Journal: Journal of Cell Science

Article Title: Epb41l5 interacts with Iqcb1 and regulates ciliary function in zebrafish embryos

doi: 10.1242/jcs.240648

Figure Lengend Snippet: EPB41L5 interaction with IQCB1. (A) The domain structure of EPB41L5 and deletion constructs used in this study. EPB41L5 contains the FERM and FA domains at the N terminus, and the evolutionarily conserved domain (CTD) at the C terminus. (B) The domain structure of IQCB1 and deletion constructs used in this study. IQCB1 contains three IQ motifs, one coiled-coil domain (CC) and the CEP290-interacting domain at the C terminus. (C) Identification of a domain in Epb41l5 required for its interaction with IQCB1. Myc-Epb41l5 and FLAG-IQCB1 were transiently expressed in HEK293 cells. After 24 h, cell lysates were immunoprecipitated by anti-FLAG antibody. The N-terminal FERM domain of Epb41l5 was required and sufficient for its interaction with IQCB1. Bands corresponding to these exogenously expressed proteins are marked by asterisks. (D) Identification of a domain in IQCB1 required for its interaction with EPB41L5. Myc-EPB41L5 and HA-IQCB1 were transiently expressed in HEK293 cells. After 24 h, cell lysates were immunoprecipitated by anti-HA antibody. The domain between amino acids 287 and 443 was required and sufficient for its interaction with EPB41L5. Bands corresponding to these proteins are marked by asterisks. HC, heavy chain; LC, light chain.

Article Snippet: Erythrocyte protein band 4.1 like 5 (EPB41L5) is an adaptor protein that contains the FERM (band 4.1, ezrin, radixin and moesin) domain ( Baines, 2006 ; Moleirinho et al., 2013 ; Tepass, 2009 ) and regulates morphogenesis of epithelial and neuroepithelial cells.

Techniques: Construct, Immunoprecipitation

Journal: Journal of Cell Science

Article Title: Epb41l5 interacts with Iqcb1 and regulates ciliary function in zebrafish embryos

doi: 10.1242/jcs.240648

Figure Lengend Snippet: EPB41L5 suppresses IQCB1 association with the ciliary base in hTERT-RPE1 cells. (A) Subcellular localization of exogenously expressed Epb41l5 in hTERT-RPE1 cells. Epb41l5 was mainly localized at the plasma membrane. Enlarged images of a portion of A are shown in a–a″. Epb41l5 did not colocalize with a ciliary protein ARL13B. (B) Subcellular localization of exogenously expressed IQCB1 in hTERT-RPE1 cells. Enlarged images of a portion of B are shown in b–b″. IQCB1 accumulated at the base of cilia labeled by ARL13B. (C) Coexpression of Epb41l5 reduced accumulation of IQCB1 at the ciliary base. Enlarged images of a portion of C are shown in c–c″ . C′ shows localization of FLAG-Epb41l5. (D) Knockdown of epb41l5 by shRNA promoted the accumulation of IQCB1 at the ciliary base. shRNA-expressing cells were labeled with GFP. Enlarged images of a portion of D are shown in d–d″. D′ shows expression of memb-GFP as a marker of epb41l5 shRNA expression. (E) Efficacy of epb41l5 shRNAs. Two independent epb41l5 shRNAs were used. epb41l5 expression was quantified by RT-qPCR. Data are mean±s.d. of n=3 experiments. (F,G) Quantification of HA-IQCB1 accumulation at the ciliary base. HA-IQCB1 was expressed alone or coexpressed with the full-length or deletion mutants of Epb41l5 (F), or coexpressed with epb41l5 shRNA alone or with exogenous Epb41l5 for shRNA rescue (G). The relative integrated fluorescence intensity in the surrounding area of the ciliary base was quantified using an ImageJ plug-in. Box indicates the interquartile range (IQR) and whiskers indicate inner fences (±1.5×IQR), the horizontal bar shows the median. **P<0.01; n.s., not significant. Scale bars: 5 μm.

Article Snippet: Erythrocyte protein band 4.1 like 5 (EPB41L5) is an adaptor protein that contains the FERM (band 4.1, ezrin, radixin and moesin) domain ( Baines, 2006 ; Moleirinho et al., 2013 ; Tepass, 2009 ) and regulates morphogenesis of epithelial and neuroepithelial cells.

