Figures and data in A delta-tubulin/epsilon-tubulin/Ted protein complex is required for centriole architecture

Figures
Tables
Additional files

5 figures, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements

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Loss of TEDC1 or TEDC2 phenocopies loss of delta-tubulin or epsilon-tubulin.

(A) Immunofluorescence staining of control (RPE1 *TP53^-/-*), *TEDC1^-/-* (RPE1 *TP53^-/-; TEDC1^-/-*), TEDC1 Rescued (RPE1 *TP53^-/-; TEDC1^-/-*; TEDC1-Halotag-3xflag), *TEDC2^-/-* (RPE1 *TP53^-/-; TEDC2^-/-*), TEDC2 Rescued (RPE1 *TP53^-/-; TEDC2^-/-*; TEDC2-V5-APEX2) cells. Top row: G1 stage cells with 2 centrioles. Bottom row: S/G2 stage cells with 4 centrioles. Blue: DAPI; Yellow: Centrin (CETN); Magenta: CP110. Images are maximum projections of confocal stacks. Scale bar: 5 µm (B) Centriole number counts of the indicated cell lines. Cells were either asynchronous, serum-starved for G0/G1, stained for PCNA for S-phase, synchronized with RO-3306 for G2/M, or mitotic figures were identified by DAPI staining. Each condition was performed in triplicate, with n=100 cells scored for each. (C) Percent of all centrioles (parental, pro-, and *de novo* centrioles) in indicated cell types positive for SASS6 staining. Each condition was performed in triplicate, with 200 cells scored for each. (D) Percent of all centrioles (parental, pro-, and *de novo* centrioles) in indicated cell types positive for CEP164 staining. Each condition was performed in triplicate, with 100 cells scored for each. (E) TEM cross-section of a centriole in a G2-phase *TEDC1^-/-* cell. Scale bar: 100 nm (F) TEM cross-section of a centriole in a G2-phase *TEDC2^-/-* cell. Scale bar: 100 nm (G) Schematic of centriole formation and loss in control and *TEDC1^-/-* or *TEDC2^-/-* cells.

Figure 1—source data 1 Raw data (centriole counts) for Figure 1B, C and D.: https://cdn.elifesciences.org/articles/98704/elife-98704-fig1-data1-v1.xlsx
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Figure 1—figure supplement 1

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Creation of *TEDC1^-/-* and *TEDC2^-/-* mutant cell lines.

(A) Gene structure of the TEDC1 locus in parental *TP53^-/-* cells and the *TEDC1^-/-* mutant. Green boxes: exons; blue lines: introns; red triangles: sgRNA binding sites; black arrow: translation start site. The *TEDC1^-/-* mutant (clone 2F4) is a compound heterozygote bearing a deletion of 227 bp on one allele and a deletion of 329 bp on the other allele. In both alleles, the ATG start site is deleted and the next ATG is not in-frame. (B) Gene structure of the TEDC2 locus in parental *TP53^-/-* cells and the *TEDC2^-/-* mutant. Green boxes: exons; blue lines: introns; red triangles: sgRNA binding sites; black arrow: translation start site. The *TEDC2^-/-* mutant (clone F5) is a compound heterozygote bearing a deletion of 19 bp on one allele flanking the ATG start site. On the other allele, there is an insertion of 306 bp corresponding to a fusion between TEDC2 and the hCLHC1 gene. In both alleles, the ATG start site is deleted, the next ATG is not in-frame, and no additional ATG start sites are found. (C) Genotyping PCR of the TEDC1 locus in parental *TP53^-/-* cells, the *TEDC1^-/-* mutant, and TEDC1 Rescued (RPE1 *TP53^-/-; TEDC1^-/-*; TEDC1-Halotag-3xflag) cells. Top: PCR for TEDC1. Bottom: PCR for Halotag. (D) Genotyping PCR of the TEDC2 locus in parental *TP53^-/-* cells, the *TEDC1^-/-* mutant, and TEDC2 Rescued (RPE1 *TP53^-/-; TEDC2^-/-*; TEDC2-V5-APEX2) cells. Top: PCR for TEDC2. Bottom: PCR for APEX2. (E) Western blot of TEDC1 protein levels in parental *TP53^-/-* cells, the *TEDC1^-/-* mutant, and TEDC1 Rescued (RPE1 *TP53^-/-; TEDC1^-/-*; TEDC1-Halotag-3xflag) cells. Total protein stain is used as a loading control. TEDC1-Halotag-3xFlag is overexpressed 73-fold above endogenous levels (average of three independent experiments). Asterisks mark non-specific bands. (F) Western blot of TEDC2 protein levels in parental *TP53^-/-* cells, the *TEDC2^-/-* mutant, and TEDC2 Rescued (RPE1 *TP53^-/-; TEDC2^-/-*; TEDC2-V5-APEX2) cells. Total protein stain is used as a loading control. TEDC2-V5-APEX2 is overexpressed 26-fold above endogenous levels (average of three independent experiments). Asterisks mark non-specific bands.

