Figure 3. Preciliary vesicle components contribute to PCNT-induced pericentrosomal crowding.

(A) Representative structured illumination microscopy images from time course experiments of RPE1 D21, T21, and Q21 cells grown on coverslips and serum depleted for 2, 4, and 24 hr. Cells were stained with GT335 to label centrioles and MYO5A. (B–D) Distribution of MYO5A intensities around the centrosome for 2 (B), 4 (C), and 24 (D) hr timepoints. All values were normalized to D21 at 0 µm. Inset in (B) shows MYO5A intensity distribution in D21 cells prior to normalization. Graphs show mean ± SD. N=3. (E) Quantitation of centrosomal MYO5A intensity in 0–1.2 µm region around the centrosome for control and siPCNT treated D21, T21, and Q21 cells. Cells were treated with siControl or siPCNT for 24 hr concurrent with serum depletion. All values were normalized to the D21 siControl average. Graph show mean ± SD. N=3. Mann-Whitney U test. (F) Quantitation of pericentrosomal MYO5A intensity in 1.2–5 µm region around centrosome for control and siPCNT treated D21, T21, and Q21 cells. Cells were treated with siControl or siPCNT for 24 hr concurrent with serum depletion. All values were normalized to the D21 siControl average. Elevated MYO5A levels are distinct from the reduction observed in the distribution analyses (D) and are likely the result of the unique conditions for each experiment. Graph show mean ± SD. N=3. Mann-Whitney U test.

Figure 3—figure supplement 1. Preciliary vesicle components contribute to PCNT-induced pericentrosomal crowding.

(A) Quantitation of whole cell MYO5A intensity for D21, T21, and Q21 cells. Graph shows mean ± SD. N=3. Mann-Whitney U test. (B) Western Blot probing for MYO5A (top panel) and India Ink stain for loading control (bottom panel) on D21, T21, and Q21 whole cell lysates. (C) Quantitation of MYO5A Western Blot band intensities normalized to D21. Graph shows mean ± SD. N=3. Mann-Whitney U test. (D–F) Distribution of centrosomal MYO5A intensities moving away from the centrosome for 2 (D), 4 (E), and 24 (F) h timepoints. Values were normalized to the D21 average. Note that the FIJI radial analysis plugin does not plot the intensity at the centroid, thus the first point on the graph is the average intensity within the first concentric circle from the centroid. Graph shows mean ± SD. N=3. (G) Representative structured illumination microscopy images of RPE1 D21, T21, and Q21 cells grown on coverslips and serum depleted for 24 hr. Cells were either treated with control or PCNT siRNA during the 24 hr serum depletion. Cells were stained with GT335 and MYO5A. (H) Quantitation of PCNT intensities in a 5 µm radial circle around the centrosome in control and PCNT siRNA treated cells normalized to the D21 siControl average. Graph shows mean ± SD. N=3. Mann-Whitney U test. (I, J) Correlation analysis between PCNT and MYO5A levels in 5 μm radius for D21, T21, and Q21 cells 24 hr after serum depletion. N=3. Pearson r value is shown. (K) Representative confocal images of RPE1 D21, T21, and Q21 cells stably expressing GFP-EHD1 grown on coverslips and serum depleted for 24 hr. Cells were stained for PCNT and Actub. Percentages represent cells with indicated phenotype across 3 N’s.