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RESEARCH |
University of Cambridge, The Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, Tennis Court Road, Cambridge CB2 1QR, UK and Department of Genetics, Downing Street, Cambridge CB2 3EH, UK
Correspondence should be addressed to M Zernicka-Goetz; Email: mzg{at}mole.bio.cam.ac.uk
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The importance of actin in positioning the first meiotic spindle is unequivocal (Longo & Chen 1985, Leader et al. 2002), but the possibility of microtubule–actin interactions has not been investigated. Although microtubule-depolymerisation drugs still allow movement of the chromosomes to the cortex, the integrity of the chromosome mass is not maintained in the absence of a spindle (Maro et al. 1986, Van Blerkom & Bell 1986), and the fact that the direction of spindle migration in untreated oocytes is always along the long axis of the spindle (Verlhac et al. 2000) suggests that the spindle microtubules (MTs) do play a role in deciding the direction of movement. Moreover, in other systems where spindle migrations and rotations have been studied in detail, interactions between the spindle MTs and the cortex play an important role (for reviews see Segal & Bloom 2001, Dujardin & Vallee 2002, Kaltschmidt & Brand 2002, Ahringer 2003). Such interactions during positioning of the mouse meiotic spindle have been discounted because of the belief that there are no astral MTs emanating from this spindle (Verlhac et al. 2000, Maro & Verlhac 2002), although previously astral MTs were seen by electron microscopy (Szöllösi et al. 1972). Recent work investigating centrosomal and spindle structure dependence on culture conditions indeed reveals some astral MTs on meiotic spindles (Sanfins et al. 2003, Sanfins et al. 2004) and after metaphase II (MII) oocyte activation (Albertini & Barrett 2004).
Some progress has been made towards understanding the molecular details of the actin-dependent spindle migration. Roles for myosin II isoforms (Simerly et al. 1998), fodrin (Schatten et al. 1986a), formin-2 (Leader et al. 2002) and the two mammalian PAR-6 homologues mPARD6a and mPARD6b (partitioning-defective family members; Gray et al. 2004, Vinot et al. 2004) have been proposed, but the interactions between the chromosomes, or the spindle, and the cortex have not been characterised. Here we present studies of proteins proposed as candidate factors involved in possible microtubule-dependent positioning of the first mouse meiotic spindle.
Candidates were investigated whose homologues have known roles in spindle orientation or organisation in other model systems: PAR-1, CLASP2 and dynactin. PAR-1 is a kinase and is part of the partitioning-defective family. Mammalian homologues are also known as ELKL-motif kinases (EMKs) or microtubule affinity-regulating kinases (MARKs) (Inglis et al. 1993, Drewes et al. 1997, Ebneth et al. 1999, Trinczek et al. 2004). The roles of PAR-1 in cell polarisation and spindle orientation have been shown in several model systems (for reviews see Ahringer 2003, Macara 2004). CLASP2 is a CLIP (cytoplasmic linker protein)-associating protein with a role in stabilising MTs (Akhmanova et al. 2001), and possibly in providing capture sites to link MTs to the membrane (for reviews see McNally 2001, Schuyler & Pellman 2001, Mimori-Kiyosue & Tsukita 2003). The involvement of dynactin in spindle orientation has been demonstrated in several species (Busson et al. 1998, Skop & White 1998, O’Connell & Wang 2000, Sheeman et al. 2003).
In this report we show specific localisation of PAR-1, CLASP2 and dynactin on mouse meiotic spindles. In addition, we have found fixation conditions allowing visualisation of astral MTs on meiotic spindles. Thus our results further support the proposition that MTs may be important in meiotic spindle positioning.
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Materials and Methods |
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RT-PCR
Pools of five to fifteen embryos or oocytes were frozen at –80 °C in PBS. Two methods were used for RT-PCR. Similar results were obtained by both methods and with two sets of primers for each gene. All products were of the expected size and were checked by sequencing, and negative controls for both RT and PCR were performed.
Method A
mRNA was extracted using Dynabeads mRNA DIRECT Micro Kit (Dynal Biotech, Oslo, Norway), mRNA not eluted from beads. RT was performed at 42 °C for 30 min, using the bead oligo(dT)25 as primer and a 2:1 mix of AMV and M-MuLV reverse transcriptases (Roche Diagnostics GmbH, Mannheim, Germany). Gene-specific PCR was performed using Taq polymerase (Roche) and a Bio-Rad MJ Research PTC-100 Peltier thermal cycler (Bio-Rad, Hemel Hempstead, UK). Up to 45 cycles of a touchdown PCR (annealing temperature decreasing from 72 °C to 60°C) were used.