Figure 1—figure supplement 1—source data 1 Original files of full uncropped, unedited blots in Figure 1—figure supplement 1E and F.: https://cdn.elifesciences.org/articles/98704/elife-98704-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2 Labeled uncropped, unedited blots in Figure 1—figure supplement 1E and F.: https://cdn.elifesciences.org/articles/98704/elife-98704-fig1-figsupp1-data2-v1.zip
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Figure 1—figure supplement 2

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Symmetrization of *TEDC1^-/-* and *TEDC2^-/-* mutant centrioles.

Original (left) and symmetrized (right) images of TEM images of *TEDC1^-/-* and *TEDC2^-/-* centrioles. The top image is the same as that in Figure 1E, the bottom image is the same as that in Figure 1F. The middle image is an additional centriole from the *TEDC1^-/-* mutant cells. Scale bars: 10 nm.

Figure 2 with 1 supplement

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TEDC1 and TEDC2 localize to centrioles.

(A) Immunofluorescence staining of TEDC1 rescue cell lines expressing TEDC1-Halotag-3xFlag in G1, S/G2, and M. Images are maximum projections of confocal stacks. Blue: DAPI; Cyan: Centrin (CETN); Magenta: TEDC1-Halotag-3xFlag (localized with anti-Flag antibody); Yellow: SASS6. Scale bar: 5 µm. (B) Immunofluorescence staining of TEDC2 rescue cell lines expressing TEDC2-V5-APEX2 in G1, S/G2, and M. Images are maximum projections of confocal stacks. Blue: DAPI; Cyan: Centrin (CETN, localized with anti-GFP antibody recognizing GFP-centrin); Magenta: TEDC2-V5-APEX2 (localized with anti-V5 antibody); Yellow: SASS6. Scale bar: 5 µm. (C) U-ExM of TEDC1 rescue cell lines expressing TEDC1-Halotag-3xFlag, arranged by procentriole length. Cyan: Acetylated tubulin; Magenta: TEDC1-Halotag-3xFlag (localized with anti-Flag antibody). Confocal image stacks were deconvolved using Microvolution; single plane images shown. Scale bar: 1 µm. (D) U-ExM of TEDC2 rescue cell lines expressing TEDC2-V5-APEX2, arranged by procentriole length. Cyan: Acetylated tubulin; Magenta: TEDC2-V5-APEX2 (localized with anti-V5 antibody). Confocal image stacks were acquired with a Yokogawa CSU-W1 spinning disk microscope and deconvolved using Microvolution; single plane images shown. Scale bar: 1 µm.

Figure 2—figure supplement 1

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Extended localization analyses of TEDC1 and TEDC2.

