Loss of Tedc1 or Tedc2 phenocopies loss of delta-tubulin or epsilon-tubulin

(A) Immunofluorescence staining of control (RPE1 p53−/−), Tedc1−/− (RPE1 p53−/−; Tedc1−/−), Tedc1 Rescued (RPE1 p53−/−; Tedc1−/−; Tedc1-Halotag-3xflag), Tedc2−/− (RPE1 p53−/−; Tedc2−/−), Tedc2 Rescued (RPE1 p53−/−; 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; Magenta: CP110. Images are maximum projections of confocal stacks. Scale bar: 5 um (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 centrioles in indicated cell types positive for SASS6 staining. Each condition was performed in triplicate, with n=200 cells scored for each. (D) Percent of centrioles in indicated cell types positive for CEP164 staining. Each condition was performed in triplicate, with n=100 cells scored for each. (E) TEM cross-section of a centriole in a G2-phase Tedc1−/−cell. (F) TEM cross-section of a centriole in a G2-phase Tedc2−/−cell. (G) Schematic of centriole formation and loss in control and Tedc1−/−or Tedc2−/− cells.

Tedc1 and Tedc2 localize to centrioles

(A) Immunofluorescence staining of Tedc1 rescue cell lines in G1, S/G2, and M. Images are maximum projections of confocal stacks. Blue: DAPI; Cyan: Centrin; Magenta: Tedc1 (localized with anti-Flag antibody); Yellow: SASS6. Scale bar: 5 um. (B) Immunofluorescence staining of Tedc2 rescue cell lines in G1, S/G2, and M. Images are maximum projections of confocal stacks. Blue: DAPI; Cyan: Centrin (localized with anti-GFP antibody recognizing GFP-centrin); Magenta: Tedc2 (localized with anti-V5 antibody); Yellow: SASS6. Scale bar: 5 um. (C) U-ExM of Tedc1 rescue cell lines, arranged by procentriole length. Cyan: Acetylated tubulin; Magenta: Tedc1 (localized with anti-Flag antibody). Confocal image stacks were deconvolved using Microvolution; single plane images shown. Scale bar: 1 um. (D) U-ExM of Tedc2 rescue cell lines, arranged by procentriole length. Cyan: Acetylated tubulin; Magenta: Tedc2 (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 um.

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 (localized with anti-Flag antibody). Scale bar: 5 um. (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 (localized with anti-V5 antibody). Scale bar: 5 um. (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. (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. (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.

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

(A) Lengths of expanded centrioles (um) from cells of the indicated cell cycle stages. Cells were synchronized in S/G2/M and S-phase cells were marked with PCNA. The expansion factor was approximately 4x (see Fig 4 - Supp fig 1). (B) U-ExM images of nuclei and centrioles from cells of the indicated cell cycle stages and genotypes, used for quantification in A). Nuclei were stained for PCNA, scale bar = 25 um. Cells in S phase had punctate PCNA staining. Centrioles were stained for acetylated tubulin, scale bar = 1 um. (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 (E) i) 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. Scale bar = 1 um. (ii) 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. Scale bar = 1 um. (F, G, H, I) U-ExM of centrioles in S and G2 phase stained for acetylated tubulin (cyan) or monoE (GT335, 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 2.8x relay and deconvolved with 10 iterations using Microvolution. Scale bars: 1 um.

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 (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 2.8x relay.

Genotyping of Tedc1−/− and Tedc2−/− mutant cell lines

(A) Gene structure of the Tedc1 locus in parental p53−/− 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 p53−/−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 p53−/− cells, the Tedc1−/− mutant, and Tedc1 Rescued (RPE1 p53−/−; Tedc1−/−; Tedc1-Halotag-3xflag) cells. Top: PCR for Tedc1. Bottom: PCR for Halotag. (D) Genotyping PCR of the Tedc2 locus in parental p53−/− cells, the Tedc1−/− mutant, and Tedc2 Rescued (RPE1 p53−/−; Tedc2−/−; Tedc2-V5-APEX2) cells. Top: PCR for Tedc2. Bottom: PCR for APEX2.

