Expression of dominant-negative EB1 or EB3 inhibits cilia formation in RPE cells. RPE cells stably expressing GFP-tagged full-length (-FL) or C-terminal EB (-C) constructs were grown to confluency and serum starved for 72 hours. (A) Western blot analysis of lysates of GFP-EB1-FL (clones 1 and 2) and GFP-EB1-C using EB1, GFP or p150^Glued (loading control) antibodies. (B,C) Similar analysis was performed for cell lines expressing GFP-tagged C-terminal EB2 (B) or GFP-tagged FL or C-terminal EB3 (C). Asterisks in panel B indicate extra bands that are probable degradation or aggregation products resulting from overexpression of the GFP-EB2-C fusion protein. Although the EB3 antibody used in panel C (Komarova et al., 2005) crossreacts with EB1 and three additional bands at ~55–70 kDa, siRNA depletion demonstrated its specificity in IFM analysis (Fig. 1Bv,vi). (D) Western blots of RPE cell lines using monoclonal antibody against GFP to compare fusion protein levels. (E) Quantification of cilia in the RPE cell lines. A GFP-expressing RPE cell line was used as control (see Fig. 3C for western blot). The analysis was done as in Fig. 1C. Error bars display the s.d. *, significantly different from GFP expressing control (P<0.001). (F) Relative molar levels of native or GFP-tagged EB1 or EB3, as determined by quantitative western blot with EB antibodies (see Materials and Methods for details). Molar levels are presented relative to native EB3 levels.

Expression of GFP-EB1-FL prevents inhibition of cilia formation upon depletion of EB3. RPE cells stably expressing GFP or GFP-tagged EB constructs (Fig. 2) were treated with siRNA, as indicated, grown to confluency and serum starved for 72 hours. (A,B) Western blots of cell lysates using antibodies as indicated. (C) RPE cell lines not treated with siRNA were analyzed by western blot with monoclonal antibody against GFP to compare GFP fusion protein levels. (D) Quantification of cilia in cells treated like those shown in A and B. Error bars display the s.d. #, significantly different from GFP-expressing cells treated with EB3 siRNA (GFP, EB3 siRNA) (P<0.05). (E) FACS analysis of untransfected RPE cells and clones GFP-EB1-FL1 and GFP-EB3-FL showing the fluorescence intensities of GFP. GFP-EB3-FL displays an approximately twofold higher average GFP fluorescence level than GFP-EB1-FL1.

EB3 localizes to the tip of motile cilia of bronchial epithelial cells. Human bronchial epithelial cells were fixed with cold methanol followed by 4% formaldehyde in PBS. The cells were stained with antibodies against EB3 (green) and acetylated α-tubulin (red) to label cilia; nuclei were visualized with DAPI (blue). The image shows a side view of a single cell. Note the presence of EB3 at the tip as well as the base of cilia.

Ultrastructural analysis of hFF cells treated with mock or EB1-specific siRNA. TEM analysis of growth-arrested control (mock; A–C) or EB1-depleted (D–I) hFF cells. (A) Longitudinal section of a basal body (bb) and attached primary cilium. Transition fibers (tf; thin double arrows), subdistal appendages (sda; thick arrows) and rootlet filaments (rf; small arrowheads) are visible. In the cilium pit two vesicles (v; large arrowheads) are present. (B,C) Grazing longitudinal sections of basal bodies. Microtubules (MT; thin arrows) radiating in a fan-like pattern from the subdistal appendages (thick arrows) are seen. (D,F) Consecutive longitudinal serial sections of a basal body bearing a stumpy cilium. Subdistal appendages (D–F; thick arrows) and transition fibers (D,E; thin double arrows) can be seen. In D, a coated vesicle constricting from the cilium-pit is visible (large arrowhead). (G–I) Consecutive longitudinal serial sections of a basal body with attached stumpy cilium. Three subdistal appendages can be seen, one of which is attached to the centriole in a more median to proximal position (H,I; thick arrows at the right side of the centriole) than the other two (G,I; thick arrows). Scale bars: A, 500 nm; I, 500 nm (applies to B–I).