(A) Immunofluorescence staining of a TEDC1 rescue cell in G2 phase expressing TEDC1-Halotag-3xFlag, super-resolution image using SoRA disk and 2.8 x relay. Maximum projection. Cyan: Centrin (CETN); Magenta: TEDC1-Halotag-3xFlag (localized with anti-Flag antibody); Yellow: SASS6. Scale bar: 0.5 µm (B) Immunofluorescence staining of a TEDC2 rescue cell in G2 phase expressing TEDC2-V5-APEX2, super-resolution image using SoRA disk and 2.8 x relay. Maximum projection. Cyan: Centrin (CETN); Magenta: TEDC2-V5-APEX2 (localized with anti-V5 antibody); Yellow: SASS6. Scale bar: 0.5 µm (C) Expansion microscopy image of TEDC1 rescue cells expressing TEDC1-Halotag-3xFlag. Expansion gel was made as described in Kong et al., 2024. The procentriole is oriented vertically. Cyan: CEP44; Magenta: TEDC1-Halotag-3xFlag (localized with anti-Flag antibody). Deconvolved using Microvolution; maximum projection. Scale bar: 1 µm. (D) Expansion microscopy image of TEDC2 rescue cells expressing TEDC2-V5-APEX2. Expansion gel was made as described in Kong et al., 2024. The procentriole is oriented vertically. Cyan: CEP44; Magenta: TEDC2-V5-APEX2 (localized with anti-V5 antibody). Deconvolved using Microvolution; maximum projection of confocal stacks. Scale bar: 1 µm. (E) Immunofluorescence staining of *TP53^-/-* cells expressing Halotag-Flag - negative control for Figure 2A. Images are maximum projections of confocal stacks and were acquired with the same exposure settings as in Figure 2A. Blue: DAPI; Cyan: Centrin; Magenta: Flag; Yellow: SASS6. Scale bar: 5 µm. (F) Immunofluorescence staining of *TP53^-/-* cells expressing V5-APEX2 - negative control for Figure 2B. Images are maximum projections of confocal stacks and were acquired with the same exposure settings as in Figure 2B. Blue: DAPI; Cyan: Centrin (localized with anti-GFP antibody recognizing GFP-centrin); Magenta: V5; Yellow: SASS6. Scale bar: 5 µm. (G) U-ExM of *TP53^-/-* cells expressing Halotag-Flag - negative control for Figure 2C. Cyan: Acetylated tubulin; Magenta: Flag. Confocal image stacks were deconvolved using Microvolution; single plane images shown. Images were acquired using the same parameters as Figure 2C. Scale bar: 1 µm. (H) U-ExM of *TP53^-/-* cells expressing V5-APEX2 - negative control for Figure 2D. Cyan: Acetylated tubulin; Magenta: V5. Confocal image stacks were deconvolved using Microvolution; single plane images shown. Images were acquired using the same parameters as Figure 2D. Scale bar: 1 µm. (I) Expansion microscopy image of *TP53^-/-* cells stained with Flag antibody, negative control for (C). Cyan: CEP44; Magenta: Flag. Confocal image stacks were deconvolved using Microvolution; image is a maximum projection of confocal stack. Scale bar: 1 µm. (J) Expansion microscopy image of *TP53^-/-* stained with V5 antibody, negative control for (D).Cyan: CEP44; Magenta: V5. Confocal image stacks were deconvolved using Microvolution; image is a maximum projection of confocal stack. Scale bar: 1 µm.

Figure 3 with 1 supplement

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TEDC1, TEDC2, TUBD1, TUBE1 form a complex in cells.

(A) Centrosomal TEDC1 localization depends on TEDC2, TUBD1, TUBE1. Immunofluorescence staining of cells expressing TEDC1-Halotag-3xFlag. Control cell is *TEDC1^-/-* mutant cells rescued with TEDC1-Halotag-3xFlag. Images are maximum projections of confocal stacks. Blue: DAPI; Cyan: SASS6; Magenta: TEDC1-Halotag-3xFlag (localized with anti-Flag antibody). Scale bar: 5 µm. (B) Centrosomal TEDC2 localization depends on TEDC1, TUBD1, TUBE1. Immunofluorescence staining of cells expressing TEDC2-V5-APEX2. Control cell is TEDC2 mutant cells rescued with TEDC2-V5-APEX2. Images are maximum projections of confocal stacks. Blue: DAPI; Cyan: SASS6; Magenta: TEDC2-V5-APEX2 (localized with anti-V5 antibody). Scale bar: 5 µm. (C) TEDC1 pulls down TEDC2 in the absence of delta or epsilon-tubulin. Western blot of input and pulldown of Halotag-Flag or TEDC2-Halotag-Flag in the indicated cell lines. Control cells are *TP53^-/-* cells expressing Halotag-3xFlag. IB: indicates the antibody used for immunoblotting. The proteins and their positions are labeled on the right. Asterisks mark non-specific bands. (D) TEDC2 pulls down TEDC1 in the absence of delta or epsilon-tubulin. Western blot of input and pulldown of TUBA1B-V5-APEX2 or TEDC2-V5-APEX2 in the indicated cell lines. Control cells are *TP53^-/-* cells expressing TUBA1B-V5-APEX2. IB: indicates the antibody used for immunoblotting. The proteins and their positions are labeled on the right. Asterisks mark non-specific bands. (E) AlphaFold-Multimer prediction of the complex (F) AlphaFold-Multimer prediction colored according to pLDDT. Very high: pLDDT > 90. High: 90 > pLDDT > 70. Low: 70 > pLDDT > 50. Very low: pLDDT <50 (G) Predicted align error of the AlphaFold Multimer prediction. Expected position error (Angstroms) is graphed.