Method B
RT-PCR was performed using SuperScript one-step RT-PCR kit with platinum Taq (Invitrogen Paisley, UK) and 40–44 cycles of PCR (optimised for each primer pair so that a product was just detectable), with annealing temperature of 55 °C. This method uses gene-specific primers for the RT step as the RT and PCR proceed without interruption.
Primers
The following primers were used: EMK 5'-CGAGTGGAG-ACGCTCAGACC-3' and 5'-CTCCCACTGCACAAAGTT-CTCG-3', accession number NM_007928
[GenBank]
, fragment amplified 2041–2298; also EMK 5'-GCATGAGGACGATG-AGCT-3' and 5'-GTAGGAATTCGAGGTGGGAATGG-3', accession number NM_007928
[GenBank]
, fragment amplified 1014–1353; CLASP2 5'-ACCTAAAACACCTGGGAATCCTG-3' and 5'-AAAAGCAATCATACTGTGCGGC-3', accession number XM_135174, fragment amplified 258–556; also CLASP2 5'-CCTTGGGGATAAAGAGCCTAC-3' and 5'-TAGACCTGGGAAACCGCAG-3', accession number XM_135174, fragment amplified 3453–4115; ß-actin 5'-GAAGTGTGACGTTGACATCCG-3' and 5'-ACTTGCGGT-GCACGATGGAGG-3', accession number X03672
[GenBank]
, fragment amplified 929–1200.
Fixation and immunostaining
Before fixation, the zonae pellucidae were removed by a brief incubation in acidic Tyrode’s solution. Fixation was performed in 3.7–5% (w/v) paraformaldehyde plus 0.03–0.1% (v/v) Triton X100, up to 0.3% (v/v) Tween 20, up to 0.15% (v/v) glutaraldehyde and up to 2% (w/v) sucrose, at 37 °C for 20–40 min in agar-coated dishes. Preservation of microtubules was particularly sensitive to detergent concentration and temperature and exact parameters needed reoptimisation for each set of experiments. For immunostaining, all steps were carried out in agar-coated 96-well plates, oocytes/embryos being moved between solutions by mouth pipetting. After fixation, oocytes and embryos were permeabilised in 0.25–0.5% Triton X100 for 20 min at room temperature, aldehyde groups were quenched with NH4Cl at 2.6 mg/ml for 10 min at room temperature, blocking was in 3% (w/v) BSA in PBS for 30 min at room temperature. Primary and secondary antibodies were diluted in 3% BSA/PBS and incubations were at 4 °C overnight or at least 1 h at room temperature. DNA was stained with 2–5 µM TOTO-3 (Molecular Probes, Eugene, Oregon, USA) in PBS, for 10 min at room temperature. Oocytes/embryos were mounted on coverslips coated with 0.01% (w/v) poly-L-lysine, in Vectashield (Vector Laboratories Inc. Peterborough, UK), and viewed with an upright confocal microscope (Bio-Rad Radiance 2000 confocal mounted on a Nikon E800 microscope). Images were created using LaserSharp software (Bio-Rad) with filter settings adjusted to minimise bleedthrough between channels, and Z-series were saved as multiple TIFF files.
Western blotting
Embryos/oocytes were collected in PBS and frozen at –80 °C, 150 were used per lane. Mouse brain extract was prepared by homogenisation in ice-cold PBS with protease inhibitors, followed by boiling in Laemmli buffer (Sigma, Poole, Dorset, UK). Oocyte/embryo samples were thawed in Laemmli buffer and boiled for 3 min prior to loading. Proteins were separated by SDS-PAGE (7.5% acrylamide) using a Mini-Protean 3 electrophoresis system (Bio-Rad), then transferred to Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Amersham, Bucks, UK) using a mini trans-blot transfer cell (Bio-Rad) for immersion transfer. Membranes were blocked overnight at 4 °C in 5% dried milk in Tris-buffered saline. Antibody incubations were performed at room temperature for 1 h. Horseradish peroxidase-conjugated secondary antibodies were detected with the ECL + Plus western blotting system (Amersham Pharmacia Biotech).
