal bodies (RFP-centrin).

Amino acids 1225-1426 of NPHP4 showed an enhanced membrane association. Basal bodies are labeled by RFP-centrin.

Confocal microscopy showing the apical and subapical actin organization in ctl MO-treated multi-ciliated cell. Ciliary rootlets and actin cytoskeleton were labeled by GFP-clamp and Alexa Fluor 568-phalloidin, respectively.

The C-terminal half of NPHP4 contains two distinct subdomains targeting NPHP4 to the basal bodies (RFP-centrin): amino acids 863-1250 and amino acids 1251-1426.

Confocal microscopy showing that the dense actin network deposited at the apical cell surface of multi-ciliated cells was comprised of two distinct layers: 1) the apical actin layer that forms meshwork-like structure immediately below the apical plasma membrane, and 2) the subapical actin layer that forms evenly distributed filament-like structures aproximately one micrometer below the apical actin layer. Actin cytoskeleton, basal bodies, and ciliary rootlets were labeled using Alexa Fluor 568-phalloidin, an anti-gamma tubulin antibody (light blue), and GFP-clamp, respectively.

High speed confocal microscopy revealed reduced motility and uncoordinated beating of cilia (mGFP) in nphp4 MO-treated multi-ciliated cell.

Confocal microscopy of the Xenopus multi-ciliated cell revealed that basal bodies (RFP-centrin) failed to migrate to the apical cell surface and remained within the cytoplasm in nphp4 SB MO-treated embryos. Apical cell surface was labeled by mGFP.

Confocal microscopy of the ctl MO-treated Xenopus multi-ciliated cell showing that apically migrated basal bodies (RFP-centrin) are embedded in the dense actin cytoskeleton (Alexa Fluor 488-phalloidin) deposited at the apical cell surface.

Confocal microscopy for the multi-ciliated cells shows that GFP-Daam1 accumulated at the apical cell surface. At least some proportion of GFP-Daam1 localized to the transition zone, immediately distal of the basal body (RFP-centrin).

Depletion of nphp4 by a translation-blocking MO (nphp4 ATG MO) targeting nphp4 mRNA resulted in a reduction of intact cilia that penetrated the surface of cell. Apical cell surface is labeled by mGFP.

Amino acids 1050-1250 of NPHP4 are not sufficient for the localization to the basal bodies (RFP-centrin).

Confocal microscopy revealing reduced nucleation of the subapical actin network in Daam1 MO-treated multi-ciliated cell. The apical actin network remained unaffected. Ciliary rootlets and actin cytoskeleton were labeled by GFP-clamp and Alexa Fluor 568-phalloidin, respectively.

Confocal microscopy of the ctl MO-treated Xenopus multi-ciliated cell showing the polarization of basal bodies, as revealed by the relative position of the basal bodies (RFP-centrin) and the ciliary rootlets (GFP-clamp).

Time-lapse confocal microscopy in combination with Lifeact-GFP revealing the dynamic rearrangement of actin cytoskeleton in multi-ciliated cell at the initial stage of ciliogenesis. Basal bodies were labeled by RFP-centrin.

Confocal microscopy revealed that the localization of GFP-NPHP4 to the basal body (RFP-centrin) was not affected by the depletion of inturned.

Confocal microscopy for actin cytoskeleton (Alexa Fluor 568-phalloidin), ciliary rootlets (GFP-clamp), and basal bodies (gamma-tubulin). In nphp4-deficient cells, subapical actin was poorly nucleated and failed to provide the connection between the neighboring basal bodies.

Scanning electron microscopy of the Xenopus epidermis revealed the ciliogenesis defects in nphp4 SB MO-treated embryos

Confocal microscopy showing that GFP-INTURNED preferentially localizes to the transition zone, distal to the basal bodies (RFP-centrin).

Confocal microscopy of Xenopus multi-ciliated cells showing the localization of GFP-NPHP4 to the basal bodies (RFP-Centrin).

Amino acids 1251-1324 of NPHP4 are not sufficient for the localization to the basal bodies (RFP-centrin).

Confocal microscopy of the Xenopus multi-ciliated cell showing that basal bodies (RFP-centrin) were apically migrated and distributed across the apical cell surface in cto MO-treated embryos. Apical cell surface was labeled by mGFP.

Confocal microscopy showed that basal bodies (RFP-centrin) were embedded in a dense actin network (Alexa Fluor 488-phalloidin) deposited at the apical cell surface of mature multi-ciliated cell.

Flag-tagged NPHP4 and INTU were co-expressed with V5-tagged human DAAM1. After IP with anti-Flag, DAAM1 co-precipitated only in the presence of INTU

Confocal microscopy in combination with injection of mRNA encoding for GFP-NPHP4 (200 pg) revealed the broad localization of GFP-NPHP4 extending from the basal body (RFP-centrin) to the presumptive transition zone distal to the basal body.

Scanning electron microscopy of the Xenopus epidermis revealed the ciliogenesis defects in nphp4 ATG MO-treated embryos.

Confocal microscopy for actin cytoskeleton (Alexa Fluor 568-phalloidin), ciliary rootlets (GFP-clamp), and basal bodies (gamma-tubulin). In control cells, actin filaments in the subapical actin layer connect a basal body with the ciliary rootlet of the neighboring basal body.

Confocal microscopy in combination with lower dose injection of mRNA for GFP-NPHP4 (70 pg) revealed a more confined localization of GFP-NPHP4 to the presumptive transition zone. Basal bodies were labeled by RFP-centrin.

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