Myosin-10 independently influences mitotic spindle structure and mitotic progression.

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Cytoskeleton (Hoboken). Author manuscript; available in PMC 2017 June 22. Published in final edited form as: Cytoskeleton (Hoboken). 2016 June ; 73(7): 351–364. doi:10.1002/cm.21311.

Myosin-10 independently influences mitotic spindle structure and mitotic progression Joshua C. Sandquista,b,*, Matthew E. Larsonc, and Ken J. Hinea aBiology

Department, Grinnell College, 1116 8th Avenue, Grinnell, IA 50112 USA

cProgram

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in Cellular and Molecular Biology and the Medical Scientist Training Program, University of Wisconsin-Madison, 1525 Linden Drive, Madison, WI 53706, USA

Abstract

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The iconic bipolar structure of the mitotic spindle is of extreme importance to proper spindle function. At best, spindle abnormalities result in a delayed mitosis, while worse outcomes include cell death or disease. Recent work has uncovered an important role for the actin-based motor protein myosin-10 in the regulation of spindle structure and function. Here we examine the contribution of the myosin tail homology 4 (MyTH4) domain of the myosin-10 tail to the protein’s spindle functions. The MyTH4 domain is known to mediate binding to microtubules and we verify the suspicion that this domain contributes to myosin-10’s close association with the spindle. More surprisingly, our data demonstrate that some but not all of myosin-10’s spindle functions require microtubule binding. In particular, myosin-10’s contribution to spindle pole integrity requires microtubule binding, whereas its contribution to normal mitotic progression does not. This is demonstrated by the observation that dominant negative expression of the wild-type MyTH4 domain produces multipolar spindles and an increased mitotic index, whereas overexpression of a version of the MyTH4 domain harboring point mutations that abrogate microtubule binding results in only the mitotic index phenotype. Our data suggest that myosin-10 helps to control the metaphase to anaphase transition in cells independent of microtubule binding.

Keywords myosin; mitosis; spindle; microtubule; cytoskeleton

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INTRODUCTION The well-known job of the mitotic spindle is the separation of a cell’s genetic contents into two identical pools prior to cytoplasmic division (Compton 2000). The bipolar structure of the spindle is essential to this function and errors in spindle formation come at a high cost for cells and organisms. In conditions when a spindle has formed improperly, or simply not yet fully formed, the spindle assembly checkpoint (SAC) is active and prevents the onset of anaphase until a bipolar spindle has formed with all of the cell’s chromosomes properly *

Corresponding author: 641-269-4254 (tel.), 641-269-4285 (fax), [email protected]. bformer affiliation: Department of Zoology, University of Wisconsin-Madison, Madison, WI 53706 The authors declare no conflict of interest in this work.

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attached to kinetochore fibers and aligned between the two spindle poles (Taylor et al. 2004). The fates of cells that undergo a prolonged activation of the SAC (i.e. longer than normal metaphase) can vary by cell type or condition of SAC activation. Cells stuck at metaphase may die in mitosis or ‘slip’ out of mitosis and experience one of a variety of fates as a tetraploid cell (Rieder and Maiato 2004). Alternatively, some cells attempt anaphase in the presence of improperly formed spindles, e.g. multipolar spindles, which results in the inequitable distribution of chromosomes, a condition termed aneuploidy (Kwon et al. 2008; Lingle et al. 2002). In turn, aneuploidy has been linked to genomic instability, which is generally accepted to be a major factor contributing to oncogenesis (Fujiwara et al. 2005; Godinho et al. 2009; Lengauer et al. 1997; Storchova and Kuffer 2008).

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The cellular systems controlling and monitoring spindle formation and function are multifaceted and well studied, yet incompletely understood (Castanon and Gonzalez-Gaitan 2011; Kwok and Kapoor 2007; Manning and Compton 2008). Work in a variety of systems ranging from frog oocytes to cultured mammalian cells to intact vertebrate epithelia has shown that the unconventional myosin, myosin-10 (Myo10), is an important player in modulating multiple aspects of the structure and function of both meiotic and mitotic spindles. In frog oocytes, perturbation of Myo10 function negatively affects nuclear anchoring and meiotic spindle formation (Weber et al. 2004). In the epithelium of the developing frog embryo, Myo10 depletion results in a suite of mitotic spindle phenotypes including abnormal spindle movements, spindle elongation, and pole fragmentation (Woolner et al. 2008). Further, several studies have demonstrated that perturbing Myo10 function in cultured cells (two and three-dimensional cultures) and intact epithelial tissues causes a loss of normal spindle positioning/orientation (Kwon et al. 2015; Liu et al. 2012; Toyoshima and Nishida 2007; Woolner and Papalopulu 2012).

