Fig 2: Structured illumination microscopy (SIM) images and characterizations of a-taxilin and ?-taxilin at the centrosomes.(A) Immunostained a-taxilin (green) and centrin-3 (magenta) in RPE-1 cells transfected with ODF2-Scarlet (red). Scale bar, 1 µm. The cartoons to the right of each set of images graphically depict the merge images. (B) Immunostained ODF2 (red) and centrin-3 (magenta) in RPE-1 cells transfected with GFP-?-taxilin (green). Scale bar, 1 µm. (C) Immunostained a-taxilin (red) and ninein (magenta) in RPE-1 cells transfected with centrin-3-GFP (green). Scale bar, 1 µm. (D) Immunostained ?-taxilin (red) and ninein (magenta) in RPE-1 cells transfected with centrin-3-GFP (green). Scale bar, 1 µm. (E) Immunostained a-taxilin (red) and CEP170 (magenta) in RPE-1 cells transfected with centrin-3-GFP (green). Scale bar, 1 µm. (F) Immunostained ?-taxilin (red) and CEP170 (magenta) in RPE-1 cells transfected with centrin-3-GFP (green). Scale bar, 1 µm. (G–H) Schematic showing full-length (FL) and deletion mutants (?) of a-taxilin (G) and ?-taxilin (H). N, N terminus; M, middle; C, C terminus; +, positive; -, negative; SDA, subdistal appendages; PE, proximal end. (I) Immunofluorescence images of HA-tagged FL a-taxilin and deletion mutants (green) in (G) and CEP170 (red) in RPE-1 cells. Scale bar, 1 µm. (J) Immunofluorescence images of CEP170 (red) in RPE-1 cells transfected with GFP-tagged FL ?-taxilin and deletion mutants (green) in (H). Scale bar, 1 µm. Arrowheads in (I) and (J) show a-taxilin and ?-taxilin SDA localizations, respectively.

Fig 3: Models showing a-taxilin and ?-taxilin localization and assembly in the subdistal appendage (SDA).(A) A 3D model of a mother centriole, illustrating the localization of various SDA proteins, including ODF2, CCDC68, CCDC120, ?-taxilin, a-taxilin, ninein, and CEP170 in order of increasing diameter size. (B) Close view of the SDA structure from (A). (C) Model showing the hierarchical relationships of SDA proteins with ODF2 at the SDA root: (1) ODF2 recruits ?-taxilin to the SDA, which in turn hierarchically recruits a-taxilin and then CEP170; (2) CCDC68 is recruited to the SDA, which further recruits CEP170 (Huang et al., 2017); (3) ODF2 recruits ninein and CEP170 via CCDC120 (Huang et al., 2017); (4) ODF2 recruits ninein via TCHP (Ibi et al., 2011). DA, distal appendage.

Fig 4: Specific localizations of subdistal appendage (SDA) proteins, including a-taxilin and ?-taxilin.(A) Representative simulated emission depletion (STED) super-resolution images showing top views of the subdistal appendage (SDA) protein distribution patterns. For ODF2, a-taxilin, ninein and CEP170, RPE-1 cells were immunostained with appropriate antibodies. For CCDC68, CCDC120 and ?-taxilin, RPE-1 cells overexpressed with 3×FLAG-tagged CCDC68, CCDC120 or ?-taxilin full-length were immunostained with FLAG antibody. Scale bar, 500 nm. (B) Diameter analysis of SDA proteins showing ring size diameter. Data are Mean ± SD. n = 7, box = 25th and 75th percentiles. (C) Representative two-color STED super-resolution images showing side view of the SDA proteins (green) and the centriole proximal end protein C-Nap1 (red). Scale bar, 500 nm. (D) A scatter plot describing the distance of SDA proteins relative to C-Nap1. Data are Mean ± SD. n = 11, box = 25th and 75th percentiles. (E) Relative localization of SDA proteins in radial and lateral directions of the mother centriole. The upper dotted lines reveal the slanted arrangement of distal appendage (DA) and the lower dotted lines represent the triangular SDA structure, respectively. Data are Mean ± SD. Figure 2—source data 1.The diameter of subdistal appendage (SDA) proteins, including a-taxilin and ?-taxilin. Figure 2—source data 2.The longitudinal positions of subdistal appendage (SDA) proteins, including a-taxilin and ?-taxilin.

Fig 5: a-Taxilin recruits CEP170 to the subdistal appendage (SDA).(A) Simulated emission depletion (STED) images of RPE-1 cells immunostained with a-taxilin (green) and CEP170 (red). Scale bar, 500 nm. The cartoons to the right of the images graphically depict the merge images. (B) Schematic showing the full-length (FL) a-taxilin and the truncated mutants (N terminus, [M] Middle, deleted C terminus). +, positive; -, negative. (C) Lysates of HEK-293T cells co-overexpressing HA-tagged a-taxilin-FL or the indicated truncated mutants in (B) with 3×FLAG-CEP170 were immunoprecipitated (IP) with anti-FLAG and immunoblotted (IB) with anti-HA and anti-FLAG antibodies. EV, empty vector. (D) In vitro binding of GST-a-taxilin-M from (B) (expressed in E. coli and purified) with 3×FLAG-CEP170 (expressed in HEK-293T cells and purified). The GST-a-taxilin-M was stained with Coomassie brilliant blue (CBB) and the 3×FLAG-CEP170 was pulled down and IB using FLAG antibody. (E) Confocal images of immunostained CEP170 (red) and ?-tubulin (magenta) in control- or a-taxilin-siRNA treated RPE-1 cells, and those cells rescued with siRNA-resistant GFP-tagged a-taxilin-FL or the a-taxilin M2 deletion mutant (?261–300 aa) (green). Scale bar, 5 µm. (F) Comparisons of CEP170 fluorescence intensities at the centrosomes in (E). Statistical significance was determined with one-way ANOVA with three replicates. Data are Mean ± SEM. n > 100; *, p < 0.05; ***, p < 0.001; n.s., not significant. Figure 5—source data 1.Data of centrosomal CEP170 fluorescence intensity in control and a-taxilin siRNA treated RPE-1 cells, and rescued by overexpressed full-length a-taxilin or the a-taxilin M2 deletion mutant (Data provided as Mean ± SEM). Figure 5—source data 2.Full immunoblots labeled and unlabeled for Figure 5C and D.