Figure 3—source data 1 Original files of full uncropped, unedited blots in Figure 3C and D.: https://cdn.elifesciences.org/articles/98704/elife-98704-fig3-data1-v1.zip
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Figure 3—source data 2 Labeled uncropped, unedited blots in Figure 3C and D.: https://cdn.elifesciences.org/articles/98704/elife-98704-fig3-data2-v1.zip
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Figure 3—figure supplement 1

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AlphaFold-Multimer and AlphaFold3 predictions.

(Ai) Rotated view of the AlphaFold-Multimer prediction from Figure 3E (120 degrees around the y-axis) (**Aii**) Rotated view colored according to pLDDT. Very high: pLDDT > 90. High: 90 > pLDDT > 70. Low: 70 > pLDDT > 50. Very low: pLDDT <50 (**Aiii**) Rotated view of the AlphaFold-Multimer prediction from Figure 3E (240 degrees around the y-axis) (**Aiv**) Rotated view colored according to pLDDT. Very high: pLDDT >90. High: 90>pLDDT > 70. Low: 70>pLDDT > 50. Very low: pLDDT <50 (Bi) AlphaFold3 prediction of the complex (**Bii**) AlphaFold3 prediction colored according to pLDDT. Very high: pLDDT >90. High: 90>pLDDT > 70. Low: 70>pLDDT > 50. Very low: pLDDT <50 (**Biii**) Predicted align error of the AlphaFold3 prediction. Expected position error (Angstroms) is graphed. (**Biv**) Structural alignment between the AlphaFold3 prediction (magenta) and the AlphaFold-Multimer prediction (cyan). Using ChimeraX v1.7.1 Matchmaker, the RMSD between 450 pruned atom pairs is 0.538 angstroms (across all 475 pairs: 0.979). (Ci) AlphaFold-Multimer prediction of TEDC1, TEDC2, TUBA1A, TUBB (**Cii**) AlphaFold-Multimer prediction from (Ci) colored according to pLDDT. Very high: pLDDT >90. High: 90>pLDDT > 70. Low: 70>pLDDT > 50. Very low: pLDDT <50 (**Ciii**) Predicted align error of the AlphaFold-Multimer prediction from (Ci). Expected position error (Angstroms) is graphed. (Di) AlphaFold Multimer prediction of *Xenopus* TEDC1, TEDC2, TUBD1, TUBE1 (**Dii**) AlphaFold-Multimer prediction from (Di) colored according to pLDDT. Very high: pLDDT >90. High: 90>pLDDT > 70. Low: 70>pLDDT > 50. Very low: pLDDT <50 (**Diii**) Predicted align error of the AlphaFold-Multimer prediction from (Di). Expected position error (Angstroms) is graphed.

Figure 4 with 5 supplements

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Mutant centrioles elongate in G2 but fail to recruit central core proteins and have an expanded proximal region.