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Myo10 forms an amino-terminal globular head (or motor) domain that harbors actin-binding and ATPase activities, giving the protein the ability to bind filamentous actin (F-actin) and generate actin-based force. The elongated tail of Myo10 contains several protein-interaction domains including, but not limited to, pleckstrin homology (PH), myosin tail homology 4 (MyTH4), and Band 4.1/ezrin/radixin/moesin (FERM) domains (Berg et al. 2000). Collectively, these domains harbor the ability to bind phospholipids (Plantard et al. 2010), microtubules (Hirano et al. 2011; Weber et al. 2004), TPX2 (Woolner et al. 2008), integrins (Zhang et al. 2004), Mena/VASP (Tokuo and Ikebe 2004) and other proteins. Moreover, there is evidence of cooperation between Myo10 tail domains as binding to DCC requires an intact MyTH4-FERM cassette, the tandem of adjacent MyTH4 and FERM domains separated by a short linker region (Wei et al. 2011).

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The MyTH4 domain is of particular interest in examining Myo10’s spindle functions, as this domain contains a patch of basic amino acids that mediates binding to microtubules via electrostatic interaction with the acidic tails of tubulin (Hirano et al. 2011; Kwon et al. 2015). As such, it is reasonable to expect that the MyTH4 domain would form the basis for Myo10’s spindle localization (Woolner et al. 2008), although this has yet to be directly demonstrated. Moreover, Myo10’s head and MyTH4 domains together give the protein a unique ability to mediate cross talk between the F-actin and microtubule polymer systems. Indeed, recent work demonstrates that Myo10 performs important functions near the cell

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cortex where astral microtubules and F-actin meet and Myo10’s function here depends on its ability to bind microtubules via the MyTH4 domain (Kwon et al. 2015). In this case, Myo10 appears to act in a manner analogous to but distinct from dynein, presumably by Myo10 binding subcortical and/or cortical F-actin via its motor domain and astral microtubules via its MyTH4 domain.

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While there is strong evidence for such myosin-based control of astral microtubules at the cell cortex in different organisms (Hwang et al. 2003; Kwon et al. 2015; Kwon et al. 2008), this does not rule out the possibility of direct action of Myo10 on F-actin closer to the body of the spindle. For example, both Myo10 and F-actin have been observed directly on or around the spindle (Woolner et al. 2008). Further, other myosins and actin-binding proteins have been shown to localize to the spindle or spindle poles (for examples see (Wang et al. 2008; Wu et al. 1998), for review see Sandquist et al. 2011). Interestingly, Myo10 has been shown to be responsible for the spindle localization of the actin-binding protein adducin (Chan et al. 2014). Considering Myo10 and F-actin are both observed near the plasma membrane and the spindle, it is important to determine which pools of Myo10 and F-actin contribute to the various spindle functions of Myo10. Here we sought to examine the functions of Myo10 directly at the mitotic spindle. We focused on the MyTH4 domain, hypothesizing that Myo10’s functions at the spindle would depend on the microtubule binding mediated via this domain. Our data reveal multiple distinct functions for the MyTH4 domain.

RESULTS Myo10 localizes to the mitotic spindle

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Prior studies demonstrated that Myo10 localizes to the mitotic spindle in frog epithelial cells (Woolner et al. 2008). Consistent with previous results, immunostaining for endogenous Myo10 with an antibody directed against the head domain (Weber et al. 2004) demonstrates that Myo10 localizes in a cloud around the spindle and also concentrates on the spindle in a region near the spindle poles (Figure 1A, middle panel). We also observe Myo10 in the nucleus of interphase cells (Figure 1A, right panel), which is consistent with previous findings (Woolner et al. 2008). In order to determine the stability of Myo10’s interaction with the spindle, embryos were permeabilized in a mild TX-100 buffer prior to fixation. This pre-fixation permeabilization (hereafter pre-permeabilization) step releases soluble cytoplasmic proteins, leaving proteins tightly associated with stable cellular structures. Figure 1B shows that pre-permeabilization successfully reduced the cytoplasmic signal in Xenopus epithelial cells, leaving Myo10 signal that tightly overlaps with the microtubule staining of the mitotic spindle. These results extend prior findings and suggest that Myo10 exhibits a stable interaction with the mitotic spindle. Further, the nuclear Myo10 signal is preserved in pre-permeablized interphase cells (Figure 1B, right panel). We next sought to characterize the dynamics of Myo10’s localization to the spindle during the cell cycle. Accordingly, embryos were microinjected with mRNA encoding N-terminally tagged GFP-Myo10 (full-length protein) and imaged live with confocal microscopy. In order to first verify that GFP-Myo10 faithfully reports on the distribution of the endogenous


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protein, GFP-Myo10-expressing embryos were fixed and immunostained with GFP antibodies. Figure 1C shows that in cells fixed without pre-permeabilization GFP-Myo10 distributes in a cloud around the spindle similar to endogenous Myo10. Notably, exogenous GFP-Myo10 appears brighter around the cell cortex compared to the endogenous protein. Moreover, GFP-Myo10 in pre-permeabilized cells closely associates with the mitotic spindle and shows distinct signal near the spindle poles (Figure 1C, fourth panel). In contrast, the soluble cytoplasmic signal for 3xGFP (three GFP proteins fused in tandem) is dramatically reduced in pre-permeabilized cells, verifying that the majority of soluble protein is removed with this technique.