Antibodies
Primary antibodies
The following primary antibodies were used: mouse anti-dynactin p50 from BD Transduction Laboratories (BD Biosciences Pharmingen, Oxford, UK), used at 1:40 for immunostaining and 1:250 for western blotting; CLASP2 no. 2358 rabbit antiserum was a gift from A Akhmanova (Akhmanova et al. 2001), used at 1:300 for immunostaining and 1:500 for western blotting; rabbit anti-EMK was a gift from T V Kurzchalia (Böhm et al. 1997), used at 1:50 for immunostaining and 1:1000 for western blotting; tubulin rat monoclonal clone YL1/2 from Abcam (Cambridge, UK), used at 1:1000 for both immunostaining and western blotting; rabbit anti-GFP (green fluorescent protein) from Abcam used at 1:4500 for western blotting. Controls were ChromPure purified whole molecule mouse IgG (Jackson ImmunoResearch Laboratories, Stratech Scientific Ltd, Soham, Cambs, UK) and rabbit pre-immune serum as appropriate.
Secondary antibodies
The following secondary antibodies were used: AlexaFluor 488 goat anti-rat IgG (1:600), AlexaFluor 488 donkey anti-mouse IgG (1:500) and AlexaFluor 568 goat anti-mouse (1:500) (Molecular Probes); rhodamine Red-X-conjugated Fab fragment donkey anti-rabbit IgG (1:200), FITC-conjugated Fab fragment donkey anti-rabbit (1:400) and Texas Red dye-conjugated Fab fragment goat anti-rat IgG (1:500) (Jackson ImmunoResearch Laboratories); horseradish peroxidase-conjugated anti-rat, anti-mouse and anti-rabbit (1:10 000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Overexpression of dynactin-p50
p50-T7, a plasmid containing human dynactin-p50 (accession number NM_006400
[GenBank]
), was a gift from T Hyman (Wittmann & Hyman 1999). In order to produce a stable mRNA in vitro, with an N-terminal GFP tag, p50 was transferred into pßGFP/RN3P (Zernicka-Goetz et al. 1996, 1997). p50 was amplified using the following primers: 5'-CGCGTCG-ACGCGGACCCTAAATACG-3' and 5'-CGAAGCTTT CACTTTCCCAGCTTCTTCATC-3'. The PCR product was cut with HindIII, blunted with Klenow polymerase (Roche) at 1 unit/µg DNA, then cut with SalI. GFPal (pßGFP/RN3P with an extra multiple cloning site) was cut with StuI then SalI and ligated to the p50 PCR product. The fusion construct was sequenced to check the sites of ligation and fidelity of PCR. The resulting plasmid was linearised with SfiI and in vitro transcription was performed with T3 polymerase using the mMessage mMachine kit for production of capped RNA (Ambion, Austin, Texas, USA). The mRNA was phenol/chloroform-extracted and precipitated with isopropanol, washed in 70% ethanol then resuspended in nuclease-free water for injection at 1 µg/µl. Microinjection of oocytes was performed using negative capacitance as previously described (Weber et al. 1999).
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). Western blotting (Fig. 1B) showed the presence of EMK protein in mouse oocytes from GV to MII stage, and in the zygote and two-cell embryo. Interestingly, two isoforms of EMK were originally isolated (Böhm et al. 1997) and both were seen in the control brain extract lane, but only the higher weight band (85 kDa) could be seen in the mouse oocytes and early embryos. These two isoforms probably arise from differential splicing (Espinosa & Navarro 1998). The larger, 170 kDa 
-isoform of CLASP2 (Akhmanova et al. 2001) was detected by western blot in all stages from GV oocytes to two-cell embryos, the brain extract contained the lower weight ß-isoform in addition, as previously reported (Akhmanova et al. 2001). The p50 subunit of dynactin was also present at similar levels in mouse oocytes, zygotes and two-cell embryos, two bands of approximately 50 kDa being detected. This antibody was also specific for an additional intense band of about 85 kDa in oocytes injected with GFP-p50, which was also recognised by an anti-GFP antibody (data not shown).
