Yasunaga et al., http :// /cgi /content /full /jcb /DC1

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1 Supplemental material JCB Yasunaga et al., http :// /cgi /content /full /jcb /DC1 THE JOU RNAL OF CELL BIO LOGY Figure S1. Spatial and temporal expression of nphp4, and localization of its protein product in Xenopus. (A) RT-PCR was performed using the lysates from the embryos of indicated stages. Expression of nphp4 was detected at all the stages tested. The housekeeping gene odc was used as a control. No RT, without reverse transcriptase. (B) Expression of nphp4 was studied by whole-mount in situ hybridization. nphp4 was expressed in the neural folds (nf) and the presumptive eye (e) at stage 18. Bar, 0.5 mm. (C) At stage 32, nphp4 was expressed in the pronephric duct (pd), nephrostrome (ns), cloaca (c), somites (s), posterior spinal cord (sc), eye (e), branchial arch (ba), otic vesicle (ov), and fore-, mid-, and hindbrain (fb, mb, and hb, respectively). (D) In situ hybridization at stage 24 reveals the expression of nphp4 in the multiciliated cells of the Xenopus epidermis. The boxed region of the right top panel is magnified in D to demonstrate the typical punctate staining pattern of the multiciliated cells. The bottom panel shows the colocalization between nphp4 and acetylated tubulin (green). Expression of nphp4 was also detected in the brain (b), spinal code (sc), eye (e), olfactory placode (op), and cloaca (c). Bars: (top) 500 µm; (bottom) 50 µm; (D ) 100 µm. (E) At stage 32, when ciliogenesis was completed, GFP-NPHP4 (green in merge) colocalized with the basal bodies labeled by RFP-Centrin (red in merge). Maximum intensity projections of 3D confocal images are shown. Area indicated by white boxes is magnified (5.9 ) in the bottom panels. Bar, 5 µm. (F) Localization of GFP-NPHP4 was examined at stage 19, before the onset of ciliogenesis. Association of GFP-NPHP4 with apically migrating basal bodies was detected. Serial confocal images are projected in the x-z plane, with the apical cell surface indicated by a dashed white line. (right) Magnified image after processing the confocal dataset with Imaris software. Bar, 2 µm. S13

2 Figure S2. Analysis of the actin cytoskeleton in multiciliated cells. (A) GFP-tagged Nphp4 rescued the fluid flow defect caused by depletion of nphp4 in a dose-dependent fashion (t test; *, P < 0.05; **, P < 0.01). Error bars, SEM. (B) RT-PCR was performed to confirm the efficacy of the splice-blocking (SB) morpholino oligonucleotide (MO; un, uninjected; -RT, without reverse transcription; ctl, control, odc, ornithine decarboxylase). (C) Lifeact-GFP (green in merge) labels F-actin structures stained with phalloidin (red in merge). (D) At stage 20, when basal bodies (RFP-Centrin) started to reach the apical cell surface, the actin cytoskeleton was dynamically rearranged around the apically localized basal bodies, revealed by a time-lapse imaging for Lifeact-GFP. Maximum intensity projection of z series of time-lapse confocal dataset is shown. Area enclosed by white boxes is magnified in the bottom panels (3.5 magnification). (E) A single optical section at the level close to the apical cell surface is shown. Basal bodies start to reach the apical cell surface in the lower part of the cells (below the dashed line), where a dense actin cytoskeleton is nucleated. In contrast, fewer actin bundles are observed in the region devoid of basal bodies (above the dashed line). (F) At stage 31, when ciliogenesis was completed, basal bodies labeled by RFP-Centrin (red in merge) were enclosed in a dense network of actin cytoskeleton labeled by phalloidin (green in merge) at the apical surface of the cell. Maximum intensity projection (top) or projection in the x-z plane (middle) of confocal datasets are shown. Bottom panels show magnified image for the area enclosed by white boxes (6.7 magnification). (G) The cortical actin cytoskeleton is comprised of two distinct pools. The first pool forms a web-like structure just underneath the apical plasma membrane (apical), whereas the second (subapical) pool is characterized by evenly distributed short filament-like structures 1 µm below the first pool. Maximum intensity projections of serial confocal images are shown on the left panel. Single optical sections at the level of apical and subapical actin pool for the area enclosed by a black box are magnified (4.2 ) in the middle and right panels, respectively. Bars, 5 µm. (H) GFP-tagged Daam1 rescued the fluid flow defect caused by depletion of nphp4 in a dose-dependent fashion (t test; *, P < 0.05; ***, P < 0.001). Error bars, SEM. S14 JCB 2015

3 Figure S3. Identification of binding partners for NPHP4 and their functional analysis in multiciliated cells. (A) V5-tagged human NPHP4 was cotransfected with Flag-tagged CD2AP (negative control), fly cappuccino (capu), human SPI RE, or human INT URNED (INTU) in HEK 293T cells. After immunoprecipitation (IP) with an anti-flag antibody, NPHP4 coprecipitated with capu and INTU (WB, Western blot). (B) After cotransfection 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 coprecipitated with capu. Numbers indicate the first and last amino acid of the NPHP4 truncations. (C) Flag-tagged NPHP4 and INTU were coexpressed with V5-tagged human DAAM1. After IP with anti-flag, DAAM1 coprecipitated only in the presence of INTU. (D) Flag-tagged INTU was coexpressed with V5-tagged NPHP4 and DAAM1. Both NPHP4 and DAAM1 coprecipitated with INTU. (E) Flag-tagged DAAM1 was coexpressed with V5-tagged NPHP4 and INTU. After IP with anti-flag, INTU coprecipitated with DAAM1. However, NPHP4 coprecipitated only in the presence of INTU with DAAM1. (F) Embryos injected with formin-1 MO and formin-2 MOs presented only with a moderately decreased ciliary fluid flow across the epidermis at the doses tested (13 ng). A splice-blocking MO for formin-1 was designed against the intron 7 exon 7 boundary. The boundaries of intron 6 exon 7 and exon 11 intron 11 were targeted by formin-2 MO1 and MO2, respectively. Error bars, SEM. (G) High-resolution imaging revealed that GFP-tagged Daam1 (GFP-Daam1, green) is closely associated with actin filaments (white arrowheads, top) in the cortical actin cytoskeleton of Xenopus epidermal cells. However, not all actin filaments (labeled by phalloidin, blue) are decorated by GFP-Daam1 (white arrow, top). GFP-Daam1 is also found in the space in between basal bodies (labeled by RFP-Centrin, red) and surrounding actin filaments (white arrows, bottom). S15