Ultrastructural defects in cells lacking EB3. TEM analysis of growth-arrested, EB3-depleted hFF cells. (A) Longitudinal section of a mother centriole with subdistal appendages (sda; thick arrow). At the distal end of the centriole three vesicles are visible (v; large arrowheads). Rootlet filaments (rf) are labeled (small arrowheads). (B–D) Consecutive serial longitudinal sections of a basal body with a stumpy cilium. Vesicles (large arrowheads) are visible within the cilium. In D, the oblique sectioned daughter centriole, subdistal appendages (thick arrows) attached to the basal body and rootlet filaments (small arrowheads) can be seen. (E,F) Consecutive serial longitudinal sections of a basal body with stumpy cilium. Vesicles (large arrowheads) are accumulated within the cilium. (G) Longitudinal section of a basal body bearing a stumpy cilium. The cilium pit shows a long, tubular extension (large double arrowheads). The oblique sectioned daughter centriole is visible to the right. (H–J) Consecutive longitudinal serial sections of a basal body with stumpy cilium. The cytoplasmic area around the tip of the cilium looks distorted and many vesicles (large arrowheads) are present. In J, vesicles (large arrowhead at the bottom of the panel) can be seen, which are localized in the region of the attachment site between the transition fibers (tf; thin double arrows in I) and the membrane of the cilium pit. (K–M) Consecutive serial cross sections of a mother centriole. The transition fibers (tf; thin double arrows) attached to the distal end of the centriole are clearly visible (K). At least six subdistal appendages are associated with the centriole in a subdistal position (L, sda; thick arrow); one additional subdistal appendage is associated with the centriole in a more median to proximal position (M, thick arrow). (N) Longitudinal section of a basal body. At the left side of the centriole two subdistal appendages (sda; thick arrows) are associated, above each other, with the centriole; at the right side only one subdistal appendage (thick arrow) is present. Scale bars: J, 500 nm (applies to A-J); N, 200 nm (applies to K–N).

Cells lacking EB3 contain abnormally long rootlet filaments. (A–F) TEM analysis of growth-arrested, EB3-depleted hFFs. (A–C) Consecutive serial cross sections of a centriole displaying an elaborate and extended rootlet filament (rf) system (small arrowheads). The second centriole is sectioned in an oblique angle. (D–F) Consecutive longitudinal serial sections of both centrioles with the basal body bearing a stumpy cilium. Both centrioles display an elaborate system of rootlet filaments (small arrowheads) attached to their proximal end. Scale bar: 500 nm. (G) IFM of siRNA-treated, serum-starved hFFs stained with anti-rootletin antibody (Bahe et al., 2005). Abnormally long rootlets are seen in EB3-depleted cells (arrows in vii-ix). (H) Different RPE cell lines (see Fig. 2) analyzed by IFM with rootletin antibody. Unfocused rootletin staining (arrows) is seen in cell lines expressing GFP-EB1-C or GFP-EB3-C. Similar dispersal of phosphorylated β-catenin staining was observed in the same cell lines (data not shown). Scale bar: 3 μm (applies to G and H). (I,J) Quantification of the results shown in G and H, respectively. The x-axis depicts area of rootletin staining in μm², whereas the y-axis indicates percent of cells (44–61 cells analyzed per sample). In I, mock-, EB1 siRNA- and EB3 siRNA-treated cells are represented by black, light gray and dark gray columns, respectively. In J, GFP-, GFP-EB1-C and GFP-EB3-C expressing cells are represented by black, light gray and dark gray columns, respectively. Note that depletion of EB3 (I) or expression of GFP-EB1-C or GFP-EB3-C (J) causes an increase in the area of rootletin staining in many cells.