Techniques: Clinical Proteomics, Membrane, Labeling, Knockdown, shRNA, Expressing, Marker, Quantitative RT-PCR, Fluorescence

Journal: Journal of Cell Science

Article Title: Epb41l5 interacts with Iqcb1 and regulates ciliary function in zebrafish embryos

doi: 10.1242/jcs.240648

Figure Lengend Snippet: Identification of domains required for IQCB1 localization control by Epb41l5. (A,B) Full-length IQCB1 was coexpressed with the N-terminal FERM-FA domain of Epb41l5 (A) or the C-terminal fragment of Epb41l5 (B). The accumulation of IQCB1 at the ciliary base was examined. Enlarged images of portions of A–A″ and B–B″ are shown in a–a″ and b–b″, respectively. The N-terminal FERM-FA domain suppressed IQCB1 accumulation at the ciliary base, whereas the C-terminal fragment did not alter IQCB1 localization. (C,D) IQCB1Δ(287–443) was expressed alone (C) or coexpressed with full-length Epb41l5 (D). Enlarged images of portions of C–C″ and D–D″ are shown in c–c″ and d–d″, respectively. Although IQCB1Δ(287–443) accumulated at the ciliary base, Epb41l5 coexpression did not alter the IQCB1Δ (287–443) accumulation. (E) IQCB1(287–443) did not accumulate at the ciliary base. Enlarged images of portions of E–E″ are shown in e–e″. (F) Quantification of IQCB1Δ(287–443) accumulation at the centrosome. IQCB1Δ(287–443) was expressed alone or coexpressed with Epb41l5. The relative integrated fluorescence intensity in the surrounding area of the centrosome was quantified using an ImageJ plug-in. Box indicates the interquartile range (IQR) and whiskers indicate inner fences (±1.5×IQR), the horizontal bar shows the median. n.s., not significant. Scale bars: 5 μm.

Article Snippet: Erythrocyte protein band 4.1 like 5 (EPB41L5) is an adaptor protein that contains the FERM (band 4.1, ezrin, radixin and moesin) domain ( Baines, 2006 ; Moleirinho et al., 2013 ; Tepass, 2009 ) and regulates morphogenesis of epithelial and neuroepithelial cells.

Techniques: Control, Fluorescence

Journal: Journal of Cell Science

Article Title: Epb41l5 interacts with Iqcb1 and regulates ciliary function in zebrafish embryos

doi: 10.1242/jcs.240648

Figure Lengend Snippet: Abnormal cilia and LR patterning defects in epb41l5-deficient embryos. (A,B) Cilia in the pronephric duct in moeb476 mutants. Cilia and the pronephric epithelium were labeled using GFP-Arl13b and anti-β-catenin immunostaining, respectively. In wild-type embryos, pronephric cilia formed bundles and individual cilia were not distinguishable (A,A′). Although cilia formed in moeb476 mutants, individual cilia were easier to distinguish (B,B′). (C,D) KV cilia were immunostained with acetylated tubulin (AcTub). Cilia formation was normal in epb41l5-MOATG morphants. (E,F) KV epithelium was labeled with membrane-tethered GFP (memb-GFP) and nuclear dye DAPI. Formation was normal in epb41l5-MOATG morphants. (G,H) Randomized LR patterning in epb41l5-MOATG and epb41l5-MOSpD morphants. spaw expression was examined by in situ hybridization. Individual embryos were scored as either left, right, bilateral or absent. **P<0.01. Scale bars: 10 μm (A–F), 200 μm (G).

Article Snippet: Erythrocyte protein band 4.1 like 5 (EPB41L5) is an adaptor protein that contains the FERM (band 4.1, ezrin, radixin and moesin) domain ( Baines, 2006 ; Moleirinho et al., 2013 ; Tepass, 2009 ) and regulates morphogenesis of epithelial and neuroepithelial cells.

Techniques: Labeling, Immunostaining, Membrane, Expressing, In Situ Hybridization

Journal: Journal of Cell Science

Article Title: Epb41l5 interacts with Iqcb1 and regulates ciliary function in zebrafish embryos

