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Article |
Myosin-10 and actin filaments are essential for mitotic spindle function
Correspondence to Sarah Woolner: sarah.woolner{at}manchester.ac.uk
Mitotic spindles are microtubule-based structures responsible for chromosome partitioning during cell division. Although the roles of microtubules and microtubule-based motors in mitotic spindles are well established, whether or not actin filaments (F-actin) and F-actin–based motors (myosins) are required components of mitotic spindles has long been controversial. Based on the demonstration that myosin-10 (Myo10) is important for assembly of meiotic spindles, we assessed the role of this unconventional myosin, as well as F-actin, in mitotic spindles. We find that Myo10 localizes to mitotic spindle poles and is essential for proper spindle anchoring, normal spindle length, spindle pole integrity, and progression through metaphase. Furthermore, we show that F-actin localizes to mitotic spindles in dynamic cables that surround the spindle and extend between the spindle and the cortex. Remarkably, although proper anchoring depends on both F-actin and Myo10, the requirement for Myo10 in spindle pole integrity is F-actin independent, whereas F-actin and Myo10 actually play antagonistic roles in maintenance of spindle length.
Abbreviations used in this paper: HMM, heavy meromyosin; LatB, latrunculin B; MO, morpholino; Myo10, myosin-10.
© 2008 Woolner et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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Introduction |
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In the above examples, the key site of actomyosin–microtubule interaction is the cortex, and its major role is thought to be the anchoring or transport of spindle microtubules that extend there. However, whether actomyosin is an important component of the mitotic spindle interior or if actomyosin–microtubule interactions play additional roles in mitotic spindle function and assembly is the subject of an old and intense controversy. A series of fluorescence and electron microscopy studies in the 1970s described the presence of F-actin in the mitotic spindle (Schloss et al., 1977; Forer et al., 1979), and more recent work has found that various actin poisons and myosin inhibitors have varying effects on spindle structure and function (Fabian and Forer, 2007; Forer et al., 2007). However, the studies showing localization were challenged as artifacts of the methods used to prepare the samples (Barak et al., 1981), and inhibition of what was then thought to be the only metazoan actin-based motor, myosin-2, was shown to prevent cytokinesis without having any effect on spindle assembly and function (Kiehart et al., 1982). Finally, and most tellingly, morphologically normal and apparently functional spindles are routinely produced in Xenopus laevis egg extracts under conditions where F-actin assembly is prevented (Mitchison et al., 2005).
In contrast to mitotic spindles, meiotic spindles not only require F-actin for cortical anchoring, but, in many cases, also require F-actin for assembly (Gard et al., 1995; Kim et al., 2000; Sun et al., 2001; Sardet et al., 2002). Consistent with a role for actomyosin in the meiotic spindle, Myosin-10 (Myo10), an unconventional myosin, was recently shown to localize to meiotic spindles and to be required for their proper assembly (Weber et al., 2004). Myo10 has the unusual property of being able to bind to both F-actin, via a motor domain in its head (Homma et al., 2001; Homma and Ikebe, 2005); and microtubules, via a C-terminal MyTH4/FERM domain cassette (Weber et al., 2004). Intriguingly, Myo10 was also recently implicated in mitotic spindle positioning in cultured mammalian cells (Toyoshima and Nishida, 2007). Because Myo10 can bind both F-actin and microtubules, is required for meiotic spindle assembly, and is involved in mitotic spindle positioning, it is a strong candidate to mediate F-actin–microtubule interactions in mitotic spindles. We have therefore analyzed the role of both Myo10 and F-actin in mitotic spindle assembly and function in the epithelium of the vertebrate X. laevis. Using a combination of live cell imaging, morpholino (MO)-mediated Myo10 knockdown, and gene replacement, we show that Myo10 localizes to the mitotic spindle and is required for proper spindle anchoring, spindle pole integrity, spindle length control, and mitotic progression. Furthermore, we reveal the existence of dynamic actin cables within the mitotic spindle and show that F-actin and Myo10 play both overlapping and distinct roles during mitosis.