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The above results indicate that GFP-Myo10 accurately reflects the distribution of endogenous Myo10. Figure 2 shows stills taken from a movie of GFP-Myo10-expressing cells (see also Supplemental Movie 1). These images reveal that GFP-Myo10 localizes around and in the nucleus during interphase, supporting the nuclear localization shown above and reported previously in fixed samples (Woolner et al. 2008). Then at nuclear envelope breakdown (NEB) the GFP-Myo10 intensifies around the developing spindle, and the protein continues to surround the spindle throughout mitosis, often showing areas of concentration near one or both spindle poles (Figure 2, final three panels). When the spindle pole approaches the cortex throughout mitosis, but especially during anaphase, there appears to be dynamic exchange of GFP-Myo10 between the spindle and cortical pools of protein (Figure 2, final panel). The MyTH4 domain is sufficient for Myo10 spindle localization

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We were interested to determine what domain of Myo10 directs its localization to the spindle. Several recent studies showed that the Myo10 tail binds microtubules and the microtubule-binding domain has been located within the myosin tail homology 4 (MyTH4) domain (Hirano et al. 2011; Kwon et al. 2015; Weber et al. 2004). We reasoned that this domain would be important for Myo10’s localization to the microtubule-based spindle. Indeed, the isolated MyTH4 domain of Myo10 with an amino terminal fusion to GFP, GFPMyTH4, localizes in a cloud around the spindle, as do various different fragments of the Myo10 tail that contain the MyTH4 domain (Figure 3A and data not shown). Similar to fulllength GFP-Myo10, the soluble cloud of GFP-MyTH4 is reduced upon prepermeabilization, leaving signal that closely overlaps with spindle microtubules (Figure 3B, middle panel). Frequently, puncta of GFP-MyTH4 signal are observed around and in association with the spindle, especially in pre-permeabilized cells. Puncta of endogenous Myo10 are also observed in pre-permeabilized cells (Figure 1B).

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In order to determine the importance of MyTH4’s microtubule binding capacity to its spindle localization, we generated a version of the MyTH4 domain containing two point mutations (shown to decrease microtubule binding, which we call GFP-MyTH4-DD (R1638D and R1641D, see Hirano et al., 2011 and Figure 5E)). We expressed this mutated protein and determined its localization in pre-permeabilized cells (Figure 3B). The image shows that while the signal for GFP-MyTH4-DD persists in the pre-permeabilized cells, the mutated protein does not localize as closely to the mitotic spindle as wild-type GFPMyTH4. Rather GFP-MyTH4-DD appears more flatly distributed throughout the cytoplasm


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around the spindle, consistent with reduced association with the microtubules of the mitotic spindle. Most conspicuously, GFP-MyTH4 less readily forms puncta around the spindle. In order to verify that the distribution of GFP-MyTH4-DD is not an artifact of using a fragment of Myo10 we generated a construct for full-length Myo10 containing the same microtubulebinding mutations (GFP-Myo10-DD). Figure 4 shows that like the fragment, GFP-Myo10DD exhibits reduced spindle association compared to full-length GFP-Myo10. A line scan across the cell and through both spindle poles shows sharp peaks of GFP-Myo10-DD at the cell cortex (Figure 4C) and very low signal across the spindle region. In contrast, wild-type GFP-Myo10 exhibits strong signal both at the poles and along the length of the spindle, with an observable concentration of poleward spindle signal. GFP-MyTH4 expression causes spindle phenotypes


In examining GFP-MyTH4-expressing cells we observed a set of spindle phenotypes similar to those seen upon depletion of Myo10 protein (Woolner et al. 2008), including multipolar spindles, an increased number of cells with mitotic figures, and abnormal spindle movements (Figures 3A, 3C and 5A). With respect to the later phenotype, even overtly normal-looking bipolar spindles in GFP-MyTH4-expressing cells exhibited abnormal spindle movements compared to control cells. While the spindles of wild-type cells move in an oscillatory way during the minutes leading up to anaphase, the spindles of GFP-MyTH4expressing cells bounce back and forth from one side of the cell to the other more erratically and often slide sideways along the cortex, a behavior that is not observed in wild-type cells (see Figure 3C and Supplemental Movies 2 and 3). Moreover, spindles of GFP-MyTH4expressing cells spend much more time near the cortex than those of wild-type cells. Specifically, in movies of wild-type cells the spindle was observed to touch the cortex 46 ± 8% of the 3.5 minutes leading up to anaphase, while spindles in GFP-MyTH4 expressing cells spent 75 ± 7% of the same period touching the cortex (p = 0.011, n=9–12). Perhaps the most conspicuous phenotype observed upon expression of GFP-MyTH4 is the formation of multipolar spindles (Figures 3A and 5A). Larger Myo10 tail fragments containing the MyTH4 domain such GFP-PH-MyTH4-FERM (see Figure 5E) also produce multipolar spindles (Figure 5D and data not shown), though GFP-MyTH4 does so most readily, perhaps due to higher expression of this smaller fragment. In addition to multipolar spindles, GFP-MyTH4 expression produces a total increase in the presence of mitotic figures. Specifically, the total mitotic index (percent of cells in prophase through telophase) for control cells is 10.7 ± 0.9%, while that for GFP-MyTH4-expressing cells, when including multipolar spindles, is 25.6 ± 3.2% (Figure 5B, gray bars).