(A) Lengths of expanded centrioles from cells of the indicated cell cycle stages. Lengths were adjusted for the gel expansion factors. Cells were synchronized in S/G2/M and S-phase cells were marked with PCNA. For each genotype, the differences between S and G2 phase centriole lengths are statistically significant (p<0.0001, Welch’s t-test). (B) U-ExM images of centrioles stained for alpha-tubulin and acetylated tubulin. (C) U-ExM of centrioles in S or G2 phase stained with monoE (GT335) antibody. (D) U-ExM of control centrioles in S or G2 phase stained with acetylated tubulin and polyE antibodies (Ei) U-ExM of centrioles in G2-phase stained with acetylated tubulin (cyan) and POC5 (magenta) antibodies. POC5 is present in the central core of control procentrioles and *de novo* centrioles and absent from mutants. (**Eii**) U-ExM of centrioles in G2 phase stained with acetylated tubulin (cyan) and WDR90 (magenta) antibodies. WDR90 is present in the central core of control procentrioles and *de novo* centrioles, and absent from mutants. (**F–I**) U-ExM of centrioles in S and G2 phase stained for alpha tubulin (cyan) or acetylated tubulin (Ac Tub, cyan) and the following antibodies in magenta: (F) SASS6, (G) CEP135, (H) STIL, (I) CPAP. In control centrioles, these proteins are limited to the proximal end. In mutant centrioles, these proteins are present at the proximal end in S phase centrioles and elongate throughout the entire centriole in G2 phase. Images were acquired with a Yokogawa CSU-W1 SoRA with 2.8 x relay and deconvolved with 10 iterations using Microvolution. Scale bars: 1 µm.

Figure 4—source data 1 Data for Figure 4A.: https://cdn.elifesciences.org/articles/98704/elife-98704-fig4-data1-v1.xlsx
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Figure 4—figure supplement 1

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Extended analyses of mutant centriole architecture and U-ExM gel expansion factor.

(**A–F**) U-ExM of centrioles in S and G2 phase stained for acetylated tubulin (cyan) and the following proteins in magenta: (A) CEP44, (B) CETN2, (C) CP110, (D) CEP120, (E) gamma-tubulin, (F) CEP295. Scale bars = 1 µm. Images were acquired with a Yokogawa CSU-W1 SoRA with 2.8 x relay and deconvolved with 10 iterations using Microvolution. (G) Measurements of the widths of parental centrioles from each experiment as a readout of expansion factor, including the cell cycle analyses in Figure 4A and B. Centriole widths were a mean of 1.0 um, corresponding to a fourfold expansion factor.

Figure 4—figure supplement 1—source data 1 Data for Figure 4—figure supplement 1G.: https://cdn.elifesciences.org/articles/98704/elife-98704-fig4-figsupp1-data1-v1.xlsx
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Figure 4—figure supplement 2

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Total protein levels of centrosomal proteins are unchanged in mutant cells.

(A) Western blot of control (RPE1 *TP53^-/-*), *TEDC1^-/-*, *TEDC2^-/-, TUBD1^-/-, TUBE1^-/-, SASS6^-/-* cell lysates, immunoblotted for SASS6. Total protein stain (Revert) serves as a loading control. (B) Western blot of control (RPE1 *TP53^-/-*), *TEDC1^-/-*, *TEDC2^-/-, TUBD1^-/-, TUBE1^-/-* cell lysates, immunoblotted for STIL. Total protein stain (Revert) serves as a loading control. (C) Western blot of control (RPE1 *TP53^-/-*), *TEDC1^-/-*, *TEDC2^-/-, TUBD1^-/-, TUBE1^-/-* cell lysates, immunoblotted for CPAP. Total protein stain (Revert) serves as a loading control. (D) Western blot of control (RPE1 *TP53^-/-*), *TEDC1^-/-*, *TEDC2^-/-, TUBD1^-/-, TUBE1^-/-* cell lysates, immunoblotted for POC5. Total protein stain (Revert) serves as a loading control.

Figure 4—figure supplement 2—source data 1 Original files of full uncropped, unedited blots in Figure 4—figure supplement 2.: https://cdn.elifesciences.org/articles/98704/elife-98704-fig4-figsupp2-data1-v1.zip
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Figure 4—figure supplement 2—source data 2 Labeled uncropped, unedited blots in Figure 4—figure supplement 2.: https://cdn.elifesciences.org/articles/98704/elife-98704-fig4-figsupp2-data2-v1.zip
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Figure 4—figure supplement 3

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**Quantification of CEP135 centriolar localization through S and G2 phases**.