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-tubulin showed that Myo10 localized to a region just beside the -tubulin domain (Fig. 1 b). The localization pattern displayed by Myo10 (in the nucleus at interphase and moving to the spindle poles during mitosis) is reminiscent of spindle assembly factors such as TPX2 and NuMA (Merdes et al., 1996; Wittmann et al., 2000). Indeed, triple labeling for 
-tubulin, Myo10, and TPX2 demonstrated colocalization of Myo10 and TPX2 in the nucleus and in overlapping domains at spindle poles (Fig. 1 c).
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-tubulin at 12, 16, and 24 h after microinjection, and mitotic spindles in the outermost epithelial layer of the embryo were assessed for defects. At 12 h, no discernible mitotic phenotype was seen; however, by 16 h, Myo10 morphants showed a clear multipolar spindle phenotype, which was further enhanced by 24 h (Fig. 2, b and c). At 24 h, an increase in the number of normal bipolar spindles was also seen in the morphants compared with water-injected controls (Fig. 2 c). The increase in bipolar spindles in morphants likely reflects a delay in mitotic progression (see following section). Further analysis of the morphant spindles revealed a third spindle phenotype; Myo10 morphant spindles were significantly longer than controls, both in terms of absolute length (not depicted) and when corrected for cell size (Fig. 2 d). To test if other aspects of spindle organization were affected in morphants, propidium iodide was used to visualize chromosomes. As in controls, most of the chromosomes were localized near to the metaphase plate in morphant metaphase spindles (Fig. 2 e). Moreover, Myo10 morphant spindles in the early stages of the phenotype, exhibiting increased length and one or two extra poles, were observed to have similar chromosomal content to controls (Fig. 2 e), which indicates that these phenotypes were not an indirect result of a previous failed cell division. To ensure that these phenotypes represent Myo10 depletion, two control experiments were performed: the morphant phenotype was rescued by microinjection of full-length Myo10 (see "The head and tail of Myo10..." and Fig. 5), and embryos were microinjected with a mispair control MO. The control MO differed from the Myo10 MO by just five mispairing nucleotides, and gave a reduced level of Myo10 knockdown (Fig. 2 a) and a correspondingly reduced level of phenotype compared with the morphant (Fig. 2 c), which indicates that Myo10 is the relevant target of the MO. Although these controls demonstrated the specificity of the MO, it was possible that knockdown of Myo10 in the embryo caused spindle defects indirectly by, for example, disrupting nuclear architecture or previous cytokinetic events. To test if the effect on spindle structure was direct, we used the X. laevis egg extract system to assemble spindles in vitro in the absence or presence of a Myo10 antibody. Although control spindles displayed normal bipolar morphology, spindles assembled in the presence of the Myo10 antibody appeared longer and exhibited multipolar phenotypes similar to those in the Myo10 morphants (Figs. 2 f and S2, available at http://www.jcb.org/cgi/content/full/jcb.200804062/DC1).
Myo10 morphant spindles are initially bipolar and then undergo pole fragmentation
The fact that chromosomes localized near the metaphase plate in Myo10 morphant spindles suggested that these spindles initially assembled normally. To explore this possibility, live confocal imaging was used. Using GFP–-tubulin to visualize mitotic spindles, we found that it was possible to follow mitosis live in the cells of X. laevis embryonic epithelium by time-lapse confocal microscopy (Fig. 3 a and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200804062/DC1).
