Making the Connection: Ciliary Adhesion Complexes Anchor Basal Bodies to the Actin Cytoskeleton  Ioanna Antoniades, Panayiota Stylianou, Paris A. Skourides  Developmental Cell  Volume 28, Issue 1, Pages 70-80 (January 2014) DOI: 10.1016/j.devcel.2013.12.003 Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 FA Proteins FAK, Paxillin, and Vinculin Associate with Basal Bodies in Ciliated Cells (A and A′) Multiciliated cell expressing GFP FAK and centrin2 RFP. Each basal body is associated with a GFP FAK punctum at its posterior (in relation to the tadpole’s anterior-posterior axis). GFP FAK signal displays a gradient within the cell (in respect to the anterior-posterior axis), with elevated signal at the basal bodies localized at the cell’s posterior. (B and B′) Multiciliated cell expressing GFP Paxillin and centrin2 RFP. GFP Paxillin exhibits a similar localization and distribution throughout the cell, like GFP FAK. (C and C′) Multiciliated cell expressing GFP Vinculin and centrin2 RFP. Localization and distribution of GFP Vinculin resembles that of GFP FAK and GFP Paxillin. (D) Immunofluorescence staining with a p-S732 FAK antibody in centrin2 CFP-injected embryos. Endogenous FAK, associated with the basal bodies, is phosphorylated on Serine 732. (E) Immunofluorescence staining of endogenous Vinculin in centrin2 RFP-expressing multiciliated cells, gives a similar localization as GFP Vinculin. (F and F′) GRP ciliated cells of a stage 17 embryo expressing GFP FAK and centrin2 RFP, immunostained for acetylated tubulin. (G and G′) Neural tube cross section of a stage 24 embryo coexpressing GFP FAK and centrin2 RFP and immunostained for acetylated tubulin. GFP FAK associates with the basal bodies in both GRP and neural tube ciliated cells. (H and I) Immunofluorescence staining of endogenous Serine 732 phosphorylated FAK (H) and Paxillin (I) in NIH 3T3 cells, costained for γ tubulin and acetylated tubulin. Endogenous FAK and Paxillin localize at the base of primary cilia next to the basal body in mammalian ciliated cells. See also Figure S1. Developmental Cell 2014 28, 70-80DOI: (10.1016/j.devcel.2013.12.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 FA Proteins Present an Anterior-Posterior Gradient in Multiciliated Cells (A–B) Intensity color-coded maximum intensity projections of centrin2 RFP (A) and GFP Vinculin (A′) reveals the presence of a Vinculin gradient in multiciliated cells. The signal of centrin2 RFP is uniform (A), while the GFP Vinculin signal is stronger at the cell’s posterior (A′). In (B), multiciliated cells coexpressing GFP Vinculin and centrin2 RFP are shown. The anterior-posterior gradient is maintained at the tissue level. The arrows indicate cells’ posterior where the signal for GFP Vinculin appears elevated. (C) Multiciliated cell coexpressing mKate2 FAK, clamp GFP, and centrin2 CFP. FAK is localized next to the basal body at the end of the region marked by clamp GFP. (D and E) Confocal optical sections of a multiciliated cell showing that GFP Paxillin and mKate2 FAK colocalize. GFP Paxillin is more concentrated at the apical-most region of the basal bodies (D), while mKate2 FAK is more concentrated slightly below (E). (F–G′) Intensity coding of ROIs of (D) and (E). Paxillin exhibits highest intensity at 0.00 μm, as shown in (F) and (F′), whereas FAK exhibits highest intensity at 0.38 μm, as shown in (G) and (G′). Developmental Cell 2014 28, 70-80DOI: (10.1016/j.devcel.2013.12.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 FA Proteins Form CA Complexes Connecting Basal Bodies to the Actin Network (A) Basal bodies (labeled with centrin2 RFP) are docked at the apical surface of multiciliated cells, in the same plane as apical actin (visualized with GFP utrophin). Each basal body is found at the center of an actin ring. (B) mKate2 FAK is in contact with the apical actin network. (C) Coexpression of centrin2 RFP and GFP FAK followed by phalloidin staining suggests that FAK connects basal bodies to the apical actin network. (D and D′) Multiciliated cell expressing GFP FAK, marking CAs, and mKate2 actin, showing the apical actin network, before (D) and after (D′) acceptor photobleaching. (D″) mKate2 (acceptor) intensity drops after photobleaching, while GFP (donor) intensity rises, showing that FRET is taking place between GFP and mKate2 and suggesting that FAK is interacting with actin. (E and E′) Multiciliated cell expressing centrin GFP and mKate2 actin before (E) and after (E′) acceptor photobleaching. (E″) mKate2 intensity drops after photobleaching, but the GFP intensity remains unchanged, suggesting that no FRET is taking place between GFP and mKate2, indicating the absence of a direct interaction between centrin and actin. See also Figure S2. Developmental Cell 2014 28, 70-80DOI: (10.1016/j.devcel.2013.12.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 CA Complexes Link Basal Bodies and Ciliary Rootlets to the Subapical Actin Network (A–A″′) Optical sections of an intercalating multiciliated cell expressing GFP Paxillin and centrin2 RFP showing that Paxillin is associated with basal bodies during their migration to the apical surface. (B) 3D reconstruction (y-z) of optical sections of the intercalating cell in (A)–(A″′). (C and C′) Multiciliated cell expressing GFP FAK and stained with phalloidin. GFP FAK exhibits the highest density at two focal planes. The first one corresponds to the plane of the apical actin network (C), while the second one appears slightly below, at the plane of the subapical actin network (C′). GFP FAK is in close association with both pools of actin. (D) Maximum intensity projection of a region of a multiciliated cell coexpressing centrin2 RFP (to label basal bodies), clamp RFP (to label the striated rootlet), and GFP FAK. GFP FAK exhibits two intensity maxima: one at the level of the cell’s apical surface, adjacent to the basal bodies, and a second one deeper, at the end of the ciliary rootlets. (D′) Confocal image from a mechanically sectioned (along the apicobasal axis) multiciliated cell coexpressing centrin2 CFP, clamp RFP, and GFP FAK. The arrows mark the two maxima of GFP FAK, one associated with the basal body and one associated with the end of the striated rootlet. (E) Optical section of a multiciliated cell coexpressing centrin2 CFP and mKate2 FAK and labeled with phalloidin (apical surface is at the top). The cell was initially mechanically sectioned, approximately along its anterior-posterior axis. The subapical actin, known to connect each basal body with the end of the striated rootlet of the cilium behind (Werner et al., 2011), appears to project from the CAs (red-labeled with mKate2 FAK) and form the characteristic discontinuous network. (F) Optical section of a multiciliated cell expressing mKate2 FAK and labeled with phalloidin (apical surface is at the top). The cell was initially mechanically sectioned, approximately along its left-to-right axis. The subapical actin appears to connect CAs of neighboring basal bodies (along the left-right axis), creating continuous loops. Again, the subapical actin appears to project from the CAs. (G) Multiciliated cell coexpressing GFP FRNK and centrin2 RFP. GFP FRNK associates with the basal bodies in a similar way as the full-length protein. It is localized at the posterior site of each basal body and exhibits an anterior-posterior gradient within the cell. (H) Multiciliated cell coexpressing Paxillin C GFP and centrin2 RFP. Paxillin C GFP localizes at the posterior site of basal bodies and displays a gradient with respect to the anterior-posterior axis of the cell. Developmental Cell 2014 28, 70-80DOI: (10.1016/j.devcel.2013.12.