Mutant centrioles have over-elongated CEP135. (A) control procentrioles, n=29 centrioles; (B) *de novo* centrioles, n=42 centrioles; (C) *TUBD1^-/-*, n=32 centrioles; (D) *TUBE1^-/-*, n=30 centrioles; (E) *TEDC1^-/-*, n=23 centrioles; (F) *TEDC2^-/-*, n=36 centrioles. For each panel, representative U-ExM images of centrioles in S and G2 phase are shown. These are the same centrioles as shown in Figure 4G and were stained for alpha-tubulin (cyan), acetylated tubulin (yellow), and CEP135 (magenta). Scale bars = 1 µm. Graphs: Each column represents a centriole, for which the proximal and distal positions of CEP135 (magenta), acetylated tubulin (yellow) and alpha-tubulin (cyan) are displayed. Centrioles were arranged from shortest to longest. Numbers were adjusted for expansion factor. Lines of best fit were added for CEP135 position: control procentrioles (dashed), *de novo* centrioles (dotted), and mutants (solid).

Figure 4—figure supplement 3—source data 1 Data for Figure 4—figure supplement 3.: https://cdn.elifesciences.org/articles/98704/elife-98704-fig4-figsupp3-data1-v1.xlsx
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Figure 4—figure supplement 4

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Quantification of SASS6 centriolar localization through S and G2 phase.

Mutant centrioles have over-elongated SASS6. (A) control procentrioles, n=53 centrioles; (B) *de novo* centrioles, n=44 centrioles; (C) *TUBD1^-/-*, n=39 centrioles; (D) *TUBE1^-/-*, n=44 centrioles; (E) *TEDC1^-/-*, n=34 centrioles; (F) *TEDC2^-/-*, n=30 centrioles. Each column represents a centriole, for which the proximal and distal positions of SASS6 (magenta) and alpha-tubulin (cyan) are displayed. Centrioles were arranged from shortest to longest. Numbers were adjusted for expansion factor. Lines of best fit were added for SASS6 position: control procentrioles (dashed), *de novo* centrioles (dotted), and mutants (solid).

Figure 4—figure supplement 4—source data 1 Data for Figure 4—figure supplement 4.: https://cdn.elifesciences.org/articles/98704/elife-98704-fig4-figsupp4-data1-v1.xlsx
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Figure 4—figure supplement 5

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Quantification of centriole widths and lengths.

Mutant centrioles have smaller widths compared to controls. (A) control procentrioles, n=82 centrioles; (B) *de novo* centrioles, n=86 centrioles; (C) *TUBD1^-/-*, n=64 centrioles; (D) *TUBE1^-/-*, n=74 centrioles; (E) *TEDC1^-/-*, n=56 centrioles; (F) *TEDC2^-/-*, n=62 centrioles. Centriole widths and lengths measured by alpha-tubulin antibody are graphed, adjusted for expansion factor. A line of best fit was added (red).

Figure 4—figure supplement 5—source data 1 Data for Figure 4—figure supplement 5.: https://cdn.elifesciences.org/articles/98704/elife-98704-fig4-figsupp5-data1-v1.xlsx
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Figure 5

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Mutant centrioles elongate further in mitosis before fragmenting.