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Interestingly, not all of the reported Myo10-depletion phenotypes were induced by GFPMyTH4 expression. In particular, previous work shows that Myo10-depletion results in an increase in the pole-to-pole length of bipolar spindles (Woolner et al. 2008). However, the mean ± SE pole-to-pole lengths of bipolar spindles as a percent of cell diameter measured here in control, Myo10-depleted, and GFP-MyTH4-expressing cells are 59 ± 1.6%, 77 ± 1.2%, and 57 ± 1%, respectively (n > 50 spindles each condition, p< 0.001 for control v. Myo10-depleted, p = 0.27 for control v. GFP-MyTH4).


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Some GFP-MyTH4 phenotypes are independent of microtubule binding


The above results reveal that GFP-MyTH4 expression recapitulates some, but not all, of the spindle phenotypes caused by Myo10-depletion. The most parsimonious explanation for this result is that GFP-MyTH4 functions as a dominant negative, blocking some, but not all, of endogenous Myo10’s functions in the cell. In order to determine whether microtubule binding is required for the MyTH4 domain’s dominant negative effects, we examined GFPMyTH4-DD expressing cells for spindle phenotypes. Figure 5 shows that when GFPMyTH4-DD is expressed at levels equivalent to or higher than GFP-MyTH4 (Figure 5C) it does increase the percentage of cells in mitosis, though very few multipolar spindles are observed. Specifically, figure 5B shows that while GFP-MyTH4-DD increases the mitotic index of normal bipolar spindles to 20.7 ± 1.9%, including the rare multipolar spindles in the count does not significantly change the mitotic index (22.0 ± 2.0%). In contrast, the mitotic index in GFP-MyTH4-expressing cells when not including multipolar spindles is 17.3 ± 1.4%, while the mitotic index including multipolar spindles is 25.6 ± 3.2%, a statistically significant difference (p = 0.027).

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The above result suggests that the multipolar and mitotic index phenotypes are distinct, and that the former depends on microtubule binding while the latter does not. However, we did observe instances when the GFP-MyTH4 phenotypes paralleled the GFP-MyTH4-DD phenotypes. That is, some GFP-MyTH4-expressing embryos, particularly those with lower expression, exhibit an elevated mitotic index in the absence of multipolar spindles. In order to examine this observation further, we injected varying amounts of GFP-MyTH4 mRNA in order to produce a sort of dose-response curve GFP-MyTH4 overexpression phenotypes. Figure 6A shows a separation of the multipolar and mitotic index phenotypes based on expression level of GFP-MyTH4. Specifically, low levels of GFP-MyTH4 increased the mitotic index without causing overt structural spindle phenotypes (see Figures 6A and 6C). In contrast, the majority of spindles in embryos highly expressing GFP-MyTH4 were multipolar, with medium levels of GFP-MyTH4 producing a mix of bipolar and multipolar spindles (see Figures 6A and 6B). Importantly, low levels of GFP-MyTH4-DD still produce an elevated mitotic index (see Figures 6A and 6C). Perturbing Myo10 function causes a delay in anaphase onset

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In order to determine at what stage in mitosis GFP-MyTH4 and GFP-MyTH4-DD expressing cells were getting stuck, we counted the number of cells in each of the following stages: late G2, prophase, metaphase, post-metaphase. This was done with images of embryos expressing low or medium levels of GFP-MyTH4 or GFP-MyTH4-DD. Figure 6B shows that in the medium expression conditions both GFP-MyTH4 and GFP-MyTH4-DD increase the number of metaphase cells whereas only GFP-MyTH4 produces a significant number of multipolar spindles. The prevalence of the other mitotic stages is not different from controls for either construct. In the low expression condition, both constructs still cause an elevation in the number of metaphase spindles but neither produces a significant increase in multipolar spindles. These results suggest that the MyTH4 domain can somehow retard progression through mitosis, ostensibly at the metaphase to anaphase transition, without binding to microtubules or causing overt structural phenotypes. To further support this observation, movies were acquired for control cells and cells expressing low levels of GFP-


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MyTH4 or GFP-MyTH4-DD, and the lifetime of the metaphase plate in each condition was determined. The survival plots in figure 7 show that both GFP-MyTH4 and GFP-MyTH4DD expressing cells take longer to transition from metaphase to anaphase than controls. It is important to note that these cells do eventually progress into anaphase, generally within 30 minutes. In contrast, multipolar spindles are generally not observed to attempt anaphase in the lengths of movies recorded, with some lasting more than two hours.