Live imaging revealed four features of the Myo10 morphant phenotype that were not apparent from fixed cell analysis. First, in the initial stages of the morphant phenotype, spindles assembled normally with two poles, although these spindles were longer than controls (Videos 2 and 3). Second, supernumerary poles formed via fragmentation of the original poles, and rather than a wholesale fragmentation, fragmentation was quantal, with only one supernumerary pole forming from each original pole at a time (Fig. 3 b and Videos 2–4). Third, morphant spindles delayed in metaphase for significant periods of time: morphant metaphase took a mean 46.0 ± 8.0 min (n = 5), with 25.4 ± 3.2 min of that time spent as a bipolar spindle, before any pole fragmentation, compared with a metaphase duration of 6.6 ± 0.5 min (n = 4) in controls. The morphant metaphase delay could explain the increase in bipolar spindles seen in the fixed analysis of the morphant (Fig. 2, b and c). Fourth, as the morphant phenotype progressed, cytokinesis failures began to manifest, both in cells where spindles were elongated but remained bipolar (Video 5) and also in those where multipolar spindles, after a long delay, entered a disorganized anaphase and attempted cytokinesis (Video 2). As a result of these aberrant divisions, binucleate cells were often seen at later time points, and when these cells entered mitosis, spindles were multipolar from the onset of assembly (Videos 2 and 6).
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-tubulin localized to every supernumerary pole in morphant multipolar spindles, irrespective of whether the spindle had recently undergone pole fragmentation (in spindles with one or two extra poles; Fig. 3 c) or was a more established multipolar spindle (three or more extra poles; Fig. 3 c). Morphant spindles were also stained to assess the localization of the spindle assembly factor TPX2. Intriguingly, TPX2 localization was more diffuse in morphant spindles compared with controls, with TPX2 no longer showing a strong concentration at the spindle pole but instead spreading down the spindle microtubules toward the metaphase plate (Fig. 3 d). This result indicated that Myo10 might be required to recruit or maintain TPX2 at the spindle pole during mitosis. To test for an interaction between Myo10 and TPX2, a GST pull-down assay was performed using the MyTH4/FERM domain cassette of Myo10 and full-length TPX2. This assay demonstrated that Myo10 binds directly to TPX2 via its MyTH4/FERM cassette (Fig. 3 e).
Abnormal spindle movement and anchoring in Myo10 morphants
Mitotic spindles in control embryos typically underwent a great deal of abrupt movement in the cell during metaphase, such that they rotated with a jerky motion, dramatically changing their position in very short periods of time (Fig. 4 a and Video 1).
In contrast, Myo10 morphant spindles, even before they sprouted supernumerary poles, rotated with a much smoother motion (Fig. 4 b and Videos 2 and 3). Rotation rate measurements showed that control spindles underwent only slight rotation before metaphase, displayed the greatest changes in rotation rate during metaphase, and rotated slower once anaphase began (Fig. 4 c, blue). In comparison, morphant spindles never reached the same extremes in rotation rate but instead gently rotated throughout their extended metaphase; similar movement continued after spindle pole fragmentation and remained unchanged right up to an aberrant anaphase (Fig. 4 c, red).
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-tubulin. Treatment of water-injected embryos with LatB caused cells of the epithelium to round up but did not increase the number of bipolar spindles, promote spindle pole fragmentation, or significantly affect mean spindle length (Fig. 6, a, c, and d).
However, LatB treatment resulted in impaired anchoring such that spindles oriented perpendicular to the plane of the epithelium, unlike the parallel arrangement in controls (Fig. 6 a). Treatment of Myo10 morphant embryos with LatB had no significant effect on the number of bipolar or multipolar spindles (Fig. 6 c). However, a startling affect on spindle length was seen when Myo10 morphants were treated with LatB: the longer spindles seen in the morphant were rescued by disrupting F-actin (Fig. 6, b and d), which suggests that Myo10 and F-actin function antagonistically to maintain spindle length.
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-tubulin revealed that F-actin was present in control mitotic spindles. Dynamic actin cables were seen surrounding and running the length of the spindle as it assembled, and these cables remained associated with the spindle through early mitosis (Fig. 7 a, arrows; and Video 7, available at http://www.jcb.org/cgi/content/full/jcb.200804062/DC1).
