Expression and localization of two axonemal tubulins, TBA-5 and TBB-4 and characterization of their missense mutations. (a–b) Models of the tba-5 and tbb-4 gene. Two tba-5 missense mutations, qj14 and dyf-10, and the deletion mutation, tm4200 (a) and a tbb-4 missense mutation dyf-12(sa127) and a deletion mutation, tbb-4(OK1461) (b) are shown. (c–d) Inner (c) and outer (d) views of structure of predicted TBA-5 and TBB-4 heterodimer based on porcine brain tubulin dimer structure, 1JFF. The three point mutation sites (P360, A19 and L253) and the loop that contains P360 are shown in green. (e–h) Dyf assays of dpy-6 and dpy-6;tba-5(tm4200);tbb-4(OK1461) show no obvious defects in ciliary structure. Bar=10 μm. (i–j) A transgene tba-5p::tba-5::GFP is expressed in amphid neurons (i) and phasmid neurons (j) in tba-5(qj14). Bar=10 μm. (k–n) Cilium formation was rescued in amphids and phasmids of tba-5(qj14), by expression of the transgene, tba-5p::tba-5::GFP, demonstrated by the pseudocolor green in cilia and dendrites (k–l) and by the red color in Dyf assays (m–n). Bar=5 μm. (o–r) TBB-4::YFP restored the ciliary length of amphids (o) and phasmids (p) in tbb-4(sa127) and localized to the entire cilia nearly homogeneously. TBA-5::GFP restores the lengths of amphid (q) and phasmid (r) cilia in tba-5(qj14) and extended from the distal regions of middle segments to the distal tips of distal segments. The cartoons in the right depict the structure of the cilia and dendrites in amphids and phasmids. Bar=5 μm. Arrows point to transition zones with cilia oriented upward. A, axon; C, cilia; CB, cell body; D, dendrite.

Dynamics of axonemal MTs at the middle segment and distal segment tips. (a–c) Cilia expressing TBB-4::YFP were photobleached in different regions and recovery was recorded for entire cilia (a), tips of middle segments (b) and distal segments (c) in phasmids. In each case, images are shown before, at (0 s) and after photobleaching. The arrows point to the recovery regions. The cartoon at the upper left for each set represents the region of cilia that was analyzed; the photobleached region is shown by a black square and the region used for recovery analysis is shown by a red square. Bar=5 μm. (d–e) The kinetics of FRAP recovery at the tips of middle segments (d) and the distal segments (e) were fit with a single exponential equation (red line). The fluorescence intensity is normalized to the prebleach. (f) EBP-2::GFP proteins are more concentrated at the tips of middle segments and distal segments. Bar=5 μm. (g) A line scan along the cilia in (f) is shown. (h) Dynamics of EBP-2::GFP in dendrites where the EB1 homolog tracks the tips of the MTs. The arrows point to the comets. Bar=5 μm. (i) A kymograph of EBP-2::GFP comets from (h) is shown. Horizontal bar=10 μm and vertical bar=10 s. The labels are: BB, basal body (equivalent to TZ); CB, cell Body; D, dendrite; DS, distal segment; MS, middle segment; TZ, transition zone.

Characterization of the dyf-6, ift-81, ift-74, tba-5 and tbb-4 mutants. (a) Cartoon illustrating the structure of the phasmid endings. (b–c) The DYF-1::GFP marker was used to visualize the phasmid ciliary morphology of wild type, three IFT-B mutants (b) and two tubulin mutants (c). (d) The DYF-1::GFP marker was used to visualize the phasmid ciliary morphology of the double mutants, dyf-6;klp-11, ift-81;klp-11 and ift-74;klp-11, tba-5/klp-11, tbb-4/klp-11, demonstrating that dyf-6, ift-81 and ift-74 are distinct from tba-5(qj14) and tbb-4(tm324). (e) Four different markers were used to visualize the phasmid ciliary morphology in the tubulin mutants. In b-e, bar=5 μm. Arrows point to transition zones with cilia oriented upward. See also Figures S3, which shows the ciliary morphology of these mutants in the phasmid and amphids using other markers. (f) Electron micrographs of amphid middle segments in wild-type (left), tba-5(qj14) (middle), and tbb-4(sa127) (right) adult animals. Arrows in the wild-type section point to singlet microtubules that occur less frequently in tbb-4(sa127) mutants. (g) Same as (f) except the amphid distal segment is shown. White arrow heads point to the empty distal channel. In (f) and (g), bar=200 nm.

Tubulin point mutants are temperature sensitive. (a) Wild type and the two deletion mutants, tbb-4(OK1461) and tba-5(tm4200), were nearly 100% stained in the amphid and phasmid neurons at 15°C, 20°C and 25°C. tbb-4(sa127) and tba-5(qj14) worms were not stained when grown at 15°C. But at 25°C, around 50% of the amphids and 20% phasmids are stained while tba-5(dyf-10) has very little temperature effect. n indicates the number of amphids or phasmids. (b) Visualized with a TBB-4::YFP tubulin marker, tba-5(qj14) possessed only the middle segment of the amphid and phasmid cilia at 15°C, but full-length cilia could be seen in tba-5(qj14) at 25°C. Bar=5 μm. Arrows point to transition zones with cilia oriented upward. (c) Visualized with an OSM-6::GFP marker, tbb-4(sa127) possessed only the middle segment of the amphid and phasmid cilia at 15°C, but full-length cilia could be seen in tbb-4(sa127) at 25°C. Bar=5 μm. Arrows point to transition zones with cilia oriented upward. The wild type image of the TBB-4::YFP marker in b and c was the same as used in Fig. 1e and Fig. S3b.

Analysis of TBB-4::YFP transport rate in cilia. Kymographs of DYF-1::GFP and TBB-4::YFP in IFT assays under exactly same conditions except that TBB-4::YFP was photobleached with a mercury lamp before recording to reduce the background. (a) DYF-1::GFP represents the IFT transport in cilia, and the IFT tracks are clear and thick in the kymograph. (b) The tracks of TBB-4::YFP in cilia are faint and thin compared to IFT tracks, e.g. in (a). (c) For comparison, OSM-9::GFP, which is proposed to be transported by IFT was used as control. All the recorded movies were processed using the basic filters (Sharpen (High) and Low pass) before creating kymographs. K is the kymograph that was created along the cilia and K′ is a drawing of the kymograph lines in K. In (a–c), Horizontal bar=2.5 μm and vertical bar=5 s. (d–f) Modeling of MT dynamics in a cilium. Dynamic instability and the delivery of tubulin subunits via IFT can constrain the length fluctuations of MTs in both the middle (blue) and the distal (black) segments to a narrow range (d). In silico FRAP of the cilium shown in (d) for both the middle (e) and distal (f) segments indicates similar recovery curves to the experimental results (Figure 4d, e). The fluorescence intensity is normalized to the prebleach. The labels are: DS, distal segment; MS, middle segment.