Figure 3. Centriolar satellites are... | Rockefeller University Press

Figure 3.

$Centriolar satellites are conserved in Drosophila but do not concentrate near centrosomes or cilia, nor move in a directed manner. (A) PCM1 concentrates in the vicinity of centrosomes (marked with γ-tubulin) in vertebrate cells. The radial profile of the normalized distribution of PCM1 shows the mean ± SEM. N = 27 centrosomes. (B) CMB localization in Drosophila S2 cells. Unlike PCM1, CMB does not concentrate near the centrosome (marked with CNN), but is found throughout the cytoplasm. The radial profile shows the mean ± SEM. N = 39 centrosomes. (C) Top: Schematics showing cells and tissues used to examine CMB localization/dynamics. Bottom: Immunofluorescence micrographs show CMB localizing to cytoplasmic foci in all tissues examined. SAS-4/CNN was used to visualize centrioles/centrosomes. (D) Time-lapse recording of Drosophila S2 cell expressing CMB-GFP. Time is shown in min:s. CMB particles display little movement. (E) Corresponding trajectory analysis showing the position of individual particles over time. (F) Histogram plotting frequency of instantaneous speed (µm/s) of all particles tracked in eight different cells. N = 140 particles. The majority of particles moves at slow speeds (0.2–0.3 µm/s), consistent with diffusion. (G) MSD as a function of time. Trajectories with at least 11 frames from eight cells were analyzed. All tracks analyzed are shown in gray (N = 99 tracks). Weighted MSD (mean ± SD) of all diffusive particles follows a linear fit (turquoise line), reflecting the overall Brownian diffusion. (H) Stacked column plot (mean ± SEM) showing all tracks analyzed in G assigned to different categories as described in Materials and methods. The majority of tracks analyzed display the Brownian diffusion (54.2% ±3.9) or subdiffusion/anomalous diffusion (41.1% ±4.3). Only a minor fraction displays directed/active (1.8% ±0.7) or confined movement (2.9 % ±1.1). (I) Centriolar (SAS-4) and centrosomal (CNN) proteins colocalize with CMB on cytoplasmic foci, although immunofluorescence signal is weak compared with that at centrosomes (marked with γ-tubulin). Colocalization of CMB with SAS-4 and CNN was assessed by Pearson’s correlation coefficient. Randomized control has one of the channels rotated 90°. Centrosomes were excluded from analysis. Error bars are the mean ± SD. N = 50 cells per condition. A Mann–Whitney test was used to test statistical significance; ****P < 0.0001. Scale bars, 10 µm (A–C), 5 µm (D and I), 1 µm (A–C, insets, I, magnified views). See also Fig. S3. MSD, mean squared displacement.$

Centriolar satellites are conserved in Drosophila but do not concentrate near centrosomes or cilia, nor move in a directed manner. (A) PCM1 concentrates in the vicinity of centrosomes (marked with γ-tubulin) in vertebrate cells. The radial profile of the normalized distribution of PCM1 shows the mean ± SEM. N = 27 centrosomes. (B) CMB localization in Drosophila S2 cells. Unlike PCM1, CMB does not concentrate near the centrosome (marked with CNN), but is found throughout the cytoplasm. The radial profile shows the mean ± SEM. N = 39 centrosomes. (C) Top: Schematics showing cells and tissues used to examine CMB localization/dynamics. Bottom: Immunofluorescence micrographs show CMB localizing to cytoplasmic foci in all tissues examined. SAS-4/CNN was used to visualize centrioles/centrosomes. (D) Time-lapse recording of Drosophila S2 cell expressing CMB-GFP. Time is shown in min:s. CMB particles display little movement. (E) Corresponding trajectory analysis showing the position of individual particles over time. (F) Histogram plotting frequency of instantaneous speed (µm/s) of all particles tracked in eight different cells. N = 140 particles. The majority of particles moves at slow speeds (0.2–0.3 µm/s), consistent with diffusion. (G) MSD as a function of time. Trajectories with at least 11 frames from eight cells were analyzed. All tracks analyzed are shown in gray (N = 99 tracks). Weighted MSD (mean ± SD) of all diffusive particles follows a linear fit (turquoise line), reflecting the overall Brownian diffusion. (H) Stacked column plot (mean ± SEM) showing all tracks analyzed in G assigned to different categories as described in Materials and methods. The majority of tracks analyzed display the Brownian diffusion (54.2% ±3.9) or subdiffusion/anomalous diffusion (41.1% ±4.3). Only a minor fraction displays directed/active (1.8% ±0.7) or confined movement (2.9 % ±1.1). (I) Centriolar (SAS-4) and centrosomal (CNN) proteins colocalize with CMB on cytoplasmic foci, although immunofluorescence signal is weak compared with that at centrosomes (marked with γ-tubulin). Colocalization of CMB with SAS-4 and CNN was assessed by Pearson’s correlation coefficient. Randomized control has one of the channels rotated 90°. Centrosomes were excluded from analysis. Error bars are the mean ± SD. N = 50 cells per condition. A Mann–Whitney test was used to test statistical significance; ****P < 0.0001. Scale bars, 10 µm (A–C), 5 µm (D and I), 1 µm (A–C, insets, I, magnified views). See also Fig. S3. MSD, mean squared displacement.

This Feature Is Available To Subscribers Only