DISCUSSION

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Although the mitotic spindle is generally thought of as a microtubule-based structure, several studies over the years in different biological systems have revealed more or less direct roles for the actin-based cytoskeleton in influencing spindle structure and/or function (see (Sandquist et al. 2011) for a review). As a prime example of this, several recent studies and the work presented here demonstrate that myosin-10 (Myo10) is a major factor in spindle function, either at the spindle (Woolner et al. 2008) or near the cell cortex (Kwon et al. 2015; Toyoshima and Nishida 2007; Weber et al. 2004). That Myo10 has spindle functions can be rationalized by the fact that this protein also binds microtubules, although how Myo10 uses its microtubule-binding properties to alter spindle function is not clear in all cases. Accordingly, we examined the importance of the MyTH4 domain to Myo10’s spindle functions.


We show here that the MyTH4 domain by itself localizes to the spindle and that microtubule binding is important for tight association with the spindle (Figure 3). It seems likely that this domain contributes to the spindle localization of the full-length protein. However, while the GFP-MyTH4-DD mutant does not exhibit the tight spindle association seen with GFPMyTH4, it does persist in a cloud around the spindle, and so we cannot rule out the possibility that other factors or binding partners contribute to Myo10’s spindle localization. For example, Myo10’s MyTH4-FERM cassette binds TPX2, a known spindle associated protein (Woolner et al. 2008). Or, more generally, soluble proteins can be concentrated around the spindle via an organelle-exclusion envelope (Schweizer et al. 2015). In the latter model, a membrane-based system would exclude organelles, resulting in a region around the spindle that is relatively enriched with cytoplasm and soluble proteins, causing the diffuse cloud of soluble GFP-MyTH4-DD observed around spindle with no directly spindleassociated signal apparent (Figure 3B). The most conspicuous difference in GFP-MyTH4 and GFP-MyTH4-DD localization is the absence of puntcal staining for the latter. As the puncta of GFP-MyTH4 and endogenous Myo10 often coincide with microtubules (Figures 1 and 3), one explanation for the lack of GFP-MyTH4-DD puncta is that the Myo10 puncta are manifestations of some manner of specific association with microtubules that is reduced by the DD mutations. The importance of Myo10’s actin-binding motor domain to its spindle localization and functions is not fully understood. When expressed in Myo10-depleted cells a tailless fragment of Myo10 (approximately equivalent to heavy meromyosin of myosin-2) partially rescues the elongated spindle phenotypes (Woolner et al. 2008). However, it is not clear from these studies where in the cell this fragment is acting (e.g. spindle or cortex). Future work examining the distribution of the motor domain of Myo10 with and without point


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mutations altering actin-binding or actin-contracting activity could be telling with regards to the importance of Myo10’s actin-based motor activity to its spindle localization and various functions. Further, nothing is known about the importance of the putative nuclear Myo10 (Figure 2). Several myosins are known to localize within the nucleus (for reviews see (Simon and Wilson 2011; Woolner and Bement 2009)) and clear nuclear functions for some have been characterized (Philimonenko et al. 2004). Interestingly, work in Tetrahymena examining a MyTH4-FERM containing class XIV myosin showed that ectopically expressed GFP-MyTH4 localizes to the Tetrahymena macronucleus and perturbs the normal organization of intramacronulcear microtubules (Gotesman et al. 2011). Moreover, a recent study identified an actin-bundling kinesin that binds intranuclear actin in Xenopus oocytes (Samwer et al. 2013), representing another cytoskeletal motor protein that can bind both Factin and microtubules and distribute within the nucleus. The potential roles for nuclear Myo10 represent an interesting area of future study.

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Data presented here show that GFP-MyTH4 overexpression reproduces some but not all of the spindle phenotypes observed in Myo10-depleted cells. Specifically, both Myo10depletion and GFP-MyTH4 overexpression induce the formation of multipolar spindles, increase mitotic index, and cause abnormal spindle movements (Figures 3 and 5 and (Woolner et al. 2008)), while GFP-MyTH4 expression does not increase spindle length like Myo10-depletion does (see text in Results section). These observations demonstrate specificity of the dominant negative effects and imply that different regions of the protein are specifically responsible for different functions. This is consistent with the observations that headless and tailless versions of Myo10 can rescue different Myo10-depletion phenotypes (Woolner et al. 2008).