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) (100%, n = 7). Comparison with the MT network in the GV oocyte (Fig. 2B) revealed similarity, although incompatibility of fixation conditions prevented co-staining. When the first meiotic spindle formed, EMK accumulated on the spindle (Fig. 2C) (82%, n = 11). This localisation continued through the first division, with EMK on the MTs of the spindle during anaphase (Fig. 2D) and cytokinesis (100%, n = 4). Similarly at MII, EMK clearly associated with the spindle (Fig. 2L) (68%, n = 44). At this stage, EMK co-localisation with MTs was evident, as the peripheral position of the MII spindle allowed better MT fixation. Use of rabbit pre-immune serum demonstrated the specificity of this immunostaining (compare Fig. 2J with 2K). In the zygote, EMK was again located in a network pattern, and densely around the nuclear envelopes (Fig. 2E) (100%, n = 9), a pattern that could also be observed at two-cell stage (Fig. 2H) (100%, n = 17). EMK accumulated around the condensing chromosomes early in prophase (Fig. 2H) (100%, n = 4), then during the first two embryonic divisions EMK was located in the region of highest MT density, on the spindles and in their vicinity (Fig. 2F, G and I) (100%, n = 15). Thus EMK localised specifically to the spindles during meiotic and mitotic divisions in mouse oocytes and early embryos. Cytoplasmic staining was also evident at all stages examined, indicating that not all EMK relocates to forming spindles. The accumulation of EMK in areas of most active MT function could be consistent with a role regulating MT dynamics.
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) (64%, n = 47). This specific localisation was even more apparent at MII spindle poles (Fig. 3B and C) (63%, n = 46). Throughout meiosis there was also diffuse punctate staining of CLASP2. At both metaphases there was some detectable enrichment on the chromosomes, probably reflecting the kinetochore staining seen in other cell types (e.g. Maiato et al. 2003). The staining pattern was shown to be specific by use of rabbit pre-immune serum as a negative control (compare Fig. 3C with 3D).
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). These dots are likely to be the kinetochores, as kinetochore localisation of dynactin has been previously reported in mammalian cell lines (Echeverri et al. 1996). This kinetochore stain was specific, as shown by using purified whole molecule mouse IgG as a negative control (compare Fig. 4A with 4B). In maturing oocytes there are many non-spindle cytoplasmic asters, which persist until just after fertilisation. Dynactin-p50 localised to the centres of these asters (Fig. 4C and D). In zygotes there was slight accumulation of dynactin-p50 around both pronuclei (Fig. 4F), consistent with its proposed role in pronuclear migration in bovine zygotes (Payne et al. 2003). On mitotic spindles, as well as accumulating on kinetochores, dynactin-p50 localised to the spindle poles (Fig. 4G and H) (100%, n = 16). This kinetochore and spindle pole staining persisted through anaphase and until the nuclei reformed (Fig. 4J and K) (73%, n = 15). A similar pattern of dynactin-p50 localisation was observed upon microinjection of GFP-p50 mRNA to overexpress this subunit. However, in addition to the kinetochore and cytoplasmic MTOC staining, there was a further large accumulation of dynactin-p50 on the poles of the MII spindle (Fig. 4L). This suggests a differential affinity of dynactin-p50 for subsets of MTOCs.
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). When fixation in 5% paraformaldehyde plus Triton X100, Tween 20 and sucrose (see Materials and Methods) was optimal, approximately 70% (23/33) of meiotic spindles had visible astral MTs. Fixation in 3.7% paraformaldehyde plus Triton X100 also showed astral MTs in more than one third (11/28) of spindles fixed. These preparations also showed that the barrel-shaped first meiotic spindles can have mutiple MTOCs at the spindle poles, making the spindles more triangular or trapezoid in shape (for example Fig. 5B) (27%, n = 107).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The accumulation of EMK on meiotic and mitotic spindles suggests that also in mouse oocytes and embryos it might act as a regulator of MT dynamics, as has been shown in other model systems (Drewes et al. 1997, Ebneth et al. 1999, Shulman et al. 2000, Trinczek et al. 2004). The localisation pattern is, however, different from that seen in C. elegans embryos, Drosophila oocytes and in polarised mammalian epithelial cells, where there is an accumulation of PAR-1 at a specific region of the cortex (Guo & Kemphues 1995, Böhm et al. 1997, Shulman et al. 2000). This cortical accumulation creates a region of MT destabilisation, important for localising proteins in these polarised cells (Shulman et al. 2000, for review see Pellettieri & Seydoux 2002). The differences in localisation suggest that, in the non-epithelial oocyte and early embryo, EMK might be involved in regulating spindle MT dynamics, rather than the dynamics of the MTs which organise the polarisation of the whole cell. The observed localisation on MTs is more like the MT-plus-centrosomal localisation shown for MARK4 (Trinczek et al. 2004). MARK4 kinase activity was shown to have a bundling effect on MTs (Trinczek et al. 2004). Such an effect on the spindle MTs by EMK in mouse oocytes and embryos could be important for proper spindle structure. Recently, another member of the PAR family of proteins, mPARD6, was reported to localise to the mouse meiotic spindles (Vinot et al. 2004). Taken together with the pattern of EMK/PAR-1 localisation shown here, this further suggests that the PAR proteins may have different roles in mouse oocytes because, in other systems where PAR proteins have a role in mediating cell polarity, PAR-1 and PAR-6 are found in mutually exclusive locations (for reviews see Ahringer 2003, Macara 2004).
