Fig 2: CCDC66-positive centriolar satellites exhibit both diffusive and microtubule-mediated directional motility. (a) Representative fluorescence images of satellites from time-lapse videos of RPE1::GFP-CCDC66 cells (top-left and bottom-left panels) and corresponding trajectories of satellites as a function of time (top-right and bottom-right panels). CCDC66-positive satellites were identified and tracked using the single-particle tracking algorithms detailed in Materials and Methods. Satellites were classified into persistent (magenta) and diffusive (green) motility groups using a persistence ratio cutoff value of 0.5. (b) The distribution of the persistence ratio (direct distance(D)/total distance(T)) was used to determine the different motility groups. Persistence histogram (gray bars) were fitted with a single or double Gaussian function (black line). (c) Average speed and direct distance of satellites. (d) The distribution of satellite instant speed. The changes at higher instant speed values were shown in the inset. (e) Time-colored trajectories of persistent (top-left) and diffusive (bottom-left) satellites and analysis of their directionality to the centrosome (right-hand graphs). Corresponding distribution of distance from/to centroids were plotted to determine the directed motility of satellites. Centroids were marked as C, indicating the localization of the centrosome. Negative values indicate movement towards the centrosome and positive values indicate movement away from the centrosome. (f) A representative fluorescence image of RPE1::GFP-CCDC66 cells stained with SIR-Tubulin. Images were overlaid to determine microtubule mediated movement of satellite that move persistently. The right-hand panel indicates the time dependent movement of satellites. (g) Representative satellite (green) exhibits bimodal motility by alternating between persistent and diffusive movements. Still images from Movie 6 at the indicated time points were shown. The bottom right-hand panel indicates the time dependent movement of the satellite.

Fig 3: Centriolar satellites inhibit CCDC66 dynamic localization at the centrosome. (a) Effect of PCM1, CEP72 and CEP290 depletion on CCDC66 level at the centrosome. RPE1::GFP-CCDC66 cells were transfected with control, PCM1, CEP290 or CEP72 siRNAs for 48 h. Cells were then fixed and stained for GFP, PCM1 and gamma tubulin. Images represent centrosomes in cells from the same coverslip taken with the same camera settings. Scale bar, 1 µm. (b) Quantification of (a). GFP-CCDC66 fluorescence intensities were measured in a 2.5 µm2 circular area around the centrosome from two independent experiments. Levels are normalized to the mean of the control group (=1). n )0.50 cells for each group. t-test was used for statistical analysis. Standard error of mean (SEM): siControl = 0.046, siPCM1 = 0.075, siCEP290 = 0.17, siCEP72 = 0.27. (c) Effect of PCM1, CEP72 and CEP290 depletion on total cellular CCDC66 levels. Cells were transfected with control, PCM1, CEP290 or CEP72 siRNAs for 48 h. Cell extracts were cells were immunoblotted for CCDC66, PCM1, CEP290 and CEP72. Beta-actin was used as a loading control. (d) Effect of PCM1, CEP72 and CEP290 depletion on CCDC66 dynamics at the centrosome. RPE1::GFP-CCDC66 cells were transfected with control, PCM1, CEP290 or CEP72 siRNAs for 48 h. 2.5 µm2 circular area around the centrosome marked by yellow dashed circle was photobleached and imaged for 250 seconds after photobleaching. Still images represent centrosomal GFP-CCDC66 signal at the indicated times. Scale bar, 1 µm. (e) Percentage of recovery graph from (d). Individual FRAP experiments from two independent experiments were fitted into one phase association curves. n = 8 for control and PCM1, n = 8 for CEP290 and n = 6 for CEP72 depleted cells per group. Half-time of and mobile pool were calculated using recovery data. (f) mobile pool of (d). Error bars, SEM: siControl = 2.49, siPCM1 = 3.10, siCEP290 = 2.16, siCEP72 = 3.64 and (g) half-time analysis of (d). Error bars, SEM: siControl = 4.00, siPCM1 = 2.39, siCEP290 = 2.89, siCEP72 = 2.50.

Fig 4: Model CCDC66 localization and functions during mitosis and cytokinesis. The model shows CCDC66 localization during cell division and the phenotypic consequences of CCDC66 depletion on mitotic and cytokinetic MT arrays as well as chromosomes. In metaphase cells, CCDC66 (magenta) localizes to the centrosomes, the astral microtubules, the spindle microtubules, and K-fibers. CCDC66 loss results in a reduced PCM size, microtubule nucleation capacity, astral/spindle microtubule density as well as unstable K-fibers. It also causes a shift in orientation and increases metaphase plate width. During anaphase, CCDC66 localizes to the centrosomes and midzone. CCDC66 depletion causes defective central spindle formation, reduction in central spindle microtubule intensity, lagging chromosomes, and orientation shift. During cytokinesis, CCDC66 additionally localizes to the centrosomes and the midbody. CCDC66 depletion causes asymmetric cleavage furrow formation. CCDC66, coiled-coil domain-containing protein 66; PCM, pericentriolar material.

Fig 5: Ciliary CCDC66 is not dynamic and its recruitment is regulated by satellites. (a) STED analysis of ciliary CCDC66 localization. RPE1::GFP-CCDC66 cells were serum starved for 48 h, fixed with 4% PFA and stained for GFP, Arl13B (ciliary membrane marker) or acetylated tubulin (ciliary axoneme marker). Cells were imaged on a Leica TCS SP8 STED 3X confocal laser scanning microscope. Scale bar, 1 µm. (b) FRAP analysis of ciliary GFP CCDC66. RPE1::GFP-CCDC66 cells were serum starved for 48 h. Whole cilium, upper ciliary region or area between the tip and basal body indicated by yellow dashed rectangles was photobleached and cells were imaged for 250 seconds after photobleaching. Still images represent ciliary GFP-CCDC66 signal at indicated time points. Scale bar: 1 µm. (c,d) Percentage of recovery graph from (b). Individual FRAP experiments from two independent experiments (n In7 in total) were fitted into one phase association curves. (e) Effect of PCM1, CEP72 and CEP290 depletion on CCDC66 level at the cilia. RPE1::GFP-CCDC66 cells were transfected with control, PCM1, CEP290 or CEP72 siRNAs. 24 h after transfection, they were serum starved for 48 h. Cells were then fixed and stained for GFP, PCM1 and acetylated tubulin (cilia marker). Images represent cilia in cells from the same coverslip taken with the same camera settings. Scale bar, 1 µm. (f) Quantification of GFP-CCDC66 ciliary concentration from (e). GFP-CCDC66 fluorescence intensities were measured in the ciliary area defined by the acetylated tubulin ciliary marker from two independent experiments. Ciliary protein concentrations were determined by dividing fluorescence signal of the protein to the cilium length, which was quantified using acetylated tubulin staining. Levels are normalized to the mean of the control group (=1). n = 50 cells for each group. t-test was used for statistical analysis. Error bars, SEM: siControl = 0.06, siPCM1 = 0.21, siCEP290 = 0.14, siCCEP72 = 0.27.