doi: 10.1242/jcs.240648

Figure Lengend Snippet: epb41l5Δctd mutants show relatively normal epithelial morphogenesis. (A) Design of epb41l5Δctd gRNA and epb41l5-MOSpD morpholino. The exon–intron structure of the zebrafish epb41l5 gene from exon 24 to 26 is shown. The stop codon is located in exon 26. An arrowhead shows the exon–intron boundary of exon 25. Primers used for RT-PCR analysis, shown by arrows, are located in exon 24 and exon 26. (B) Three examples of gene editing in the genome of epb41l5Δctd mutants. The exon–intron boundary of exon 25 is shown by an arrowhead. (C) Analysis of epb41l5 transcripts in homozygous moeb476 mutant embryos, heterozygous epb41l5Δctd1 mutant embryos and epb41l5-MOSpD morphants. The lower band indicates epb41l5Δctd transcripts in which exon 25 is spliced out. All alleles of epb41l5Δctd mutants and epb41l5-MOSpD morphants generated the same epb41l5Δctd transcripts. (D) The predicted protein sequence of Epb41l5ΔCTD from epb41l5Δctd mutants and epb41l5-MOSpD morphants. The predicted protein sequence of Epb41l5ΔC60, a translation product of in vitro synthesized epb41l5Δc60 mRNA. Evolutionarily conserved amino acids are shown in red. Peptide sequences originated from mis-splicing of exon 25 and the subsequent frame-shift are shown in blue. (E–J) Gross morphology of homozygous moeb476 mutant embryos and homozygous epb41l5Δctd1 mutant embryos at 30 hpf (E–G) and 48 hpf (H–J). Gross morphology of epb41l5Δctd1 mutants is normal at 30 hpf (F). On the other hand, moeb476 mutants showed slight body curvature, failure of brain ventricle formation and pericardial edema at 30 hpf (arrowhead in G). At 48 hpf, epb41l5Δctd1 mutants showed slight body curvature and pericardial edema (arrowhead in I). moeb476 mutants showed severe body curvature, mosaic eye pigmentation and pericardial edema (arrowhead in J). (K–M) Retinal pigmentation at 48 hpf. epb41l5Δctd1 mutants did not show the ‘mosaic eyes’ phenotype (L) as seen in moeb476 mutants (M). (N-P) Formation of the brain ventricles and apico-basal polarity formation in the hindbrain. In the hindbrain of epb41l5-MOSpD morphants, brain ventricle inflation or accumulation of a tight junction protein ZO1 at the ventricular surface was not significantly altered (O) compared to wild type (N). On the other hand, moeb476 mutants failed to form brain ventricles aligned by ZO1 (P). Scale bars: 100 μm (E–G,K–M), 300 μm (H–J), 50 μm (N–P).

Article Snippet: Erythrocyte protein band 4.1 like 5 (EPB41L5) is an adaptor protein that contains the FERM (band 4.1, ezrin, radixin and moesin) domain ( Baines, 2006 ; Moleirinho et al., 2013 ; Tepass, 2009 ) and regulates morphogenesis of epithelial and neuroepithelial cells.

Techniques: Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Generated, Sequencing, In Vitro, Synthesized

Journal: Journal of Cell Science

Article Title: Epb41l5 interacts with Iqcb1 and regulates ciliary function in zebrafish embryos

doi: 10.1242/jcs.240648

Figure Lengend Snippet: Cilia abnormalities in embryos expressing Epb41l5ΔCTD. (A–C) KV cilia formed in control wild-type (stdMO) embryos (A), epb41l5-MOSpD morphants (B) and embryos expressing Epb41l5Δ60 (C). KV cilia were immunostained by anti-AcTub antibody at 14 hpf. (D–F′) Pronephric cilia formation. Pronephric cilia were immunostained by anti-AcTub antibody at 32 hpf. Pronephric cilia formed bundles in control wild-type embryos (D,D′). Although cilia formed, individual cilia were easier to distinguish in epb41l5-MOSpD morphants (E,E′) and embryos expressing Epb41l5ΔCTD (F,F′). (G–I) Pronephric cilia motility defects in epb41l5-MOATD (H) and epb41l5-MOSpD (I) morphants. epb41l5-MOATD or epb41l5-MOSpD morpholino were injected into embryos expressing Arl13b-GFP. At 32 hpf, live embryos were mounted in low-melting agarose. Cilia located at the posterior part of the pronephric duct were video-recorded on the confocal microscope with low-speed scanning. In wild-type control embryos (G), the confocal imaging failed to capture the movement of individual cilia, suggesting that cilia motility was high. See Movie 1. (H,I) In epb41l5-MOATD or epb41l5-MOSpD morphants, the confocal imaging captured the movement of individual cilia, suggesting that cilia motility is low. See Movies 2 and 3. (J) Representative images of cmlc2 expression at 28 hpf. The direction of heart jogging was scored as left jog, no jog or right jog. (K–M) LR patterning defects in epb41l5-MOSpD morphants (K), epb41l5Δctd mutants (L) and embryos expressing exogenous Epb41l5Δ60 (M). (N) Representative image of symmetric or asymmetric charon expression in KV. In wild-type embryos, charon expression becomes more asymmetric at 14 hpf. (O) charon expression was more symmetric in epb41l5-MOSpD morphants. Integrated density of charon expression in the left side and in the right side was quantified using ImageJ. If integrated density had more than 25% difference, we scored it as asymmetric. (P) Genetic synergy between epb41l5 and iqcb1 in LR patterning. The direction of the heart jogging was assessed. Single knockdown of iqcb1 or epb41l5 did not alter LR patterning. On the other hand, double knockdown of iqcb1 and epb41l5 resulted in LR patterning defects. (Q–T) Genetic synergy between epb41l5 and iqcb1 in body curvature. Compared to wild type (Q), single knockdown of iqcb1 or epb41l5 did not lead to body curvature (R,S). However, double knockdown of iqcb1 and epb41l5 resulted in severe body curvature (T). (U) Quantification of body curvature phenotype in iqcb1 and epb41l5 morphants. **P<0.01, *P<0.05; n.s., not significant. Scale bars: 10 μm (A–I), 100 μm (J), 50 μm (N), 300 μm (Q–T).