U-ExM of centrioles stained for monoE (GT335, cyan), CP110 (yellow) and SASS6 (magenta). (A) A prometaphase cell with centrioles formed *de novo* after centrinone washout (B) An anaphase cell with centrioles formed *de novo* (C) A prometaphase *TUBD1^-/-* cell (D) A telophase *TUBD1^-/-* cell (E) A prometaphase *TUBE1^-/-* cell (F) An anaphase *TUBE1^-/-* cell. Scale bars: 1 um. Images were acquired with a Yokogawa CSU-W1 SoRA with 2.8 x relay.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Chemical compound, drug	Sodium acrylate	AK Scientific or Sigma Aldrich	AK Sci cat# R624, Sigma cat# 408220	There is batch to batch variability in acrylate purity
Chemical compound, drug	40% Acrylamide	Sigma Aldrich	Cat# A4058
Chemical compound, drug	36–38% Formaldehyde	Sigma Aldrich	Cat# F8775
Chemical compound, drug	N,N′-Methylenebisacrylamide solution (BIS)	Sigma Aldrich	Cat# M1533
Chemical compound, drug	Ammonium Persulfate (APS)	Bio-Rad	Cat# 1610700
Chemical compound, drug	N,N,N',N'-Tetramethylethylenediamine (TEMED)	Bio-Rad	Cat# 1610800
Chemical compound, drug	Sodium dodecyl sulfate (SDS)	Sigma Aldrich	Cat# 75746
chemical compound, drug	Sodium chloride (NaCl)	Sigma Aldrich	Cat# S9888
Chemical compound, drug	Tris base	Sigma Aldrich	Cat# 93362
Chemical compound, drug	Potassium chloride (KCl)	Sigma Aldrich	Cat# P3911
Chemical compound, drug	Triton X-100	Sigma Aldrich	Cat# T8787
Chemical compound, drug	DL-Dithiothreitol (DTT)	Sigma Aldrich	Cat# D9779
Chemical compound, drug	Glycine	Thermo Fisher Scientific	Cat# BP381-5
Chemical compound, drug	Tween 20	Sigma Aldrich	Cat# P1379
Antibody	Anti-acetlyated- tubulin, clone 6-11B-1 (monoclonal mouse IgG2b)	Sigma Aldrich	Cat# T6793, RRID:AB_477585	UExM (1:1000)
Antibody	Anti-acetyl-alpha-tubulin, Lys40 (rabbit polyclonal)	Cell Signaling Technology	Cat# 5335, RRID:AB_10544694	UExM 1:100
Antibody	Anti-centrin, clone 20H5 (monoclonal mouse IgG2a)	EMD Millipore	RRID:AB_10563501	IF 1:200, UExM 1:500
Antibody	Anti-centrin3, clone 3e6 (monoclonal mouse IgG2b)	Novus Biological	RRID:AB_537701	UExM 1:1000
Antibody	Anti-CENPJ (rabbit polyclonal)	Proteintech	Cat# 11517–1-AP, RRID:AB_2244605	WB 1:1000 UExM 1:500
Antibody	Anti-Cep44 (rabbit polyclonal)	Proteintech	Cat# 24457–1-AP, RRID:AB_2879557	UExM 1:100
Antibody	Anti-Cep120 (rat polyclonal)	Gift from Moe Mahjoub		Betleja et al., 2018
Antibody	Anti-Cep135 (rabbit polyclonal)	Proteintech	Cat# 24428–1-AP, RRID:AB_2879543	UExM 1:500
Antibody	Anti-Cep295 (rabbit polyclonal)	Sigma-Aldrich	Cat# HPA038596, RRID:AB_10672720	UExM 1:1000
Antibody	Anti-CP110 (rabbit polyclonal)	Proteintech	Cat# 12780–1-AP, RRID:AB_10638480	IF 1:200, UExM 1:2000
Antibody	Anti-Flag, clone M2 (monoclonal mouse IgG1)	Sigma-Aldrich	Cat# F1804, RRID:AB_262044	WB 1:2000, UExM 1:500
Antibody	Anti-gamma-tubulin, clone GTU-88 (monoclonal mouse IgG1)	Sigma-Aldrich	RRID:AB_477584	IF 1:1000, UExM 1:500
Antibody	Anti-PCNA (monoclonal mouse IgG2a)	BioLegend	RRID:AB_314692	UExM 1:500
Antibody	Anti-POC5 (rabbit polyclonal)	Bethyl Laboratories	RRID:AB_10949152	IF 1:500
