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. 2012 Nov 1;125(Pt 21):5096-109.
doi: 10.1242/jcs.107326. Epub 2012 Aug 16.

WD40-repeat protein 62 is a JNK-phosphorylated spindle pole protein required for spindle maintenance and timely mitotic progression

Marie A Bogoyevitch  1 , Yvonne Y C Yeap, Zhengdong Qu, Kevin R Ngoei, Yan Y Yip, Teresa T Zhao, Julian I Heng, Dominic C H Ng
Affiliations

WD40-repeat protein 62 is a JNK-phosphorylated spindle pole protein required for spindle maintenance and timely mitotic progression

Marie A Bogoyevitch et al. J Cell Sci. .

Abstract

The impact of aberrant centrosomes and/or spindles on asymmetric cell division in embryonic development indicates the tight regulation of bipolar spindle formation and positioning that is required for mitotic progression and cell fate determination. WD40-repeat protein 62 (WDR62) was recently identified as a spindle pole protein linked to the neurodevelopmental defect of microcephaly but its roles in mitosis have not been defined. We report here that the in utero electroporation of neuroprogenitor cells with WDR62 siRNAs induced their cell cycle exit and reduced their proliferative capacity. In cultured cells, we demonstrated cell-cycle-dependent accumulation of WDR62 at the spindle pole during mitotic entry that persisted until metaphase-anaphase transition. Utilizing siRNA depletion, we revealed WDR62 function in stabilizing the mitotic spindle specifically during metaphase. WDR62 loss resulted in spindle orientation defects, decreased the integrity of centrosomes displaced from the spindle pole and delayed mitotic progression. Additionally, we revealed JNK phosphorylation of WDR62 is required for maintaining metaphase spindle organization during mitosis. Our study provides the first functional characterization of WDR62 and has revealed requirements for JNK/WDR62 signaling in mitotic spindle regulation that may be involved in coordinating neurogenesis.