How does the MyTH4 domain produce its dominant negative effects? Presumably it competes with endogenous Myo10 for binding to some other cellular protein, thus disrupting native protein complexes. However, what and where the GFP-MyTH4 is binding to produce the observed effects cannot be conclusively determined. The observation that overexpression of GFP-MyTH4, which localizes tightly to the spindle (Figure 3B), causes multipolar spindle formation whereas overexpression of GFP-MyTH4-DD, which has reduced spindle association (Figure 3B), produces only very few multipolar spindles is consistent with the idea that GFP-MyTH4 disrupts spindle pole integrity by directly competing with endogenous Myo10 for binding to spindle microtubules. However, we cannot rule out that GFP-MyTH4 is also acting by disrupting Myo10 at the cortex (Kwon et al. 2015). Further, it is not clear how the MyTH4 domain would specifically compete with Myo10 and not other microtubule associated proteins for binding to microtubules, unless Myo10 binds a specific subset of microtubules. With respect to that possibility, full-length Myo10 (endogenous or expressed) does concentrate toward the spindle poles and in puncta along microtubules and their tips, representing discrete pools of Myo10 that could be disrupted by GFP-MyTH4 (Figure 1). While the pool of Myo10 at the spindle pole is well positioned to have a direct effect on spindle pole integrity, it is not known how displacing endogenous Myo10 from spindle microtubules would produce the observed phenotypes. If Myo10 were indeed acting as a functional link between F-actin and microtubules, the overexpression of MyTH4 could


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disrupt this linkage. This seems to be the case at the cortex, where Myo10 appears to link astral microtubules to populations of subcortical actin and contribute to proper spindle positioning (Kwon et al. 2015). In this cell culture system, expression of neither a headless nor a tailless version of Myo10 alone can rescue the spindle orientation phenotypes resulting from Myo10-depletion, which is consistent with a role for Myo10 as a crosslinker of the Factin and microtubule polymer systems (Kwon et al. 2015). Thus, disrupting these interactions with GFP-MyTH4 could perturb the normal balance of forces in the cell that are responsible for controlling spindle position, resulting in the abnormal spindle movements observed in figure 3. However, expression of a headless version of Myo10 that is slightly larger than the one used above but still lacks the F-actin binding domain, is able to rescue Myo10-depletion-induced multipolar spindles (Woolner et al. 2008) and defects in spindle positioning along the apico-basal axis in tissue epithelial cells (Woolner and Papalopulu 2012). In addition to working in different model systems, one difference between the truncates used in the aforementioned studies is that the longer headless myosin that is able to rescue apico-basal positioning contains a domain that may mediate dimerization (Lu et al. 2012; Tokuo et al. 2007), though the importance of dimerization to the different functions of Myo10 is not fully understood. Interestingly, this larger headless version of Myo10 (which contains the IQ domain through carboxy-terminus, see Figure 5E) is similar to a naturally expressed headless version of Myo10 that is able to act as a natural dominant negative in brain tissues (Raines et al. 2012; Sousa et al. 2006). Taken together, the above results indicate that some Myo10 functions may require crosslinking of the F-actin and/or microtubule polymer networks while other Myo10 functions do not. Further, considering the multitude of binding partners for the Myo10 tail (see introduction), more studies examining the specific function of a single interaction domain of the Myo10 tail are needed.

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With respect to that goal, the most important finding from this study is the increase in mitotic index induced by moderate overexpression of GFP-MyTH4-DD (Figure 6C). That higher expression of the wild-type GFP-MyTH4 increases mitotic index is not surprising given the generally accepted model for the spindle assembly checkpoint (SAC) and the fact that higher levels of GFP-MyTH4 expression induce the formation of multipolar spindles. However, GFP-MyTH4-DD, which has reduced MT binding and does not produce multipolar spindles, still causes a delay in the metaphase to anaphase transition (Figure 7). Further, low levels of GFP-MyTH4 expression also cause a delay without producing multipolar spindles or obvious structural defects. While we cannot completely rule out the possibility that low levels of GFP-MyTH4 or GFP-MyTH4-DD cause minor structural phenotypes that trigger the SAC, these cells do undergo a normal looking anaphase.

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A recent related study suggested that spindle pole fragmentation occurs as a consequence of sustained microtubule-based tension during a prolonged metaphase (Stevens et al. 2011). In contrast, our results indicate that Myo10 perturbation induces a mitotic delay that is separable from pole fragmentation. Moreover, in movies of Myo10-depleted cells in which both NEB and pole fragmentation were observed, the average time to fragmentation after NEB is 24.4 ± 2.4 minutes (mean ± SE, n=6) (see also (Woolner et al. 2008)). In contrast, in normal cells anaphase onset typically begins 9.9 ± 0.15 minutes after NEB (n = l6). Thus, while in these depleted cells Myo10 levels are low all throughout mitosis, the poles do not actually split until after a delay has already occurred, which is consistent with the idea that a Cytoskeleton (Hoboken). Author manuscript; available in PMC 2017 June 22.