4 Video 1. Beating of cilia in ctl MO-injected Xenopus embryos. Xenopus embryos were injected with ctl MO (8 ng) and mrna for membrane-targeted GFP (mgfp) at the four-cell stage. The beating of cilia, as revealed by mgfp fluorescence, was examined at stage 32 by a confocal line-scanning microscope (LSM 5 Duo-LIVE; Carl Zeiss). Frames were taken at a rate of 108 frames/s for 4.6 s. Fast and coordinated beating of cilia was observed. Video 2. Beating of cilia in nphp4 MO-injected Xenopus embryo. Xenopus embryos were injected with 8 ng nphp4 MO and mrna for mgfp at the four-cell stage. The beating of cilia was examined at stage 32 by a confocal line-scanning microscope (LSM 5 Duo-LIVE; Carl Zeiss). Frames were taken at a rate of 108 frames/s for 4.6 s. Reduced motility and uncoordinated beating of cilia were observed. Video 3. Dynamic rearrangement of the apical actin cytoskeleton in Xenopus multiciliated cells. At the four-cell stage, Xenopus embryos were injected with mrnas encoding Lifeact-GFP (green) and RFP-Centrin (red) to label actin cytoskeleton and basal bodies, respectively. Dynamic rearrangement of actin cytoskeleton around the basal bodies was examined at stage 20 using confocal laser scanning microscope (LSM 510; Carl Zeiss). Frames were taken every 39 s for 13 min. Selected stills are shown in Fig. S2 (B and C). Table S1. Localization of NPHP4 truncations in multiciliated cells NPHP4 BB Comment 1 1,426 + Basal body, transition zone Cytoplasmic 511 1,426 + Basal body 863 1,426 + Basal body 863 1,250 + Basal body, rootlet, plasma membrane 863 1,065 (+/ ) Faintly to basal body and rootlet 1,050 1,426 + Basal body, plasma membrane 1,050 1,250 Faintly to basal body and rootlet 1,225 1,426 + Basal body, plasma membrane 1,251 1,426 + Basal body 1,251 1,324 Cytoplasmic (?) The expression of each constructs was confirmed by Western blot. Summary of localization results: +, clear localization to the basal bodies (BB); (+/ ), weak localization around the basal bodies;, negligible localization to the basal bodies. Table S2. Primers for Xenopus nphp4 and odc Name Xenopus nphp4_forward (stage-specific expression) Xenopus nphp4_reverse (stage-specific expression) Xenopus nphp4_forward (morpholino effect check) Xenopus nphp4_reverse (morpholino effect check) Xenopus odc_forward Xenopus odc_reverse Sequence 5 -GCG ACGCG TAGAT CTGCT TGTTT CTCTAT-3 5 -GCT GCGGC CGCTA TAAGA TGTAC TAGAT GCC-3 5 -ATG AAGGA CTGGG AGAAT ATTTT TTCTC-3 5 -CAT TTGGA TCTTT CAGAG TTGGA TGC-3 5 -GTC AATGA TGGAG TGTAT GGATC-3 5 -TCC ATTCC GCTCT CCTGA GCAC-3 Depicted are the primer sequences used to isolate Xenopus nephrocystin-4 (nphp4) and ornithyldecarboxylase (odc) cdnas by RT-PCR. S16 JCB 2015

5 Table S3. Antisense MO sequences Name Sequence Targeting site ctl MO 5 -CCT CTTAC CTCAG TTACA ATTTA TA-3 nphp4 ATG MO 5 -ATA TTCTC CCAGT CCTTC ATCTT GA-3 Start of translation nphp4 SB MO 5 -TAG GGTTC AGGTC GTCTT ACCTC GT-3 Exon 2 intron 2 boundary formin-1 MO 5 -TCC AAAAA TCGGT ACATA CCTTG TT-3 Exon 7 intron 7 boundary formin-2 MO1 5 -GCT TGACA ACCTG ACAAA ATGAA CA-3 Intron 6 exon 7 boundary formin-2 MO2 5 -CTC TTGCA CAAAA TACTC ACATT GC-3 Exon 11 intron 11 boundary inturned MO 5 -TCC ATATC CCCCA GCAGC ACTTC TA-3 Start of translation daam1 MO 5 -GCC GCAGG TCTGT CAGTT GCTTC TA-3 a Start of translation Depicted are the MOs to deplete Xenopus nphp4, formin-1, formin-2, inturned and daam1. ctl, control MO; ATG, translation-blocking MO; SB, splice-blocking MO. a Habas et al., Reference Habas, R., Y. Kato, and X. He Wnt/Frizzled activation of Rho regulates vertebrate gastrulation and requires a novel Formin homology protein Daam1. Cell. 107: http ://dx.doi.org / /S (01) S17