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Figures

Fig. 1.
Fig. 1.
WDR62 depletion decreased cell proliferation within the embryonic cortex. (A) Neuro2a cells were transfected with individual mouse WDR62-targeting siRNA (t1, t2, t3 and t4), a combined mouse WDR62 siRNA pool, non-targeting siRNA (Con siRNA) or not treated with siRNA (no siRNA) and immunoblotted for WDR62 and α-tubulin. (B) Images of the coronal sections of embryonic mouse cerebral cortex electroporated with control or mouse WDR62 siRNA, together with GFP to label electroporated cells. Embryos were pulse labeled with BrdU 24 h post-electroporation, and brain sections finally stained for Ki67 to identify actively proliferating cells at time of harvest (48 h post-electroporation). Magnified panels (below) of the ventricular zone (VZ) highlight proliferating (GFP+/BrdU+/Ki67+, filled arrowheads) and non-proliferating cells (GFP+/BrdU+/Ki67−, open arrowheads). (C) Quantification of cell proliferation (GFP+/Ki67+) in brain sections from control and mouse WDR62-siRNA-treated embryos. (D) The cell cycle exit index in response to WDR62 depletion was determined by counting GFP+/BrdU+/Ki67− cells and expressing this as a proportion of GFP+/BrdU+ cells. (E) BrdU labeling index in control and WDR62-siRNA-treated brain sections used in the analysis depicted in B. (F) Coronal sections of embryos electroporated with control or mouse WDR62 siRNA were stained for phospho-histone H3 (pHH3). Magnified panels (below) of the VZ highlight cells in M phase (GFP+/BrdU+/pHH3+, filled arrowheads) and non-mitotic cells (GFP+/BrdU+/pHH3−, open arrowheads). (G) The mitotic index in response to WDR62 depletion was determined by the proportion of cells double labeled for GFP and pHH3 (GFP+/pHH3+). (H) A cell cycle exit index analysis performed with pHH3 and BrdU immunostaining was determined by counting GFP+/BrdU+/pHH3− cells within the population of GFP+/BrdU+ cells (statistical analysis could not be performed because 100% of cells were GFP+BrdU+pHH3− following WDR62 siRNA treatment). (I) BrdU labeling index in control and WDR62-siRNA-treated brain sections used in the analysis depicted in (F). Scale bar: 100 µm, while all graphs plot means ± s.e.m. (*P<0.05, **P<0.01, ns denotes not statistically significant).
Fig. 2.
Fig. 2.
Accumulation of WDR62 at spindle poles during mitosis. (A) WDR62 subcellular localization in HeLa cells at different mitotic stages. DAPI-stained condensed chromatin and γ-tubulin immunostaining revealed centrosomes and spindle poles. Scale bar: 20 µm. (B) WDR62 association with centrosomes occurred prior to nuclear envelope breakdown as demonstrated by co-staining with nuclear pore complex proteins (Mab414 NPC). Scale bar: 20 µm. (C) Higher magnification images of WDR62 localization around γ-tubulin-stained centrosomes from prophase to metaphase. Scale bar: 2 µm. Insets are lower magnification images showing entire cell and mitotic stage. (D) Co-staining of WDR62 with PCM markers (NEDD1, pericentrin, CG-NAP and Cdk5rap2). (E) WDR62 co-localization with spindle pole markers (NUMA and p150Glued). Insets in D and E are high magnification images of centrosomes. Scale bars: 20 µm.
Fig. 3.
Fig. 3.
WDR62 depletion perturbs mitotic progression. (A) HeLa cells were transiently transfected with individual human WDR62-targeting siRNA (t1, t2, t3 and t4), a WDR62 siRNA pool, non-targeting siRNA (Con siRNA) or not treated with siRNA (no siRNA) and immunoblotted for WDR62. α- and γ-tubulin were blotted as controls. (B) HeLa cells transfected with WDR62 or Con siRNA were synchronized at G1/S (DTB). Cell cycle progression following thymidine release (2–14 h as indicated) was determined by immunoblotting for cyclin B1 and cdc25C phosphorylation. (C) HeLa cells transfected with WDR62 or Con siRNA were synchronized at M-phase [nocodazole (NZ) treated; 350 nM, 16 h]. Mitotic progression following NZ release (0.5–10 h as indicated) was determined by immunoblotting for cyclin B1 and phospho-cdc25C. α-tubulin was blotted for protein loading and WDR62 levels determined to confirm knockdown. (D) HeLa cells, treated with Con siRNA or WDR62 siRNA, were synchronized (DTB) and imaged under phase-contrast optics at 6-min intervals from 7 h post thymidine release. Representative images of mitotic progression are shown. White dotted lines indicate the position of the metaphase plate. (E) Duration of mitotis in WDR62-depleted and control HeLa cells, measured from nuclear envelope breakdown until two daughter cells were observed, are shown on a vertical scatter plot. Horizontal lines indicate mean values and n values are the total cells counted from three independent experiments (*P<0.01). (F) WDR62 was depleted in HeLa cells for 48 h and the proportion of cells in M phase identified by DAPI staining. Values are means ± s.e.m. from three independent experiments.
Fig. 4.
Fig. 4.
WDR62 is required for metaphase spindle maintenance and positioning. (A) HeLa cells were transfected with WDR62 or Con siRNA and synchronized (DTB). Following thymidine release (8–10 h), cells were fixed and stained for α-tubulin/γ-tubulin. Representative cells at different mitotic stages are shown. (B) The proportion of metaphase cells with abnormal spindles characterized by displaced centrosomes was quantified. (C) Individual z-stacks of γ-tubulin staining in WDR62-depleted or control HeLa cells. Overlay images show maximum intensity projections of z-stacks. All scale bars: 20 µm. (D) Orthogonal slice along spindle polarity axis viewed from a side-on perspective of γ-tubulin immunostained z-sections reveals spindle rotation in z-axis. ‘θ’ is the angle of rotation from planar orientation. (E) The angle of spindle rotation was measured in WDR62- or Con-siRNA-treated HeLa cells and plotted on a vertical scatter plot. Horizontal lines depict means ± s.e.m. and n values are total number of cells counted from two independent experiments (*P<0.05).
