Sandquist JC et al. (2018), An interaction between myosin-10 and the cell c...

XB-ART-55790

J Cell Biol 2018 Mar 05;2173:849-859. doi: 10.1083/jcb.201708072.

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An interaction between myosin-10 and the cell cycle regulator Wee1 links spindle dynamics to mitotic progression in epithelia.

Sandquist JC , Larson ME , Woolner S , Ding Z , Bement WM .

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Anaphase in epithelia typically does not ensue until after spindles have achieved a characteristic position and orientation, but how or even if cells link spindle position to anaphase onset is unknown. Here, we show that myosin-10 (Myo10), a motor protein involved in epithelial spindle dynamics, binds to Wee1, a conserved regulator of cyclin-dependent kinase 1 (Cdk1). Wee1 inhibition accelerates progression through metaphase and disrupts normal spindle dynamics, whereas perturbing Myo10 function delays anaphase onset in a Wee1-dependent manner. Moreover, Myo10 perturbation increases Wee1-mediated inhibitory phosphorylation on Cdk1, which, unexpectedly, concentrates at cell-cell junctions. Based on these and other results, we propose a model in which the Myo10-Wee1 interaction coordinates attainment of spindle position and orientation with anaphase onset.

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Species referenced: Xenopus laevis
Genes referenced: cdc25a cdk1 h2bc21 myo10 wee1 wee2
GO keywords: spindle [+]
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	Figure 1. Myo10 directly interacts with Wee1 via the MyTH4 domain. (A) In vitro binding assay between recombinant FLAG-Wee1A and empty glutathione-sepharose beads (blank) or bead-bound GST or GST-MyTH4-FERM (GST-4F). Inputs and washed glutathione pellets were boiled in SDS sample buffer and immunoblotted for GST and Wee1 by standard methods. (B) Bacterial recombinant sepharose-bound GST and GST-MyTH4 (GST-4) were mixed with concentrated extracts from stage 10 X. laevis embryos. Washed pellets (pel) and raw extract (input) were boiled in SDS sample buffer and subjected to immunoblot analysis. Input sample represents 2% of assay input and pellets each represent half of the pulldown product. Membranes were cut at the 50-kD marker and immunoblotted for Wee1 (top) or GST (bottom). The lower GST-reactive = bands in the GST-4 pel sample represent protein degradation products. (C) Same as B, except using recombinant GST-MyTH4-DD (GST-4-DD) protein. Numbers on the left side of the blots in A–C indicate molecular weight in kilodaltons. (D) Stage 10 embryos were prepermeabilized before fixation and immunostained with antibodies against α-tubulin (microtubule [MT] not in merge), Wee1B (green), and Myo10 (red). The Myo10 antibody is conjugated with Alexa Fluor 568 and was applied after the anti–rabbit secondary used to label the Wee1B antibody. Bar, 10 µm.
	Figure 2. Increasing Cdk1 activity shortens metaphase. (A) Boxplot (median ± quartiles, X indicates outlier) of metaphase duration in DMSO control or PD-166285–treated cells reveals significant decrease with Wee1 inhibition (P = 0.005, t test, n = 13 control, 7 treated). (B) Boxplot of metaphase duration in cells with or without exogenous Cdc25a demonstrates a significant decrease in metaphase duration with Cdc25a overexpression (P = 0.0008, t test, n = 7 control, 15 cdc25a).
	Figure 3. Cdk1 is enriched at cell junctions. (A) Left: Image from a live stage 10 embryo expressing eGFP-Cdk1. Image shown is z-projection of apical region showing the native GFP signal along junctions, particularly tricell junctions. Bar, 20 µm. Right: Stage 10 embryo fixed and immunostained with antibody specific for Y15 phosphorylated Cdk1 (pCdk1). Bar, 20 µm. Arrows show tricell junctions. (B) Left: Stage 10 control embryos immunostained with ZO-1 (red) and Cdk1–phosphorylated tyrosine 15 (green). Bar, 10 µm. Right: A 3D projection of the boxed region, rotated 90 degrees, with the apical surface to the right. Bar, 2.5 µm. (C) Apical projections of stage 10 embryo fixed in methanol/DMSO and immunostained with antibodies for cyclin B. Arrows show tricell junctions. Bar, 20 µm. (D) Midcell projections of stage 10 embryo fixed in formaldehyde and immunostained with antibodies for cyclin B (green) and microtubules (red). Arrowhead shows poleward spindle staining of cyclin B. Bar, 20 µm. (E) Apical projection of stage 10 embryo fixed in methanol/DMSO and immunostained with antibodies for cyclin B (green) and ZO-1 (red). Bar, 20 µm. (F) Series of still images from a movie of stage 10 embryos expressing mChe– histone H2B (red) and cyclin B–GFP (green). Time stamps indicate minutes after first frame. Bar, 20 µm. See also Video 1.