Many F-actin cables were also concentrated around the spindle poles, with some cables extending between the poles and the cell cortex (Fig. 7 b and Video 7). As spindles rotated during metaphase, the arrangement of F-actin cables mirrored this movement, as if the cables helped to determine the direction of spindle motion (Fig. 7 c and Video 8). As spindles entered anaphase, F-actin remained concentrated around the poles (Fig. 7 a and Video 8).
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Discussion |
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Our findings indicate that Myo10 and F-actin both help to anchor the spindle during mitosis. First, spindles in Myo10 morphants undergo strikingly different movements to controls, with morphant spindles moving smoothly compared with the rapid changes in rotation rate displayed by control metaphase spindles. Second, disrupting F-actin in embryos leads to spindle positioning defects, with spindles orienting perpendicular, rather than parallel, to the cortex. The fact that knocking down Myo10 eliminates jerky spindle movement suggests that Myo10 functions in spindle anchoring by providing transient links between the spindle and the cortex through brief, high-affinity interactions with F-actin, generating a stop-start movement. It may seem counterintuitive that disrupting anchoring would lead to a reduction in rotation rate, but this phenomenon has also been seen in the Caenorhabditis elegans embryo, where knockdown of dynein, a microtubule motor that is located at the cell cortex and pulls on spindle microtubules, leads to an attenuation of spindle oscillation (Pecreaux et al., 2006). It is unclear whether Myo10's spindle anchoring capacity is mediated through cortically localized Myo10, possibly functioning in concert with cortical dynein, or through Myo10 associated with the spindle. However, it is interesting to note that because we see actin cables extending between the cortex and the spindle pole, the latter possibility is highly plausible. The notion that Myo10 acts to anchor the spindle through interactions with F-actin fits well with the fact that actin is known to play a vital role in positioning the spindle during oriented cell division (Gundersen and Bretscher, 2003; Rodriguez et al., 2003; Gundersen et al., 2004) and the recent finding that Myo10 is required for spindle orientation in cultured mammalian cells (Toyoshima and Nishida, 2007).
A second function of Myo10 during mitosis is to maintain spindle pole integrity, as disrupting Myo10 either in vitro or in vivo leads to the formation of multipolar spindles. An unusual aspect of this phenotype is that spindle poles first appear to assemble normally and only fragment subsequently. This suggests that Myo10 is not required to establish bipolarity during spindle assembly but instead acts to stabilize spindle poles as mitosis progresses, although it is also possible that the late onset of the multipolar phenotype simply reflects a slow loss of Myo10 protein in morphant cells. It is interesting to note that a similar late-onset pole fragmentation phenotype is seen when Borealin, a member of the chromosomal passenger complex, is depleted in mammalian cells (Gassmann et al., 2004). Exploring a link between Myo10 and this complex could be a fruitful area for further study. Strikingly, the function of Myo10 in the spindle pole is independent of F-actin, as LatB treatment has no effect on spindle pole integrity either in control or morphant embryos. Furthermore, injection of GFP-Myo10-IQT, a Myo10 tail construct that lacks the actin-binding myosin head, specifically rescues the pole fragmentation phenotype in Myo10 morphants. A possible explanation for Myo10 tail function in spindle pole integrity is that domains in the tail act to recruit and/or retain pole proteins such as TPX2, NuMA, or -tubulin at the pole. Consistent with this idea, disruption of TPX2 has been previously shown to generate multipolar spindles (Garrett et al., 2002), and we find that TPX2 fails to localize properly to the spindle pole in Myo10 morphants. In addition, Myo10 can interact directly with TPX2 via its MyTH4/FERM domain cassette. However, it remains unclear how TPX2 functions in spindle pole focusing, and our data do not rule out the possibility that TPX2 is instead required to recruit Myo10 to the pole to ensure pole integrity; resolving this issue will be an important line of further investigation. The upstream control of TPX2 is much better understood, as, like other spindle assembly factors, TPX2 is held at the nucleus during interphase through an interaction with the nuclear importins and is released from this by Ran GTPase as mitosis begins (Gruss and Vernos, 2004). It is interesting to note the similarities between the localization patterns of Myo10 and TPX2 and consider if Myo10 localization may also be regulated by the Ran GTPase pathway. Previous work has shown that the tail of Myo15, a MyTH4/FERM myosin, binds to the nuclear importin, importin- (Liu et al., 2008), and, intriguingly, we also find by yeast two-hybrid assay that the MyTH4/FERM domain cassette of Myo10 interacts with importin- (unpublished data), which suggests that the localization of Myo10 may be controlled in a similar manner to spindle assembly factors.