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delay in anaphase onset is not a product of significant defects in spindle structure. At this point, we can only speculate as to a mechanism for how GFP-MyTH4-DD (or low GFPMyTH4) expression retards mitotic progression. As GFP-MyTH4-DD has diminished microtubule-binding capabilities, it seems likely that GFP-MyTH4-DD perturbs mitotic progression by interrupting endogenous Myo10’s interaction with something other than microtubules. Regardless of the mechanism, the work presented here represents, to our knowledge, the first demonstration of a role for a myosin motor in directly contributing to the regulation of anaphase onset.

MATERIALS AND METHODS DNA Constructs, Morpholino and mRNA Synthesis


The DNAs encoding all expressed proteins were maintained in a custom made vectors based on the pCS2+ backbone (Sokac et al. 2003). This vector contains an SP6 RNA polymerase promoter site followed by 1–3 copies of the genes encoding for eGFP or mCherry (mChe). The DNA encoding the protein probe of interest is positioned downstream that of the fluorescent protein, generating amino-terminal reporter protein fusions. Protein probes used include: GFP-α-tubulin (human), mChe-α-tubulin, mChe-histone-H2B (human), 3xmCheEMTB (microtubule binding domain of human ensconsin (Faire et al. 1999)), and GFPMyo10 and GFP-MyTH4 (X. laevis, described in (Weber et al. 2004)). The authors note that the MyTH4 domain used here lacks two amino-terminal helices referred to as the MyTH4 extension in the crystal structure by (Hirano et al. 2011). GFP-MyTH4-DD contains two amino acid substitutions that have been shown to reduce microtubule binding (R1638D and R1641D) and was generated via site-directed mutagenesis of GFP-MyTH4 using the following primers: sense, 5’-gggcgggacattctgGACtatctcGaCttccatctgaaacgg and anti-sense, 5’-ccgtttcagatggaaGtCgagataGTCcagaatgtcccgccc. The sequence of morpholino (MO) used for the depletion of Myo10 protein was 5’-tattcctccatgtctccctctgctc (Gene Tools, LLC) as describe in (Woolner et al. 2008). mRNA was synthesized in vitro from linearized pCS2+ vectors using the mMessage mMachine SP6 kit and vendor protocol (Ambion). Synthesized mRNAs were purified by phenol/chloroform extraction followed by isopropanol precipitation and suspended in nuclease free water. Embryo Preparation and Microinjection

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Female Xenopus laevis (Nasco) were induced to ovulate by injection of 800 units of human chorionic gonadotropin (MP Biomedicals) into the dorsal lymph sac 18–20 hours prior to egg collection. Eggs laid into 1× MMR (100 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.4) were fertilized within one hour of laying. To fertilize, a macerated portion of testes was mixed with eggs in 1× MMR by gentle swirling and incubated for 1–3 minutes at room temperature. The mixture was diluted 10-fold with deionized water and incubated for an additional 25 minutes at room temperature. Fertilized eggs were dejellied in 2% cysteine (in 0.1× MMR, pH 7.8) then washed three times in 1× MMR and three times in 0.1× MMR. Embryos were cultured in 0.1× MMR at 17°C or room temperature until microinjection and then at 17°C. During microinjection, embryos were bathed in 5% ficoll (in 0.1× MMR). Depending on the experiment, embryos were injected at


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the 2-cell (5 nL per cell) or 4-cell (2.5 nL per cell) stages of development, using needle concentrations of morpholino or mRNA as indicated in the figure legends. Immunofluorescence Staining


Stage 10 embryos (developed 18–20 hours at 17°C) were washed in phosphate buffered saline (PBS) then fixed in Superfix (100 mM KCl, 3 mM MgCl2, 10 mM HEPES, 150 mM sucrose, 1 mM EGTA, 3.7% paraformaldehyde, 0.1% gluteraldehyde, 0.4% NP-40, 0.2 uM taxol, pH 7.5) for 2–3 hours at room temperature with gentle shaking. For prepermeabilization, washed embryos were incubated in buffer P (60 mM PIPES, 4 mM EGTA, 0.8 mM MgCl2, 18.4% (w/v) glycerol, 0.1% TX-100, pH 6.8) for 1–3 minutes at room temperature without shaking prior to fixing. Fixed embryos were washed in PBS then dehydrated with multiple washes in methanol and incubated for at least 1 hour at room temperature or −20°C, followed by rehydration in an escalating series of PBS:methanol washes. Rehydrated embryos were bisected equatorially then quenched in 100 mM sodium borohydride in PBS for 2–3 hours at room temperature. Quenched embryos were washed in PBS and in some cases then bleached for 30–60 minutes in bleaching solution (5% formamide, 1% H2O2 in 0.5 × SSC (75 mM NaCl, 7.5 mM trisodium citrate, pH 7)). Bleached embryos were washed into PBST (PBS + 0.1% Tween-20), the blocked for at least 30 minutes in embryo block (5% goat serum, 5% DMSO in PBST). Blocked embryo hemisects were incubated in primary antibody in embryo block overnight at 4°C with rotation. Embryos were washed 6–8 hours with rotation in PBST with four buffer exchanges. Secondary antibodies (1:200–1:500 in embryo block) were incubated overnight at 4°C with rotation, and then washed as above. DNA was labeling following the post-secondary wash. Embryos were equilibrated in 2× SSC (0.3 M NaCl and 0.03 M trisodium citrate, pH 7), treated with DNase-free RNase (100 µg/ml in 2× SSC) for 20 min at 37°C, and then incubated with 5 µM propidium iodide (in 2× SCC, Invitrogen) for 5–10 minutes at room temperature. For imaging, immunostained embryos were dehydrated in 2–3 washes in methanol then immersed in BABB (1:2 benzyl alcohol:benzyl benzoate) to clear. Primary antibodies used were: anti-α-tubulin (1:200–1:500, DM1A, Sigma Aldrich), anti-GFP (Molecular Probes #A6455 or Cell Signaling Technology D5.1, #2956), anti-Myo10 (5–10 mg/mL (Weber et al. 2004)). All secondary antibodies were from Molecular Probes. Microscopy and Image Analysis