	Figure 4. MyTH4 domain expression increases junctional pCdk1 staining. (A) Uninjected control stage 10 embryos, or those expressing eGFP-MyTH4 or eGFP-MyTH4-DD or treated with 25 µM PD166285 for 30 min, were fixed and immunostained with antibodies against α-tubulin (MT, red) and pCdk1 (green). Bar, 20 µm. (B) Quantification of junctional pCdk1 signal in uninjected control embryos, or those expressing MyTH4 or MyTH4-DD or incubated for 30 min in 25 µM PD166285. y axis represents fold change in junctional pCdk1 relative to controls within the same experiment. The red line represents the mean control pCdk1 at junctions, defined as 1. A gray dot represents a single embryo, the blue dot the mean and the blue bars the 95% confidence interval. n = 10–15. All are significantly different from control (from left to right: P = 0.0004; P =0.0001; P =0.003). (C) Embryos were microinjected at the two-cell stage with mRNA encoding either GFP-MyTH4 (GFP-4) or GFP-MyTH4-DD (GFP-4-DD). At stage 10, whole-cell extracts were prepared and immunoblotted for α-tubulin (red) and pCdk1 (green). The numbers at the bottom of the blot indicate relative levels of pCdk1 in this image. Numbers on the left side indicate molecular weight in kilodaltons. (D) Stage 10 control or eGFP-MyTH4-expressing embryos were incubated for 0 or 30 min in 25 µM PD166285. The embryos were then fixed and stained for microtubules (anti–α-tubulin) and DNA (DAPI or TO-PRO3). The percentage of metaphase and anaphase cells were counted for each field of epithelium taken at 40×. In the plot, a gray dot represents a single embryo, the blue dot the mean, and the blue bars the 95% confidence interval. n = 10–20 embryos from four different experiments
	Figure 5. Wee1 inhibition rescues MyTH4-induced anaphase delay and reduces spindle oscillations. (A) Manually measured orientation of the mitotic spindle versus spindle axis at anaphase onset in cells expressing eGFP-tubulin and mTagBFP-CAAX in the presence of Myo10 morpholino (red and blue traces) or control (gray). Image shows time series of a Myo10-depleted cell expressing BFP-CAAX (blue) and eGFP-α-tubulin (green) and corresponds to the red trace. Time stamps indicate seconds before anaphase (anaphase = 0 s). Bar, 10 µm. (B) Same as A, except cells were treated with PD166285 (red and blue traces) or control (gray). Image shows time series of a PD166285-treated cell expressing mCherry–histone H2B (red) and eGFP–α-tubulin (green) and corresponds to the red trace. (C) Spindle orientation, as a function of spindle orientation at anaphase onset, as determined by the Spindlometer in control (two blue traces) and dynamitin2-CAAX-expressing (six yellow-red traces) embryos. Image shows time series of cells expressing eGFP-tubulin (green), mChe–histone H2B (red), mTagBFP-CAAX (blue), and dynamitin2-CAAX (unlabeled). Bar, 10 μm. (D) Manually measured metaphase duration in cells prepared as in D (control n = 6, Dctn2 n = 13, P = 0.0014).
	Figure S1. Interaction between X. laevis Myo10 and Wee1. (A) A Y2H using the MyTH4-FERM cassette from the tail of X. laevis Myo10 as bait identified Wee1 as a Myo10 binding partner. All hits identified more than once in the screen are shown. (B) GST-MyTH4-FERM pulls endogenous Wee1 and pWee1 out of extracts. Bacterial recombinant sepharose-bound GST and GST-MyTH4-FERM (GST-4F) were mixed with concentrated extracts from X. laevis embryos. Raw extract (input) and washed pellets were boiled in SDS sample buffer and subjected to immunoblot analysis. Here two identical gels were run and transferred to nitrocellulose and then immunoblotted (IB) with our Wee1 antibody (top) or a commercial antibody directed against Ser53- phosphorylated Wee1 (pWee1 S53).
	Figure S2. Wee1 localizes to the mitotic spindle. (A) Stage 10 embryos were prepermeabilized before fixation and immunostained with a commercial antibody against α-tubulin (microtubule [MT], red) and our Wee1 antibody (green). DNA is labeled with propidium iodide (blue). Bar, 10 µm. (B) Stage 10 embryos, without prepermeabilization, were immunostained for α-tubulin (MT, red) and with a commercial antibody against Ser53-phosphorylated Wee1 (pWee, green). Arrows indicate pole concentration of pWee. Other commercial antibodies against total Wee1 did not work in frog cells. The pWee antibody was used simply to verify Wee1 localization to the spindle, and we do not speculate on the relation of this phosphorylation to our model. See Watanabe et al. (2005) for a description of Ser53 pWee function. Bar, 10 μm
	Figure S3. Effects of kinase-dead Wee1 and cell cycle stage on junctional pCdk1 levels. (A) Stage 10 control embryos or those expressing GFP-tagged KD Wee1A were fixed and immunostained for α-tubulin (microtubule [MT], red) and Y15-phosphorylated Cdk1 (pCdk1, green). Images shows loss of junctional pCdk1 signal in KD-Wee1A expressing cells. KD mutant was generated via a K239R substitution (Leise and Mueller, 2002). Bar, 10 μm. (B) Quantification of junctional pCdk1 signal in individual control cells at different cell cycle stages (ana, anaphase; inter, interphase; meta, metaphase). Y-axis represents fold change in junctional pCdk1 relative to mean interphase cell signal in the same image. A gray dot represents a single cell, the blue dot the mean, and the blue bars the 95% confidence interval. Sample size = 61 (interphase), 30 (metaphase), and 21 (anaphase). Only mitotic cells completely surrounded by interphase cells were analyzed. ANOVA and Tukey’s post-hoc test showed statistically significant decreases in junctional pCdk1 for both mitotic populations compared with interphase cells (P = 0.0001 for both).

References [+] :

Adams, Metaphase spindles rotate in the neuroepithelium of rat cerebral cortex. 1996, Pubmed