A third role for Myo10 in mitosis is regulation of spindle length. Specifically, Myo10 depletion results in spindles that are significantly longer than controls, both in absolute terms and as a function of cell size. Remarkably, this increased spindle length is rescued by disruption of F-actin with LatB, indicating that Myo10 and F-actin somehow play opposing roles in mitotic spindle length control. How can these findings be reconciled? The simplest explanation is that actin filaments participate in mutually opposing mechanisms that control spindle length: one mechanism that lengthens spindles and is independent of Myo10 and one that shortens spindles and is dependent on Myo10 (Fig. 8). When Myo10 is depleted, the former predominates, causing spindle lengthening; when F-actin is disrupted by latrunculin treatment either with or without Myo10 depletion, both mechanisms are neutralized, so spindle lengths are similar to controls. This model is attractive not only because it explains the results of the current study, but also because it might account for previous findings in X. laevis egg extracts showing that F-actin disruption has no obvious effect on spindle length (Mitchison et al., 2005). Curiously, in the current study, addition of Myo10 antibodies to extracts resulted in spindles that appeared longer than controls, in spite of the fact that cytochalasin D was present. Although this might simply reflect the apparent fragmentation of the poles, there is another potential explanation. That is, previously, we have shown that microtubules in extracts interact with dynein immobilized on the glass substrate (Waterman-Storer et al., 2000), and it may be that this interaction produces an outward pulling force in the extract spindles, which is exaggerated when Myo10 is depleted.
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The extent to which the various Myo10 roles described above are interrelated must be considered. The differential dependence on F-actin indicates that at least some aspects of the phenotypes can be considered separately. However, it is also likely that some of them are coupled. For example, it seems plausible that the metaphase delay is related to the spindle length defect, in that it is not hard to imagine that improper length control, and any subsequent loss of tension, would elicit the spindle assembly checkpoint (Nicklas et al., 1995; Zhou et al., 2002). Further, it is also probable that at least some of the deficits that result from Myo10 depletion feed back on each other. That is, in videos where the phenotype is just starting to become manifest, excessive spindle length and metaphase delay are apparent before excessive spindle pole fragmentation (e.g., Video 2). However, at least some of the cells with elongate spindles fail to complete cytokinesis without having obviously fragmented their poles (e.g., Video 5). Such cells would thus start the subsequent mitosis with additional poles. This condition would, presumably, further delay metaphase, provide additional material for pole fragmentation, and increase the probability of another failed cytokinesis. Consequently, in just one or two cell cycles, cells could go from an initially modest phenotype, wherein spindles have just two extra poles, to the severe phenotype in which upwards of 15 extra poles are apparent (e.g., Fig. 2, b and e).