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 FAK Knockdown Leads to Defects in Ciliogenesis (A–C) Surface view (maximum intensity projection) of the epidermis of stage 31/32 Xenopus tadpoles stained with phalloidin and immunostained for acetylated tubulin, which stain actin network and cilia, respectively. In (A), cells of control embryos appear normal, with a lot of cilia projecting from their apical surface. In (B), morphant cells (of embryos injected at one ventral blastomere, at stage 5, with 4 ng of FAK MO) have fewer cilia projecting outward from their surface. In (C), coinjection of HA FAK mRNA (90 pg) with the FAK MO (4 ng) rescues the phenotype and cells project cilia normally. (D) Surface view (maximum intensity projection) of the epidermis of a stage 31/32 Xenopus embryo stained with phalloidin and immunostained for acetylated tubulin. Coinjection of HA-FAK Δ375 (45 pg) mRNA with the FAK MO does not rescue the ciliogenesis defects. (E) Quantification of the FAK MO induced ciliogenesis defects. Bar chart shows the percentage of stage 31/32 embryos that present mild, moderate, and severe defects or are normal with respect to the number of cilia projecting toward their surface. Data were collected from 130–150 cells, from ten embryos (three experiments) of each category (control, MO injected, MO and HA FAK injected, and MO and HA FAK Δ375 injected). (F–I) 3D reconstruction (x-z) of optical sections of ciliated cells expressing centrin2 RFP and clamp GFP. In (F), most basal bodies have reached the apical surface but are not docked properly in cells injected with 4 ng of FAK MO. In (G), higher amounts of FAK MO (8 ng) lead to a more dramatic phenotype, as the majority of basal bodies fail to reach the apical surface of the cell. In (H), all the basal bodies are docked at the apical surface of a control ciliated cell. In (I), coinjection of HA FAK mRNA (120 pg) with the FAK MO (8 ng) rescues the phenotype, as the basal bodies have managed to reach the apical surface and the majority of them have docked properly. (J and K) Intercalating ciliated cells of stage 17 Xenopus embryos coexpressing centrin2 RFP and GFP utrophin to label basal bodies and the actin network, respectively. In control cells (J), basal bodies are clustered deep in the cell, surrounded by an actin network, and individual basal bodies associate with actin filaments. In cells coinjected with 8 ng of FAK MO (K), the basal bodies are dispersed, and many of them do not associate with actin, which fails to form an organized network around them. Developmental Cell 2014 28, 70-80DOI: (10.1016/j.devcel.2013.12.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 Apical Actin Enrichment and Rotational Polarity Are Unaffected in FAK Morphants (A–C) Confocal images of phalloidin-stained control (A), morphant (B), and rescued (C) ciliated cells expressing centrin2 RFP plus enlarged close-ups of selected regions, shown in (A′), (B′), and (C′), and a respective deeper optical section of each, in (A″), (B″), and (C″), showing details of apical and subapical actin networks. Morphants (B), unlike controls (A) and rescued (C) cells, display reduced numbers of basal bodies at the cell surface with large areas completely devoid of basal bodies. The apical actin network is present both in morphants and controls; however, it appears less organized and less dense in regions devoid of basal bodies. The subapical actin of morphants (B″) is completely missing in areas devoid of basal bodies and appears disorganized in areas with basal bodies. (D and E) Centrin2 RFP- and clamp GFP-expressing control (D) and FAK morphant (E) multiciliated cells. Spacing is disrupted, with regions of the apical surface devoid of basal bodies in the morphant cell. However, overall rotational polarity is not significantly affected as most rootlets are oriented in the same direction. See also Figure S3. Developmental Cell 2014 28, 70-80DOI: (10.1016/j.devcel.2013.12.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 CA Complexes in Multiciliated Cells (A) A diagram showing the localization of the CA complexes with respect to the basal bodies, ciliary rootlets, and the actin cytoskeleton in multiciliated cells. CA complexes interact with the apical and subapical pools of actin. The upper red circle shows an x-z projection of confocal optical sections of CA complexes and basal bodies. CAs are marked with GFP FAK (green) and the basal bodies with centrin2 RFP (red). (B) Proposed arrangement of the subapical actin network in multiciliated cells in relation to the basal bodies, rootlets, and CAs. Developmental Cell 2014 28, 70-80DOI: (10.1016/j.devcel.2013.12.003) Copyright © 2014 Elsevier Inc. Terms and Conditions