Gravin regulates centrosome function through PLK1

Abstract

We propose to understand how the mitotic kinase PLK1 drives chromosome segregation errors, with a specific focus on Gravin, a PLK1 scaffold. In both three-dimensional primary prostate cancer cell cultures that are prone to Gravin depletion and Gravin short hairpin RNA (shRNA)-treated cells, an increase in cells containing micronuclei was noted in comparison with controls. To examine whether the loss of Gravin affected PLK1 distribution and activity, we utilized photokinetics and a PLK1 activity biosensor. Gravin depletion resulted in an increased PLK1 mobile fraction, causing the redistribution of active PLK1, which leads to increased defocusing and phosphorylation of the mitotic centrosome protein CEP215 at serine-613. Gravin depletion further led to defects in microtubule renucleation from mitotic centrosomes, decreased kinetochore-fiber integrity, increased incidence of chromosome misalignment, and subsequent formation of micronuclei following mitosis completion. Murine Gravin rescued chromosome misalignment and micronuclei formation, but a mutant Gravin that cannot bind PLK1 did not. These findings suggest that disruption of a Gravin-PLK1 interface leads to inappropriate PLK1 activity contributing to chromosome segregation errors, formation of micronuclei, and subsequent DNA damage.

FIGURE 1:

Gravin loss is associated with increased micronuclei formation in primary prostate cancer cells. (A) Immunoblot analysis showing decreased Gravin expression in PCa1 and PCa3 cancer cells in comparison with RWPE-1 and normal patient prostate epithelium cells (26Na). (B) Control (RWPE-1), PCa1, and PCa3 3-D acini cultures fluorescently labeled for p-H3 (green) and actin (red). (C) Control (RWPE-1), PCa1, and PCa3 3-D acini labeled for DAPI (gray) displaying micronuclei within a single cell (yellow arrowhead). Confocal micrographs for B and C are presented as maximum projections. Bar, 5 μm. (D, E) Quantification of the mitotic index (D) and cells (%) with micronuclei (E) for control (RWPE), PCa1, and PCa3 in 3-D acini was calculated. n = 30 organoids over n = 3 experiments ± SEM, Student‘s t test p = 0.0099 (D) and p < 0.0001 (E). (F) Immunoblot analysis of Gravin expression in RWPE-1 cells expressing a control GAPDH shRNA or a Gravin shRNA. Tubulin was used as loading control. (G) Control shRNA and Gravin shRNA RWPE-1 3-D acini cultures stained for DAPI displaying micronuclei within a single cell (yellow arrow). Bar, 5 μm. (H) Quantification of Gravin shRNA and control shRNA treated cells with micronuclei (%) over n = 3 experiments ± SEM. Student‘s t test p = 0.0097.

FIGURE 2:

Gravin loss disrupts PLK1 dynamics predominately at mitotic centrosomes. (A) Model depicting Gravin (orange) binding PLK1 (yellow) at phosphorylated T766 (Canton et al., 2012). (B) Maximum projection of structured illumination microscopy (SIM) micrographs of Gravin (a‘) and PLK1 (b‘) localizing to mitotic centrosomes (orange arrows) in metaphase cells. Magenta arrow represents PLK1 localization at kinetochores in b‘. (C) Representative images of fluorescence recovery after photobleaching (FRAP) of GFP-PLK1 (Fire LUT, ImageJ) RPE cells at mitotic centrosomes, kinetochores, and cytokinetic midbodies. Bar, 5 μm. (D) Quantification presented as box-and-whisker plot of half-life for GFP-PLK1 in control and Gravin-shRNA treated cells (+ indicates mean, n > 20 cells across n = 3 experiments, ANOVA indicates significance of p < 0.0001). (E) A curve was fitted using one-phase decay of PLK1 fluorescence recovery at kinetochores (left) and mitotic centrosomes (right) in metaphase cells treated with control or Gravin shRNAs (n > 20 cells over n = 3 experiments). (F) GFP-PLK1 at a single metaphase mitotic centrosome in control shRNA- or Gravin shRNA-treated cells rescued with full-length wild-type Gravin, or T766A mutant Gravin prior to and 3 s after bleaching events. Confocal micrographs at a single mitotic centrosome are shown (Fire LUT, Image J, bar indicates gradient of integrated fluorescence intensity values, A.U.). Bar, 2 μm. (G) Integrated intensity profiles for GFP-PLK1 at a single mitotic centrosome before and 3 s after bleaching events are presented. (H, I) The average (H) half-life (t_1/2) and (I) immobile fraction of GFP-PLK1 at metaphase spindle poles was calculated and presented as box-and-whisker plots with + indicating mean (n > 20 cells over n = 3 experiments). One-way ANOVA indicates significance between p < 0.001 (H) and p < 0.0001 (I).