Article Snippet: Erythrocyte protein band 4.1 like 5 (EPB41L5) is an adaptor protein that contains the FERM (band 4.1, ezrin, radixin and moesin) domain ( Baines, 2006 ; Moleirinho et al., 2013 ; Tepass, 2009 ) and regulates morphogenesis of epithelial and neuroepithelial cells.

Techniques: Expressing, Control, Injection, Microscopy, Imaging, Knockdown

Journal: Journal of Cell Science

Article Title: Epb41l5 interacts with Iqcb1 and regulates ciliary function in zebrafish embryos

doi: 10.1242/jcs.240648

Figure Lengend Snippet: EPB41L5 overexpression reduces CEP290 accumulation at the centrosome. (A–D) EPB41L5 suppresses the CEP290–IQCB1 interaction. FLAG-CEP290, HA-IQCB1 and myc-EPB41L5 were transiently expressed in HEK293 cells. After 24 h, cell lysates (C) were immunoprecipitated either by anti-FLAG antibody (A) or anti-HA antibody (B). Co-immunoprecipitated proteins and lysate were analyzed by SDS–PAGE and western blotting. EPB41L5 coexpression reduced the amount of HA-IQCB1 co-immunoprecipitated by FLAG-CEP290 (A). EPB41L5 coexpression reduced the amount of FLAG-CEP290 co-immunoprecipitated by HA-IQCB1 (B). (D) Quantification of the band intensities in A–C. Band intensity of HA-IQCB1 and FLAG-CEP290 coexpressed with or without myc-EPB41L5 was quantified. (E–I) EPB41L5 suppresses CEP290 accumulation at the centrosome. Full-length EPB41L5 and deletion mutant forms of EPB41L5 were transiently expressed in hTERT-RPE1 cells. The localization of endogenous CEP290 was examined. Cells expressing memb-GFP (E,E′) or GFP-EPB41L5 (F–I′) were outlined using the Magic Wand tool in Adobe Photoshop. CEP290 localization was not altered in cells expressing control memb-GFP (E). Centrosomal localization of CEP290 was reduced in cells expressing full-length Epb41l5 (F), Epb41l5ΔCTD (G) or Epb41l5(FERM-FA) (H), but was not altered in cells expressing Epb41l5(Cfrag) (I). (J) Quantification of CEP290 accumulation at the centrosome in cells expressing full-length or deletion mutant forms of Epb41l5. CEP290 accumulation was categorized as either not affected or reduced. (K–M) Model showing how EPB41L5 could modulate ciliary function. (K) Summary of domains required for interaction between EPB41L5, IQCB1 and CEP290. Domains for the EPB41L5 and CEP290 interaction have not been identified. (L) EPB41L5 binding to IQCB1 or CEP290 promotes displacement of IQCB1 or CEP290 from the centrosome. Coexpression of either IQCB1 or CEP290 does not inhibit EPB41L5 association with CEP290 or IQCB1, respectively. (M) In wild-type embryos, CEP290 promotes IQCB1 association with the centrosome. Aberrant EPB41L5 activity suppresses IQCB1 and CEP290 interaction, which may further promote displacement of IQCB1 from the centrosome. This may lead to the misplacement of other centrosomal proteins required for ciliary functions, such as cilia motility. **P<0.01; n.s., not significant. Scale bars: 10 μm.

Article Snippet: Erythrocyte protein band 4.1 like 5 (EPB41L5) is an adaptor protein that contains the FERM (band 4.1, ezrin, radixin and moesin) domain ( Baines, 2006 ; Moleirinho et al., 2013 ; Tepass, 2009 ) and regulates morphogenesis of epithelial and neuroepithelial cells.

Techniques: Over Expression, Immunoprecipitation, SDS Page, Western Blot, Mutagenesis, Expressing, Control, Binding Assay, Activity Assay