Antibody	Anti-POC5 (rabbit polyclonal)	Thermo Fisher Scientific	Cat# A303-341A, RRID:AB_10971172	WB 1:1000 UExM 1:500
Antibody	Anti-polyglutamylation, clone GT335 (monoclonal mouse IgG1)	AdipoGen	Cat# AG-20B-0020, RRID:AB_2490210	UExM 1:500
Antibody	Anti-polyglutamylate-chain, polyE (rabbit polyclonal)	AdipoGen	Cat# AG-25B-0030, RRID:AB_2490540	UExM 1:500
Antibody	Anti-SASS6 (monoclonal mouse IgG2b)	Santa Cruz	Cat# sc-81431, RRID:AB_1128357	IF, WB, UExM 1:200
Antibody	Anti-STIL (rabbit polyclonal)	Abcam	Cat# ab89314, RRID:AB_2197878	WB 1:2000 UExM 1:500
Antibody	Anti-V5 (monoclonal mouse IgG2a)	Thermo Fisher Scientific	Cat# R960-25, RRID:AB_2556564	WB 1:1000 UExM 1:100
Antibody	Anti-WDR90 (rabbit polyclonal)	Thermo Fisher Scientfic	Cat# PA5-61943, RRID:AB_2649628	UExM 1:100
Antibody	Goat anti-mouse IgG1, 488	Thermo Fisher Scientific	Cat# A21121, RRID:AB_2535764	UExM 1:1000
Antibody	Goat anti-Mouse IgG2a, 488	Thermo Fisher Scientific	Cat# A-21131, RRID:AB_2535771	UExM 1:1000
Antibody	Goat anti-Mouse IgG2b, 488	Thermo Fisher Scientific	Cat# A-21141, RRID:AB_2535778	UExM 1:1000
Antibody	Goat anti-rabbit IgG (H+L), 488	Thermo Fisher Scientific	Cat# A-11034, RRID:AB_2576217	UExM 1:1000
Antibody	Goat anti-Mouse IgG1, 568	Thermo Fisher Scientific	Cat# A-21124, RRID:AB_2535766	UExM 1:500
Antibody	Goat anti-Mouse IgG2a, 568	Thermo Fisher Scientific	Cat# A-21134, RRID:AB_2535773	UExM 1:500
Antibody	Goat anti-Mouse IgG2b, 568	Thermo Fisher Scientific	Cat# A-21144, RRID:AB_2535780	UExM 1:500
antibody	Goat anti-rabbit IgG (H+L), 568	Thermo Fisher Scientific	Cat# A-11036, RRID:AB_10563566	UExM 1:500
Antibody	Goat anti-Mouse IgG3, 594	Thermo Fisher Scientific	Cat# A-21155, RRID:AB_2535785	UExM 1:500
Antibody	Goat anti-rat IgG (H+L), 594	Thermo Fisher Scientific	Cat# A-11007, RRID:AB_10561522	UExM 1:500
Antibody	Goat anti-Mouse IgG1, 647	Thermo Fisher Scientific	Cat# A-21240, RRID:AB_2535809	UExM 1:500
Antibody	Goat anti-Mouse IgG2a, 647	Thermo Fisher Scientific	Cat# A-21241, RRID:AB_2535810	UExM 1:500
antibody	Goat anti-Mouse IgG2b, 647	Thermo Fisher Scientific	Cat# A-21242, RRID:AB_2535811	UExM 1:500
Antibody	Goat anti-rabbit IgG (H+L), 647	Thermo Fisher Scientific	Cat# A32733, RRID:AB_2633282	UExM 1:500
Antibody	Rabbit Anti-TUBD1 (rabbit polyclonal)	Sigma-Aldrich	Cat# HPA027090, RRID:AB_1858457	WB 1:1000
Antibody	Rabbit Anti-TUBE1 (rabbit polyclonal)	Sigma-Aldrich	Cat# HPA032074, RRID:AB_10601216	WB 1:1000
Antibody	rabbit anti C14orf80 (rabbit polyclonal)	Sigma-Aldrich	Cat# HPA039049, RRID:AB_2676320	WB 1:1000
Antibody	rabbit anti C16orf59 (rabbit polyclonal)	Sigma-Aldrich	Cat# HPA055389, RRID:AB_2732595	WB 1:1000
Antibody	680 Donkey anti rabbit (H+L)	Thermo Fisher Scientific	Cat# A10043, RRID:AB_2534018	WB 1:20,000
Antibody	800 Donkey anti rabbit (H+L)	Li-COR	Cat# 926–32213, RRID:AB_621848	WB 1:20,000
Antibody	680 Donkey anti mouse (H+L)	Thermo Fisher Scientific	Cat# A10038, RRID:AB_11180593	WB 1:20,000
Antibody	800 Donkey anti mouse (H+L)	Li-COR	Cat# 926–32212, RRID:AB_621847	WB 1:20,000