TTC26/DYF13 is an intraflagellar transport protein required for transport of motility-related proteins into flagella
- University of California, San Francisco, United States
- University of Tokyo, Japan
- Texas A&M University, United States
- The Jikei University School of Medicine, Japan
- Osaka University, Japan
- Max Planck Institute for Heart and Lung Research, Germany
- Faculty of Science, Gakushuin University, Japan
Figures
Figure 1
TTC26 is a conserved ciliary protein.
(A) Phylogenetic tree of TTC26 in various ciliated organisms. All protein sequences were obtained from RefSeq (NCBI). The scale bar represents 0.1 substitutions per nucleotide site. (B) …
Figure 2 with 1 supplement
Knockdown of ttc26 in zebrafish induces shorter cilia and motility defect.
(A) Representative images of heart-looping orientations of control (Left: normal) and ttc26 knockdown (Right: reversed) fish at 2 dpf. myl7-GFP transgenic embryos were injected with control or ttc26 …
Figure 2—figure supplement 1
ttc26 knockdown zebrafish shows phenotypes typical of defective cilia.
(A) Expression of TTC26 in control and ttc26 knockdown zebrafish. 3 dpf embryos were lysed with 1× SDS sample buffer and detected by Western blotting with TTC26 and actin antibodies. Control, AUG-MO …
Figure 3
C. reinhardtii dyf13 mutant has short flagella and motility defects.
(A) Sequences of the DYF13 gene (RefSeq accession: XM_001698717) in wild-type (CC125) and dyf13 mutant cells. Red underlines show places of mutations. A one-base substitution (c.57C >T) does not …
Figure 4
TTC26/DYF13 is an IFT complex B protein.
(A) Kymographs of GFP-tagged mouse TTC26 and IFT88 moving in cilia of mouse IMCD3 cells. These kymographs were assembled from Videos 7 and 8. Bars: 10 s (horizontal), 5 μm (vertical). (B) The mean …
Figure 5
DYF13 is a part of IFT complex B.
(A) The sucrose density gradient fractions of wild-type flagella. Flagellar matrix was fractionated through a 10–25% sucrose density gradient. The sucrose density gradient fractions were separated …
Figure 6
DYF13 does not influence IFT particle movement.
(A) Kymographs of KAP-GFP in control and dyf13 mutant C. reinhardtii cells. These kymographs were assembled from Videos 9 and 10. Bars: 5 s (horizontal), 5 μm (vertical). (B) The mean IFT speeds of …
Figure 7
DYF13 does not influence localizations and amounts of other IFT proteins.
(A) Immunofluorescence images of wild-type and dyf13 mutant cells. The cells were stained with antibodies to acetylated tubulin (red) and IFT46, IFT70, and IFT122 (green). Bar: 10 μm. (B) Western …
Figure 8 with 1 supplement
dyf13 mutant reduces the levels of inner dynein arm components in its flagella.
(A) Elution patterns of inner-arm dyneins of oda1 single and oda1dyf13 double mutant C. reinhardtii cells. Inner-arm dyneins were extracted with high salt from outer-armless axonemes of oda1 and oda1…
Figure 8—figure supplement 1
dyf13 mutant does not change the levels of outer dynein arm components in its flagella.
(A) Elution patterns of outer-arm and inner-arm dyneins of wild-type and dyf13 mutant C. reinhardtii cells. The elution positions of each dyneins are indicated by αβ, γ (outer-arm), and a–g …
Videos
Video 1
Fluorescent videomicroscopy of fluid flow in the KV of control embryos.
A dorsal view of the KV from a live control embryo at 8–10-somite stage. Fluorescent beads were microinjected into the KV. Images were collected at 5 fps for a total duration of 1 min. Playback is …
Video 2
Fluorescent videomicroscopy of fluid flow in the KV of ttc26 knockdown embryos.
A dorsal view of the KV from a live ttc26 knockdown embryo at 8–10-somite stage. Fluorescent beads were microinjected into the KV. Images were collected at 5 fps for a total duration of 1 min. …
Video 3
High-speed DIC videomicroscopy of KV cilia from control embryos.
A dorsal view of the KV from a live control embryo at 8–10-somite stage. DIC imaging was performed on an inverted microscope with a high-speed camera. Images were collected at 1000 fps for a total …
Video 4
High-speed DIC videomicroscopy of KV cilia from ttc26 knockdown embryos.
A dorsal view of the KV from a live ttc26 knockdown embryo at 8–10-somite stage. DIC imaging was performed on an inverted microscope with a high-speed camera. Images were collected at 1000 fps for a …
Video 5
High-speed DIC videomicroscopy of C. reinhardtii dyf13 mutant cell.
DIC imaging was performed on an inverted microscope with a high-speed camera. Images were collected at 1000 fps for a total duration of 0.16 s. Playback is set at 20 fps (1/50 speed). The dyf13 …
Video 6
High-speed DIC videomicroscopy of C. reinhardtii wild-type cell.
DIC imaging was performed on an inverted microscope with a high-speed camera. Images were collected at 1000 fps for a total duration of 0.16 s. Playback is set at 20 fps (1/50 speed). The wild-type …
Video 7
Fluorescent videomicroscopy of TTC26-GFP in a cilium of IMCD3 cells.
Images were collected at 10 fps for a total duration of 1 min. Playback is set at 20 fps (2 × speed). TTC26-GFP moves bi-directionally along the cilium. Orientation of cilia in these movies was …
Video 8
Fluorescent videomicroscopy of IFT88-GFP in a cilium of IMCD3 cells.
Images were collected at 10 fps for a total duration of 1 min. Playback is set at 20 fps (2 × speed). IFT88-GFP moves bi-directionally along the cilium.
Video 9
Fluorescent videomicroscopy of KAP-GFP in C. reinhardtii control cell.
Images were collected at 20 fps for a total duration of 20 s. Playback is set at 20 fps (real-time speed). KAP-GFP moves toward the tip of flagella in the control fla3 mutant cell.
Video 10
Fluorescent videomicroscopy of KAP-GFP in C. reinhardtii dyf13 mutant cell.
Images were collected at 20 fps for a total duration of 20 s. Playback is set at 20 fps (real-time speed). KAP-GFP moves toward the tip of flagella in the dyf13 mutant cell.
Video 11
Fluorescent videomicroscopy of IFT27-GFP in C. reinhardtii control cell.
Images were collected at 20 fps for a total duration of 25 s. Playback is set at 20 fps (real-time speed). IFT27-GFP moves toward the tip of flagella in the control pf18 mutant cell.
Video 12
Fluorescent videomicroscopy of IFT27-GFP in C. reinhardtii dyf13 mutant cell.
Images were collected at 20 fps for a total duration of 25 s. Playback is set at 20 fps (real-time speed). IFT27-GFP moves toward the tip of flagella in the dyf13 mutant cell.
Additional files
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Supplementary file 1
Results of proteomic analyses.
(A) List of identified IFT proteins from tandem affinity purification of TTC26. (B) List of identified proteins from 2D-DIGE proteomic analysis of wild-type and dyf13 mutant flagella. Ratios 1 and 2 indicate the fold change in protein abundance quantified in each spot, with negative ratio indicating a reduction in quantity relative to wild-type flagella in each experiment.
- https://doi.org/10.7554/eLife.01566.025