FIGURE 3:

Gravin-depleted cells redistribute active PLK1, causing increased phosphorylation events at mitotic centrosomes. (A) Model of PLK1 biosensor showing how increased PLK1 activity causes a conformational change in the biosensor through phosphorylation of c-jun (green) and binding of the FHA2 domain (magenta), resulting in a loss of FRET. When there is increased phosphatase activity, the PLK1 biosensor is in a relaxed state, allowing FRET. (B) Equation for determining the inverse FRET ratio by dividing the YFP^EX→YFP^EM by the CFP^EX→YFP^EM. When the inverse FRET ratio is calculated, high ratios correspond with high levels of PLK1 activity while low ratios correspond to low levels of PLK1 activity. (C) Relative PLK1 activity shown as an inverse FRET ratio (Fire LUT, ImageJ, bar indicates gradient of FRET ratio values) and DIC images taken from nocodazole synchronized and released cells in metaphase. Control shRNA, Gravin shRNA, and Gravin shRNA cells plus BI2536 are shown. Bar, 5 μm. (D) Quantification of the inverse FRET efficiency nocodazole-released cells in metaphase that were treated with control shRNA, Gravin shRNAs, and/or BI2536 (BI) is presented as a scatterplot. n > 30 cells over n = 3 experiments, median with interquartile range shown, one-way ANOVA p < 0.0001. (E) PLK1-FRET-PACT biosensor localization in metaphase cells. Arrows depict mitotic centrosomes (Fire LUT). Bar, 5 μm. (F) The inverse FRET-efficiency of the PLK1-FRET-PACT probe was quantified from nocodazole synchronized and released metaphase cells treated with control shRNA, Gravin shRNA, and/or BI2536 (BI) and presented as a scatterplot. Representative single mitotic centrosome inverse FRET ratios shown below graph (FIRE-LUT). n > 50 cells over n = 4 experiments, median with interquartile range shown, one-way ANOVA p = 0.0371. (G) Isolated mitotic centrosomes from control shRNA and Gravin shRNA HEK293 cells immunolabeled for centrin (white) and phosphoserine/phosphothreonine (pS/pT; FIRE LUT). Bar, 2 μm. (H) Quantification of pS/pT intentsity at mitotic centrosomes in G presented as a scatterplot. n = 27 centrioles for each treatment, median with interquartile range shown, Student‘s t test p < 0.0001. (I, J) FLAG IP of Mock or FLAG-CEP215 transfected control shRNA- or Gravin shRNA ± BI2536-treated HEK293 cells as indicated. Protein expression and immunoprecipitation was analyzed by immunoblot for Gravin, CEP215, pS/pT, and GAPDH (as loading control). Black arrowhead indicates pS/pT band at 250 kDa. (K) Quantification of pS/pT intensities normalized over total FLAG-CEP215 IP (n = 3 experiments ± SEM, one-way ANOVA p = 0.0353). (L) Immunoblot analysis of FLAG-CEP215 and FLAG-CEP215-S613A IP from HEK293 cells. (M) Quantification of pS/pT expression levels from FLAG-IP normalized over total FLAG-CEP215 (n = 3 experiments ± SEM, Student‘s t test p = 0.0264). (N) Working model depicting that phosphorylated Gravin (orange) inhibits PLK1 (yellow) from phosphorylating CEP215 (purple) during mitosis.

