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. 2023 Jan 19;12:e78202. doi: 10.7554/eLife.78202

Figure 4. Increased HSA21 ploidy disrupts mother centriole uncapping in a PCNT-dosage-dependent manner.

(A) Representative structured illumination microscopy images of RPE1 D21, T21, and Q21 cells grown on coverslips and serum depleted for 24 hr. Cells were stained with GT335 and the centriole capping protein CP110. Percentages represent cells with indicated phenotype across 3 N’s. (B) Quantitation of CP110 centriole capping for control and siPCNT treated D21, T21, and Q21 cells. Graph show mean ± SD. N=3. Two-tailed unpaired t-test. (C) Selected tomographic slices and 3D models showing ciliary vesicle formation in D21, T21, and Q21 cells. T21 and Q21 have smaller ciliary vesicles (arrows) and the ciliary vesicle in Q21 cell is offset from the center of the mother centriole. Models show mother centriole (yellow), daughter centriole (magenta) ciliary vesicle (large red structure at the distal end of the mother centriole), smaller vesicles (red spheres), and smooth tubular membranes (blue-green). (D) Quantitation of membrane surface area at the centrosome in electron tomograms for D21, T21, and Q21 cells. D21 cells showed increased small vesicles (red spheres), smooth membranes (blue-green), and ciliary vesicle membrane (red structures) compared to T21 and Q21 cells. Ciliary vesicles in Q21 cells were not quantified due to low frequency (n of 1) and unusual offset of that ciliary vesicle. N=2 reconstructed cells per cell line. Scale bars are 200 nm. Movies of the complete volume and rotating models can be found in Figure 4—videos 1–3.

Figure 4—source data 1. Values for biological and technical replicates for graphs in Figure 4 and Figure 4—figure supplement 1.

Figure 4.

Figure 4—figure supplement 1. Increased HSA21 ploidy disrupts mother centriole uncapping, but not general membrane trafficking.

Figure 4—figure supplement 1.

(A) Representative structured illumination microscopy images of RPE1 D21, T21, and Q21 cells grown on coverslips and serum depleted for 24 hr. Cells were stained with GT335 and the centriole capping protein CEP97. Percentages represent cells with indicated phenotype across 3 N’s. (B) Quantitation of CP110 centriole capping for D21, T21, and Q21 cells 24 hr after serum depletion. Graph shows mean ± SD. N=3. Two-tailed paired t-test. (C) Quantitation of CEP97 centriole capping for D21, T21, and Q21 cells. Graph shows mean ± SD. N=3. Two-tailed paired t-test. (D) Quantitation of PCNT intensities in a 5 µm radial circle around the centrosome in control and PCNT siRNA treated cells normalized to the D21 average. Graph shows mean ± SD. N=3. Mann-Whitney U test. (E) Representative structured illumination microscopy images of RPE1 D21, T21, and Q21 cells grown on coverslips and serum depleted for 2, 4, 8, 24, and 48 hr. Cells were stained with γ-tubulin, Actub, and RAB8. Percentages represent cells with indicated phenotype across 3 N’s. (F) Quantitation of cells with RAB8 ciliary vesicle throughout the time course. Graph shows mean ± SD. N=3. (G) Representative confocal images of RPE1 D21, T21, and Q21 cells grown on coverslips and serum depleted for 24 hr. Cells were stained with Hoechst 33342, PCNT, Golgin97, and RAB8. (H) Quantitation of RAB8 Golgi intensity normalized to the D21 average. Graph shows mean ± SD. N=3. Mann-Whitney U test.
Figure 4—video 1. Movie of D21 cell from Figure 4 showing the electron tomogram volume, then model projecting from the image, then the model turning without image.
Download video file (18.7MB, mp4)
Mother centriole (yellow), daughter centriole (magenta), vesicles (red spheres), smooth tubular membrane (blue-green), and ciliary vesicle (red structure at distal end of mother centriole).
Figure 4—video 2. Movie of T21 cell from Figure 4 showing the electron tomogram volume, then model projecting from the image, then the model turning without image.
Download video file (24MB, mp4)
Mother centriole (yellow), daughter centriole (magenta), vesicles (red spheres), smooth tubular membrane (blue-green), and ciliary vesicle (red structure at distal end of mother centriole).
Figure 4—video 3. Movie of Q21 cell from Figure 4 showing the electron tomogram volume, then model projecting from the image, then the model turning without image.
Download video file (14.5MB, mp4)
Mother centriole (yellow), daughter centriole (magenta), vesicles (red spheres), smooth tubular membrane (blue-green), and ciliary vesicle (red structure at distal end of mother centriole). In the Q21 cell, the ciliary vesicle is slightly offset from the end of the mother centriole.