RanGTP and importin β regulate meiosis I spindle assembly and function in mouse oocytes (2019)
Figure 7

Figure 7. Ran and importin β regulate MT assembly and MTOC coalescence in MI spindles

(A). Maximum intensity z-stack projections of confocal IF images (DNA stain, indirect IF for tubulin and TPX2) in untreated oocytes (control), oocytes expressing RanT24N or injected with importin β(71-876). The representative of at least 5 experiments is shown.

(B-D) Maximum intensity z-stack projections of IF images in control and importin β(71-876)-injected cells were used to quantify the spindle vs. whole-cell area fraction (B) and the ratios of the average signal in the spindle vs. cytoplasm for the tubulin (C) and TPX2 (D) staining. Means ± SDs, with t-test in (B, C) and Mann-Whitney test in (D), oocyte numbers indicated in brackets.

(E) Ratios of the spindle-associated fractions of TPX2 vs. tubulin were calculated in maximum intensity z-stack projections of IF images. Means ± SDs, Mann-Whitney test, oocyte numbers indicated in brackets.

(F) Maximum intensity z-stack projections of IF images were used to quantify the number of ectopic MT asters. Means ± SDs, ANOVA with Kruskal-Wallis and Dunn's tests, oocyte numbers indicated in brackets.

(G) Maximum intensity z-stack projections of confocal IF images stained for DNA, pericentrin, and tubulin (directly labeled primary antibody). A representative example of at least 2 experiments is shown. Scale bar, 20 µm.

(H-I) The number and size of pericentrin foci were quantified in maximum-intensity z-stack projections of IF images stained as in (G) in at least 2 separate experiments. Means ± SDs, ANOVA with Kruskal-Wallis and Dunn's tests.

(J) Proposed model: The suppression of importin β by RanGTP is required for MT nucleation and assembly, enabling the assembly and function of the MI spindles. Through separate mechanisms, RanGTP opposes the role of importin β in preventing the coalescence of pericentrin-containing

Materials and Methods

Mouse oocyte preparation and culture

Animals were treated and used according to the guidelines of the Institutional Animal Care and Use Committee of Johns Hopkins University School of Medicine or according to the policies of the Expert Committee for the Approval of Projects of Experiments on Animals of the Academy of Sciences of the Czech Republic. In all experiments, GV oocytes were collected at room temperature from 8-10-weeks-old female mice 44-48h after injection with pregnant mare serum gonadotropin (5 IU; Sigma). Live cell FLIM imaging in Fig. 1,2 and 3 and the IF analyses in fixed cells in Fig. 5 were done with oocytes from the CF1 mice. Oocytes from the CD1 or histone H2B-EGFP transgenic mice with CD1 genetic background (Hadjantonakis and Papaioannou, 2004; Mayer et al., 2016) were used for live cell imaging in Fig. 4,5 and 6. The CF1 oocytes were kept in MEM-PVP containing IMBX(Stein and Schindler, 2011) during collection and microinjection and incubated in the IBMX-free M16 media (Sigma-Aldrich). The CD1 and histone H2B-EGFP transgenic oocytes were collected in M2 media containing 2.5 mM milrinone (Sigma-Aldrich) and cultured in MEM medium (Sigma-Aldrich) supplemented with 0.22 mM sodium pyruvate (Sigma-Aldrich), 4 mg/ml BSA (Sigma-Aldrich) penicillin, streptomycin (100 U/ml: 100 mg/ml, Sigma-Aldrich). The cultures and live imaging were performed at 37 °C in a humidified atmosphere with 5% CO2 under paraffin oil. The paraffin oil cover was omitted in all cultures involving the treatment with IPZ.

DNA and tubulin dyes

The GV oocytes were released into the M16 media containing 200 nM SiR700-DNA (Cytoskeleton) to enable visualization of chromosomes during FLIM measurements. Oocytes were stained with 100 nM SiR-tubulin (Spirochrome) for MT visualization. SiR-tubulin was added to both M2 and MEM medium.

Time-lapse confocal imaging

Time-lapse image acquisitions in Figure 4A and 5A were performed using Leica TCS SP5 with an HCX PL Apo Lambda Blue 40×1.25 oil objective with 1× zoom, and 12 confocal 7.5-µm optical sections were taken with a 1024 × 1024 pixel image resolution using 10 min time intervals as described previously (Mayer et al., 2016). For MTOCs imaging (Figure 4E and H, Fig. 6 A) in oocytes expressing mEGFP-mCDK5RAP2, H2B-mCHERRY from microinjected mRNAs and stained with SiR-tubulin we used the tracking function of the Matrix Screen module in LAS AF software (Leica Microsystems). 16 confocal 2.5-µm optical sections with a 7.75× zoom on the area of the chromosomes of individual oocytes in 256 × 256-pixel image resolution by 1000Hz speed bi-directional scan were acquired as described previously (Balboula et al., 2016).

Plasmids

The plasmids used in this study are listed in Table 1. We used previously described plasmids for the in vitro transcription of histone H2B-mCherry, mEGFP-CDK5RAP2, YFP-hTPX2, RanQ69L and RanT24N (Balboula et al., 2016; Brunet et al., 2008; Dumont et al., 2007; Kitajima et al., 2011) and plasmids for the production of the recombinant importin β(71-876) proteins in E.coli (Nachury et al., 2001). The pEGFP-N1-VAPA (1-242) was a gift from Axel Brunger (Addgene plasmid # 18874), and the pET His6 Sumo TEV LIC cloning vector (1S) was a gift from Scott Gradia (Addgene plasmid # 29659). Plasmids for YFP-hTPX2, RanQ69L, and RanT24N were kindly provided by Marie-Hélène Verlhac.

The pGEMHE2 plasmid was prepared by inserting synthetic oligo with multiple restriction cloning sites between the Nhe1 and Not1 sites in pGEMHE (Liman et al., 1992). Restriction cloning was used to insert the Rango-3 (Soderholm et al., 2011) between the BamH1 and HindIII in pGEMHE2 and RBP-3 between the Not1 and Xho1 in pGEMHE2. The WT human RCC1 α-mCherry (Hasegawa et al., 2013) was cloned between BamH1 and BsrG1 in pGEMHE1-RBP3 to create the pGEMHE2-RCC1-mCherry. The human WT Ran was cloned between BsrG1 and HindIII in pGEMHE2-RBP3 to create the pGEMHE2-EGFP-Ran. The rat VAPA (1-242) (Kaiser et al., 2005) was inserted between Nhe1 and BsrG1 in pGEMHE. The human importin β(71-876) from pET30a 6His-S-Importin β(71-876) (Nachury et al., 2001) released by EcoRV and Xho1 was cloned between the SsPI and Xho1 sites in pET-6His-SUMO TEV LIC (1C). The pGEMHE-mCherry-mTrim21 (Clift et al., 2017) was modified by cloning the V5 tag between Age1 and Xho1 sites to replacing mCherry, resulting in pGEMHE-V5-mTrim21.

The human WT Ran was cloned by PCR into pRSET-A, followed by the insertion of synthetic oligos coding for the N-terminal AviTag and a linker (sequence: PSPSTPPTSC), resulting in a construct for the E.coli expression of N-terminally biotinylated Ran protein. Restriction cloning was used to replace the WT Ran in the above with RanT24N and RanQ69L from pET30a plasmids (Kalab et al., 2002). The Q5 mutagenesis kit (NEB) was used to introduce the T42A mutation into the above descript pRSET-Avitag vectors for the WT or T24N Ran.