Specificity of Flag and V5 antibodies

(A) Immunofluorescence staining of p53−/− cells expressing Halotag-Flag - negative control for Fig 2A. Images are maximum projections of confocal stacks and were acquired with the same exposure settings as in Fig 2A. Blue: DAPI; Cyan: Centrin; Magenta: Tedc1 (localized with anti-Flag antibody); Yellow: SASS6. Scale bar: 5 um. (B) Immunofluorescence staining of p53−/− cells expressing V5-APEX2 - negative control for Fig 2B. Images are maximum projections of confocal stacks and were acquired with the same exposure settings as in Fig 2B. Blue: DAPI; Cyan: Centrin (localized with anti-GFP antibody recognizing GFP-centrin); Magenta: Tedc2 (localized with anti-V5 antibody); Yellow: SASS6. Scale bar: 5 um. (C) U-ExM of p53−/− cells expressing Halotag-Flag - negative control for Fig 2C. Cyan: Acetylated tubulin; Magenta: Tedc1 (localized with anti-Flag antibody). Confocal image stacks were deconvolved using Microvolution; single plane images shown. Images were acquired using the same parameters as Fig 2C. Scale bar: 1 um. (D) U-ExM of p53−/− cells expressing V5-APEX2 - negative control for Fig 2D. Cyan: Acetylated tubulin; Magenta: Tedc2 (localized with anti-V5 antibody). Confocal image stacks were deconvolved using Microvolution; single plane images shown. Images were acquired using the same parameters as Fig 2D. Scale bar: 1 um.

Tedc1 localization arranged by procentriole length and centriole orientation

U-ExM of Tedc1 rescue cell lines, arranged by procentriole length and centriole orientation. Procentriole lengths are indicated in each image (um). Cyan: Acetylated tubulin; Magenta: Tedc1 (localized with anti-Flag antibody). Confocal image stacks were deconvolved using Microvolution; single plane images shown. Scale bar: 1 um.

Tedc2 localization arranged by procentriole length and centriole orientation

U-ExM of Tedc2 rescue cell lines, arranged by procentriole length and centriole orientation. Procentriole lengths are indicated in each image (um). Cyan: Acetylated tubulin; Magenta: Tedc2 (localized with anti-V5 antibody). Confocal image stacks were deconvolved using Microvolution; single plane images shown. Scale bar: 1 um.

AlphaFold Multimer predictions

(A) AlphaFold Multimer prediction of TEDC1, TEDC2, TUBA1A, TUBB (B) AlphaFold-Multimer prediction from A) colored according to pLDDT. Very high: pLDDT > 90. High: 90 > pLDDT > 70. Low: 70 > pLDDT > 50. Very low: pLDDT <50 (C) Predicted align error of the AlphaFold-Multimer prediction from A). Expected position error (Angstroms) is graphed. (D) AlphaFold Multimer prediction of Xenopus TEDC1, TEDC2, TUBD1, TUBE1 (E) AlphaFold-Multimer prediction from E) colored according to pLDDT. Very high: pLDDT > 90. High: 90 > pLDDT > 70. Low: 70 > pLDDT > 50. Very low: pLDDT <50 (F) Predicted align error of the AlphaFold-Multimer prediction from D). Expected position error (Angstroms) is graphed.

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

(A, B, C, D, E, 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 um. Images were acquired with a Yokogawa CSU-W1 SoRA with 2.8x 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 Fig 4A and Fig 4B. Centriole widths were a mean of 1.0 um, corresponding to a four-fold expansion factor.

Total protein levels of centrosomal proteins are unchanged in mutant cells

(A) Western blot of control (RPE1 p53−/−), Tedc1−/−, Tedc2−/−, Tubd1−/−, Tube1−/−, Sas6−/− cell lysates, immunoblotted for SAS6. Total protein stain (Revert) serves as a loading control. (B) Western blot of control (RPE1 p53−/−), Tedc1−/−, Tedc2−/−, Tubd1−/−, Tube1−/− cell lysates, immunoblotted for STIL. Total protein stain (Revert) serves as a loading control. (C) Western blot of control (RPE1 p53−/−), Tedc1−/−, Tedc2−/−, Tubd1−/−, Tube1−/− cell lysates, immunoblotted for CPAP. Total protein stain (Revert) serves as a loading control. (D) Western blot of control (RPE1 p53−/−), Tedc1−/−, Tedc2−/−, Tubd1−/−, Tube1−/− cell lysates, immunoblotted for POC5. Total protein stain (Revert) serves as a loading control.