Fig. 5.
Fig. 5.
Centrosome integrity is reduced with WDR62 depletion. WDR62-depleted or control HeLa cells in metaphase were stained with (A) α- and γ-tubulin or (B) WDR62 and γ-tubulin. Arrows indicate γ-tubulin puncta. DAPI staining of DNA is shown in overlay images. (C) HeLa cells were transfected with WDR62 or Con siRNA together with Centrin1–GFP to label centrioles in addition to staining for γ-tubulin. (D,E) WDR62-depleted or control HeLa cells in metaphase were stained with pericentrin and (D) γ-tubulin or (E) α-tubulin. Insets in D,E are higher magnification images of the centrosomal region. All scale bars: 20 µm. (F) WDR62-depleted cells were synchronized (DTB) and treated with nocodazole (NZ, 5 nM) or Taxol (TX, 5 nM) at 7 h post-thymidine release. The number of cells in metaphase with loose/fragmented PCM, as revealed by pericentrin staining, was quantified and expressed as a proportion of total metaphase cells. Con siRNA treatment was included for comparison. n values are the total number of cells counted from three independent experiments. (G) Representative images of pericentrin staining in WDR62-depleted cells treated with nocodazole or Taxol.
Fig. 6.
Fig. 6.
WDR62 is required for spindle/centrosome maintenance after spindle bipolarity is established. (A) G1/S synchronized HeLa cells (DTB) were released into nocodazole (350 nM) before microtubules were depolymerized at 4°C (30 min). Following nocodazole washout, cells were incubated at 37°C for various time intervals before staining for WDR62 and microtubule regrowth with α-tubulin. Scale bar: 2 µm. (B) Microtubule regrowth in WDR62-depleted HeLa cells was analyzed as in A and compared to control (Con siRNA) cells. In addition, centrosomes were revealed by γ-tubulin co-staining. Scale bar: 20 µm. (C) Following MT regrowth (45 mins, 37°C) the proportion of cells with normal, abnormal or multi-polar spindles was determined. n values are total number of cells counted from three independent experiments. (D) HeLa cells synchronized to G1/S (DTB) were released into monastrol (100 µM) before fixing and immunostaining for WDR62 and α- or γ-tubulin. Scale bars: 20 µm.
Fig. 7.
Fig. 7.
WDR62 is phosphorylated during mitosis. (A) Protein lysates from asynchronous (AS), S-phase-arrested (HU, 2 mM, 16 h) or mitotically arrested (NZ, 350 nM, 16 h) HeLa cells were treated with λ-phosphatase, mock treated without enzyme or left untreated (TCL) before blotting with the indicated antibodies. Arrows indicate decreased WDR62 band migration on SDS-PAGE. (B) Protein lysates from AS-, NZ- (350 nM, 16 h) or sorbitol-treated (Sorb, 0.5 M, 60 min) HeLa cells were treated with λ-phosphatase or left untreated (TCL) before resolving on Phos-tag gels or standard SDS-PAGE followed by immunoblotting. Arrows indicated reduced mobility of phosphorylated WDR62. (C) HeLa cells were arrested at M phase (NZ, 350 nM, 16 h) before release into normal serum medium for the indicated times. WDR62 band mobility, cyclin B1, α-tubulin and phospho-cdc25C were determined by immunoblot analysis. Arrows indicate increased WDR62 gel migration and carets (<) antibody detection with time following NZ release. (D) HeLa cells were synchronized at G1/S (DTB). WDR62, cyclin B1 and cdc25C levels were then analyzed by immunoblotting. (E) Cell cycle distribution was determined by FACS at time 0 min before release and at regular time intervals following thymidine release. Representative histograms are shown.
Fig. 8.
Fig. 8.
JNK-mediated WDR62 phosphorylation is involved in spindle regulation. (A) M-phase-arrested HeLa cells (NZ, 350 nM, 16 h) were treated with JNK inhibitor VIII (20 µM) and samples immunoblotted for WDR62 and α-tubulin. (B) Schematic representation of WDR62 truncation and JNK-binding (JBD) mutants. Asterisks indicate replacement of leucines with alanines in the JBD motif. (C) JNK1-mediated phosphorylation of full-length WDR62 (WDR62-FL) and truncation mutants containing WDR62 N- (WDR62-N) or C-terminal (WDR62-C) regions as measured by in vitro kinase activity assays. Values are means ± s.d. 32P (pmol) incorporated into substrates from three independent experiments. A representative autoradiograph and Coomassie stain for protein loading are shown. (D) JNK1-mediated phosphorylation of WDR62-C-AXA was determined by in vitro kinase activity assays and compared to the WDR62-C truncation mutant. (E) Myc-tagged WDR62-C and WDR62-C-AXA were transiently expressed with HA–JNK1 in HeLa cells. HA–JNK1 was immunoprecipitated and WDR62 co-immunoprecipitation determined by immunoblotting for the myc tag. Protein expression (input) in total cell lysates was also determined. (F) GFP-labeled WDR62-FL and truncation mutants were expressed in Ad293 cells. Their subcellular localization were determined during metaphase and colocalization with α- or γ-tubulin and DAPI was examined. (G) Ad293 cells were co-transfected with WDR62 siRNA and either GFP–WDR62-FL, GFP–WDR62-FL-AXA or GFP alone. The proportion of GFP-positive cells with normal bipolar, abnormal bipolar or multipolar spindles in metaphase was determined. n values are the total number of cells counted from three independent experiments. Representative images of multipolar, abnormal or normal bipolar spindles in Ad293 cells stained for γ-tubulin and DAPI are shown. (H) Representative images of GFP–WDR62-FL-AXA spindle localization in Ad293 cells treated with WDR62 or Con siRNA and stained for γ-tubulin and DAPI. Images in F and H are single z-sections.
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