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. 2015 Aug 22;4:e07519. doi: 10.7554/eLife.07519

Figure 6. CEP90 encodes a centriolar satellite and MCPH-associated protein required to localize CEP63 to the centrosome to promote centriole duplication.

(A) HeLa total cell lysate was subjected to endogenous immunoprecipitation using antibodies to CEP63, CEP90 and a negative control. Precipitating proteins were analyzed by immunoblotting for CEP63 and CEP90. (B) Immunofluorescence images of SC and CEP90 #2 siRNA-treated HeLa cells co-stained for CEP63 (red) and Centrin (‘c’, green). (C) Total cell lysates of SC or CEP90 #2 siRNA transfected HeLa cells were analyzed by immunoblotting with antibodies to CEP63, CEP90 and Actin, which served as a loading control. Asterisk indicates the specific band. (D) S phase SC, CEP90 #1, and CEP90 #2 siRNA-treated cells were analyzed by immunofluorescence with antibodies to CEP90 (red) and Centrin (‘c’, green). (E) Total cell lysates of SC, CEP90 #1, and CEP90 #2 siRNA transfected HeLa cells were analyzed by immunoblotting for CEP90. α-tubulin served as a loading control. (F) Mean percentage of S phase SC, CEP90 #1, and CEP90 #2 siRNA-treated HeLa cells with four Centrin foci. At least 100 cells were counted per experiment (n = 3), p < 0.005 (paired t-test). (G) SC and CEP90 #2-depleted S phase cells were co-stained with Centrin (‘c’, green), CDK5RAP2 (red), CEP152 (red), WDR62 (red), and CEP63 (red). Scale bars indicate 5 μm for all images. (H) Schematic indicating that CEP90 is required for the centrosomal localization of CEP63. (I) Diagram of a simplified pedigree. Filled squares indicate individuals with MCPH. Spontaneous abortions (SAB), stillbirths (SB), and individuals of unknown gender (diamonds) are also indicated. (J) Sanger sequencing of the affected patients and their unaffected mother confirms the presence of a guanine to cytosine mutation leading to a charge reversing glutamic acid to glutamine substitution. This variant was identified by whole exome sequencing of the affected individuals and the unaffected mother. (K) We immunoprecipitated the FLAG tags of the wild-type and E89Q forms of CEP90 and blotted for endogenous CEP63 and FLAG.

DOI: http://dx.doi.org/10.7554/eLife.07519.024

Figure 6.

Figure 6—figure supplement 1. CEP90 is required to localize CEP63 to the centrosome.

Figure 6—figure supplement 1.

(A) The centrosomal fluorescence intensity of CDK5RAP2, CEP152, WDR62 and CEP63 were quantified in SC or CEP90-depleted HeLa cells. Quantification is expressed as mean percentage ±s.d. of the fluorescence intensities of SC cells. For all quantifications 30 cells were analyzed per experiment (n = 3). p < 0.005 (paired t-test) statistically significant differences are denoted by an asterisk. (B) Total cell lysate from SC and CEP90-depleted cells were subjected to immunoblotting using antibodies to CEP90, CDK5RAP2, CEP152, WDR62, and CEP63. Actin served as a loading control.
DOI: http://dx.doi.org/10.7554/eLife.07519.025
Figure 6—figure supplement 2. CEP63 interacts with WDR62 in a CEP90-dependent manner.

Figure 6—figure supplement 2.

(A) HeLa total cell lysate from SC and CEP90-depleted cells were subjected to endogenous immunoprecipitation using an antibody to CEP63 and a negative control. Precipitating proteins were analyzed by immunoblotting for CEP63 and WDR62. Total cell lysates from SC and CEP90 siRNA transfected HeLa cells were analyzed by immunoblotting with antibodies to WDR62, CEP63, and CEP90. Actin served as a loading control.
DOI: http://dx.doi.org/10.7554/eLife.07519.026
Figure 6—figure supplement 3. CEP90 human genetics.

Figure 6—figure supplement 3.

(A) Schematic diagram showing linkage and homozygosity mapping in MCPH-affected individuals MC-701 and MC-702. Regions labeled with red brackets denote homozygosity in MC-701. Blue brackets denote homozygosity in MC-702. The purple oval highlights the region of shared homozygosity between the two patients. (B) 57 Mb block of overlapping homozygosity on chromosome 13, circled in (A). (C) Calculation of logarithmic odds (LOD) score over the block of overlapping homozygosity (g.chr13:30975286-88096410 in hg19 coordinates) yields a parametric, multipoint LOD score of 1.8057. (D) Phylogenetic analysis of CEP90 (using BLAST mutual best match) identified CEP90 orthologs in unicellular organisms. Analysis of the amino-terminal portion of these proteins confirms that CEP90 is ancient and that the variant affects a highly conserved residue (E89).
DOI: http://dx.doi.org/10.7554/eLife.07519.027