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Materials and methods |
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Embryo microinjection
RNA for microinjection was made as described previously using pCS2-EGFP, pCS2-mCherry, or pStain vectors (Sokac et al., 2003). Microinjections were delivered using a PLI-100 picoinjector (Medical Systems), with embryos submerged in 0.1x MMR + 5% Ficoll. Myo10 MO (MO sequence: 5'-TATTCCTCCATGTCTCCCTCTGCTC-3'; Gene Tools, LLC) or 5-mispair control MO (5'-TATTCgTCgATcTCTCgCTCTcCTC-3', lowercase letters indicate mispairing nucleotides) were heated for 5 min at 65°C before microinjection and microinjected into two- or four-cell embryos at a needle concentration of 0.5–1 mM. For live imaging of mitotic spindles, both cells of two-cell embryos were microinjected with 5 nl of RNA for EGFP–-tubulin (needle concentration of 0.5 mg/ml) or mCherry–-tubulin (0.5 mg/ml) and EGFP-Utr-CH (0.125 mg/ml), and embryos were subsequently microinjected at the four-cell stage in all cells with 2.5 nl of Myo10 MO (0.5 mM) or nuclease-free water. For morphant rescue experiments, both cells of two-cell embryos were microinjected with 5 nl of RNA for the GFP-tagged Myo10 constructs (0.5 mg/ml) and then subsequently microinjected at the four-cell stage with 2.5 nl of Myo10 MO (0.5 mM) or nuclease-free water. After microinjection, embryos were incubated at 17°C for 12–24 h in 0.1x MMR before processing for live imaging, fixation, or Western blot analysis.
In vitro spindle assembly
Cytostatic factor (CSF)-arrested X. laevis egg extracts were prepared as described previously (Murray, 1991). X. laevis sperm chromatin was added and the extracts were subsequently cycled into interphase by the addition of 400 µM Ca2+. After 60 min, an equal volume of mitotic extract supplemented with 
90 cyclin B (1:20) was added to induce spindle assembly. Samples were fixed 45 min after extract addition, spun onto coverslips, subsequently processed for immunofluorescence with the Myo10 antibody (1:100), and visualized, all as described previously (O'Brien et al., 2005). For Myo10 antibody addition experiments, Myo10 antibody (1:10) or a nonspecific rabbit IgG (1:20) was added just before mitotic extract addition.
Immunofluorescence
For immunofluorescent analysis of microtubules only, embryos were processed as described previously (Danilchik et al., 1998). For all other staining, the methanol postfix step was omitted from this protocol. Antibodies used were: anti–-tubulin (DM1A; 1:200 dilution; Sigma-Aldrich), 5 µg/ml anti-Myo10 (Weber et al., 2004), anti-TPX2 (1:800; O'Brien and Wiese, 2006), mouse anti–-tubulin (GTU88; 1:200; Sigma-Aldrich), and 10 µg/ml rabbit anti–-tubulin (Keating and Borisy, 2000). To visualize chromosomes, embryos were first fixed and processed as above, omitting the methanol postfixation, and then permeabilized for 5 min at room temperature in PBS + 0.5% Triton X-100, equilibrated in 2x SSC (0.3 M NaCl2 and 0.03 M sodium citrate, pH 7), treated with 100 µg/ml DNase-free RNase in 2x SSC for 20 min at 37°C, and then incubated with 5 µM propidium iodide (Invitrogen) in 2x SSC for 5 min at room temperature.
LatB treatment of embryos
Embryos were microinjected with nuclease-free water or Myo10 MO (needle concentration, 1 mM) into both cells at the two-cell stage and incubated at 17°C for 20 h. Embryos were then incubated for 30 min at room temperature in 0.1% DMSO or 2.5 µM LatB in 1x MMR. All embryos were fixed immediately after treatment and stained for -tubulin as described in the previous paragraph.
Confocal imaging
For fixed tissue analysis, samples were mounted in Murray's Clear (benzyl benzoate/benzyl alcohol, 2:1) and imaged at room temperature using a microscope (Axiovert 100M ; Carl Zeiss, Inc.) with Lasersharp confocal software (Bio-Rad Laboratories) using 0.8 NA 25x or 1.4 NA 63x oil immersion objectives (Carl Zeiss, Inc.). Z series were acquired using step sizes of 0.25–1 µm and were reconstructed using Volocity software (PerkinElmer). For live imaging, embryos were mounted in 0.1x MMR (see "Egg procurement and in vitro fertilization") and imaged at room temperature using the Axiovert 100M microscope with a 1.4 NA 63x oil immersion objective. Single-plane time series were collected with intervals of 6 s and were processed using Volocity or ImageJ software.