The specific localisation of CLASP2 to spindle poles during meiosis was similar to that reported (in HeLa cells) for CLASP1 (Maiato et al. 2003) and for the Drosophila homologue Mast/Orbit (Inoue et al. 2000, Lemos et al. 2000) during mitosis. In Drosophila, the CLASP homologue has been shown to play a role in chromosome alignment, kinetochore-MT attachment and maintenance of spindle bipolarity (Maiato et al. 2002), and studies in mammalian cells have shown CLASPs to be important for regulating the dynamics of MTs (Akhmanova et al. 2001, Maiato et al. 2003). From the localisation similarities with other cell types, a similar function of CLASP2 during meiosis can also be predicted. It is also possible that CLASP2 has additional functions at the meiotic spindle poles in stabilising the MTs important for direction of spindle migration and/or anchorage at the cortex.
It has been proposed that non-spindle MTOCs present during meiosis are important for the migration of the zygotic pronuclei and for formation of the first mitotic spindles (Maro et al. 1985, Schatten et al. 1986b). The localisation pattern of dynactin shown here supports the view that there are two different groups of MTOCs in the oocyte, spindle and non-spindle MTOCs (Messinger & Albertini 1991), and that the non-spindle MTOCs mature to form the embryonic spindles, because dynactin was found only on the non-spindle MTOCs in oocytes, then on the spindle MTOCs in embryos. The localisation of p50 to meiotic spindle MTOCs when it was overexpressed points to a differential affinity for spindle and non-spindle MTOCs, with saturation on all MTOCs after overexpression. It is likely that the role of dynactin in pronuclear migration is conserved between bovine (Payne et al. 2003) and mouse zygotes, given the similar localisation around the pronuclear envelopes. This lends further support to the model of progression of MTOCs from the cytoplasm of oocytes to the pronuclear envelopes and then to mitotic spindles (Maro et al. 1985, Schatten et al. 1986b), and suggests that the dynein/dynactin complex could play a role in the process of MTOC organisation.
Our work has focused on proteins with known roles in the regulation of microtubules and found that they have specific localisation on the spindles of mouse oocytes and early embryos. Each of the proteins studied here has been previously shown to bind or regulate other microtubule-associated proteins. PAR-1 homologues regulate the affinity of some microtubule-associated proteins for MTs. CLASPs and the dynein/dynactin complex are part of the ever-growing family of plus end-associated proteins, which in many cases stabilise growing MTs. Our demonstration of astral MTs on meiotic spindles suggests that stabilisation of plus ends may be important for positioning of these spindles. Interactions between the astral MTs and the cell cortex may play roles alongside the actin-dependent migration of the first meiotic spindle to the cortex, and may be involved in tethering the second spindle during the prolonged metaphase arrest. As the meiotic divisions create an asymmetry in the egg which relates to later patterning of fertilised embryos (Gardner 1997, Weber et al. 1999, Ciemerych et al. 2000, Plusa et al. 2002, Piotrowska-Nitsche & Zernicka-Goetz 2005, Piotrowska-Nitsche et al. 2005), the study of meiotic asymmetry is important for further understanding of early embryonic development.
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Footnotes |
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Received 14 January 2005
First decision 1 March 2005
Revised manuscript received 20 May 2005
Accepted 1 June 2005
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References |
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140 kDa), dynactin-p50 50 kDa and tubulin 55 kDa.