FIGURE 4:

Gravin loss results in CEP215 disorganization and disrupted mitotic centrosome function. (A) STED (stimulated emission–depletion) micrographs of metaphase control and Gravin shRNA HEK293 cells are presented as maximum projections. Cells were immunostained for CEP215 (Fire-LUT, ImageJ, bar indicates gradient of integrated fluorescence intensity values, A.U.) and tubulin (white). Bar, 5 μm. Inserts (white boxes) depict 2× magnification of CEP215 at mitotic centrosomes. A line scan through the mitotic centrosomes is drawn and the normalized fluorescence intensity of CEP215 is plotted (right, each line represents a single line scan over n = 5 cells for each treatment). (B) Quantification of total CEP215 fluorescence intensity at mitotic centrosomes in control and Gravin shRNA–treated HEK293ad cells. n = 3 experiments, n = 60 cells, median with interquartile range shown, Student‘s t test, p < 0.0001. (C) Ratio of highest CEP215 intensity over lowest CEP215 intensity between the two mitotic centrosomes within a single cell. n = 60 cells over n = 3 experiments, median with interquartile range shown, Student‘s t test p < 0.0001. (D) A series of confocal micrographs demonstrating MT regrowth (α-tubulin, Fire-LUT, bar indicates gradient of integrated fluorescence intensity values, A.U.) at mitotic centrosomes for a time course following nocodazole washout in HEK293 cells treated with Gravin or control shRNAs. Micrographs presented as maximum projections. Bar, 5 μm. (E) The integrated α-tubulin intensity at mitotic centrosomes was quantified and presented as a scatterplot at indicated times following washout. n = 50 poles for each time and treatment, median with interquartile range shown, representative of n = 3 experiments, Student‘s t test for 0 min (p < 0.0001), 2 min (p < 0.0001), and 5 min (p = 0.60). (F) Maximum confocal projections of control shRNA and Gravin shRNA mitotic HEK293 cells treated for 5 min on ice. Cells were immunostained for acetylated tubulin (Fire-LUT, ImageJ) and DAPI (white). (G) Quantification of cells (%) lacking K-fibers in HEK293 cells treated with control or Gravin shRNAs calculated over n = 3 experiments ± SEM (Student‘s t test p = 0.0020).

FIGURE 5:

Gravin loss and increased phosphorylation of CEP215 results in higher incidence of cells containing micronuclei. (A–C) Wide-field deconvolved micrographs of Gravin shRNA, full-length Gravin rescue, and mutant T766A Gravin cells are presented as maximum projections. Cells were immunostained for DAPI (A, B, C white) and γ-H2AX (C, green). Bar, 5 μm. (D) Quantification of cells containing micronuclei (%) in Gravin shRNA–treated cells rescued with full-length Gravin or Gravin (T766A) over n = 3 experiments ± SEM; Student‘s t test p value = 0.0034. (E) Quantification of cells containing micronuclei with γ-H2AX (%) in Gravin shRNA cells rescued with full-length Gravin or Gravin (T766A) over n = 3 experiments ± SEM. (F–H) Deconvolved wide-field micrographs of Gravin shRNA–treated cells expressing FLAG-CEP215, FLAG-CEP215-S613A, or FLAG-CEP215-S613E presented as maximum projections. Cells were immunolabeled for DAPI (F, G, H, white) and FLAG (H, green). Bar, 5 μm. (I, J) Quantification of cells containing lagging chromosomes (I) and micronuclei (J) (%) in Gravin shRNA cells expressing FLAG-CEP215, FLAG-CEP215-S613A, or FLAG-CEP215-S613E. n = 3 experiments ± SEM, n > 300 cells, one-way ANOVA p = 0.0290 (I), p = 0.0003 (J). (K) Model summarizing that when Gravin is lost, a redistribution of active PLK1 leads to mitotic errors and subsequent formation of micronuclei.