Embryo lysates and immunoblotting
Embryo lysates were prepared from embryos microinjected with nuclease-free water, Myo10 MO (needle concentration, 1 mM), or 5-mispair control MO (1 mM), which had been incubated for 24 h after injection at 17°C. Embryos were homogenized in 7.5 µl of homogenization buffer (10 mM imidazol, 50 mM KCl, 2.5 mM MgCl2, 1 mM EGTA, 2.5 mM ATP, 1 mM dithiothreitol, 10 mM EDTA, 0.5% Triton X-100, and protease inhibitors) per embryo on ice using a tight dounce homogenizer. Homogenized embryos were centrifuged briefly at 4°C to remove lipids and cell debris, and lysate supernatants were transferred to fresh Eppendorf tubes and boiled with 6x SDS sample buffer. Samples were then separated by 8% SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blot according to standard protocols using 1 µg/ml anti-Myo10 and anti–-tubulin (DM1A; 1:5,000 dilution; Sigma-Aldrich).
Protein expression and in vitro binding
GST-MyTH4-FERM was expressed in Escherichia coli strain C41(DE3) and purified on glutathione-agarose beads (Sigma-Aldrich) using standard protocols. TPX2 was purified and the tag was removed as described previously (O'Brien and Wiese, 2006). Purified proteins were concentrated to 
5 mg/ml, dialyzed against a buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1 mM DTT, flash frozen in small aliquots, and stored at –80°C. For the in vitro binding assay, 2 µM of each protein (GST-MyTH4-FERM, TPX2, or GST as indicated) in a total volume of 50 µl was incubated with 25 µl of glutathione-agarose beads at 4°C on a rotator for 1 h. The beads were retrieved by a brief spin, washed three times with H100 (O'Brien and Wiese, 2006), and eluted with 1x SDS sample buffer. Supernatants were brought to 1x with 4x SDS sample buffer. Samples were boiled and subsequently loaded and separated by 10% SDS-PAGE, transferred to nitrocellulose, and Western blotted according to standard protocols.
Quantification and statistical analysis
To quantify spindle phenotypes in embryos, the number of bipolar metaphase spindles, multipolar spindles, and total cells were counted in low magnification confocal micrographs of -tubulin staining; >10 embryos were analyzed for each sample (n values are given in relevant figure legends). To determine mitotic spindle length in embryonic cells, two measurements were taken: pole-to-pole distance and total cell length, and spindle length was represented as a percentage of total cell length to allow for differences in cell size (>10 embryos and >50 spindles were analyzed for each sample). To test for a statistically significant difference between samples, unpaired Student's t tests were performed.
Online supplemental material
Fig. S1 shows cell cortex and spindle localization of Myo10. Fig. S2 shows quantification of spindles assembled in vitro in the presence of control or Myo10 antibody. Video 1 shows mitotic spindles in control embryonic epithelium. Video 2 shows mitotic spindles in Myo10 morphant epithelium. Video 3 shows mitotic spindles in Myo10 morphant epithelium. Video 4 shows spindle pole fragmentation in the Myo10 morphant. Video 5 shows failed cytokinesis in the Myo10 morphant. Video 6 shows multipolar spindle assembly in a binucleate cell in the Myo10 morphant. Video 7 shows that F-actin cables surround the mitotic spindle during assembly. Video 8 shows that F-actin cables extend between the cell cortex and the mitotic spindle during spindle rotation. Video 9 shows that F-actin organization is unaffected in Myo10 morphants. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200804062/DC1.
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Acknowledgments |
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This work was supported by a grant from the National Institutes of Health to W.M. Bement (GM52932-08), a grant from the National Science Foundation to C. Wiese (MCB-0344723) and a Predoctoral Fellowship from the University of Wisconsin-Madison Department of Biochemistry to L.L. O'Brien.
Submitted: 11 April 2008
Accepted: 6 June 2008
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References |
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