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Fixed images were collected on one of two microscopes. One was an Olympus IX81 microscope with a DSU confocal attachment (disc no. 2), a 60× objective, and a Hamamatsu Orca EM camera, using Slidebook 5 software (3i). Samples were illuminated with a HBO 200 W/2 lamp (Osram) and light was collected using filter sets from Chroma Technology Corp: FITC (49002), TRITC (49005) or Cy5 (49913). A second microscope used was a BioRad MRC1024 laser scanning confocal microscope with Bio-Rad 1024 Lasersharp confocal software (Bio-Rad), using a 63× objective and illumination with 488, 568 or 647 nm laser lines. Images were processed in ImageJ or FIJI. Images of live samples were collected with the above Bio-Rad microscope or a 3-channel confocal microscope from Prairie Technologies. Mitotic stage assessment was made based on spindle and DNA morphology, with the following definitions: G2, separated centrosomes prior to nuclear envelope breakdown (NEB); prophase, post NEB but before chromosome congression; metaphase, Cytoskeleton (Hoboken). Author manuscript; available in PMC 2017 June 22.

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full chromosome congression with no space between sister chromatids; post-metaphase, after chromosome separation but before significant cleavage furrow formation. For the survival plots, metaphase duration was calculated as the time from chromosome alignment at the metaphase plate to separation of duplicate chromosomes at anaphase onset. Initial chromosome alignment, chromosome separation, or both was not observed in censored samples. Preparation of Embryo Lysates and Western Blotting

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Stage 10 embryos were washed in PBS and then homogenized in lysis buffer (PBS, 0.5 mM PMSF, protease inhibitor cocktail (Sigma #P8340 at vendor recommendations)) by repeated pipetting in a volume of 10 microliters of lysis buffer per embryo. Homogenates were centrifuged at 21,000×g for 15 minutes. Cleared cytosplasm (middle layer) was collected and boiled for 5 minutes in SDS Sample Buffer (final concentrations: 18 mM Tris-base, 3% glycerol, 3% SDS, 3% 2-mercaptoethanol, 3 mM DTT, bromophenol blue, pH 6.8). Lysate equivalent to 0.5–1 embryo was loaded in a single lane and separated by standard SDSPAGE technique. The proteins were transferred to nitrocellulose, stained with Ponceau S., destained and then dried. Dried membranes were either stored at 4°C or immediately rehydrated for immunoblotting. Blocked membranes (Odyssey PBS blocking buffer (Li-Cor, #927-40100)) were incubated overnight at 4°C in primary antibodies in antibody buffer (blocking buffer + 0.2% Tween-20). Primary antibodies used were DM1A (Sigma, 1:8000) and anti-GFP (Cell Signaling Technology, 1:1000). Membranes were washed 3 times 5 minutes PBST then incubated one hour at room temperature in secondary antibodies in antibody buffer (all fluorophore-conjugated secondaries from Li-Cor, 1:10,000). Membranes were washed 3 times 5 minutes in PBST then scanned with Odyssey CLx Infrared Imaging System (Li-Cor).

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Data Analysis Unless otherwise noted, statistical comparisons were made via a two-way ANOVA and Tukey’s post-hoc tests using Minitab Express (Minitab Inc.). The bar charts were made in Excel (Microsoft). Survival plots were generated using the ecdf function in MatLab (Mathworks). Log-Rank tests were performed in Prism (GraphPad). Multiple comparisons of the Log-Rank test used a Bonferroni adjusted threshold for significance of 0.017.

Supplementary Material Refer to Web version on PubMed Central for supplementary material.

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Acknowledgments JCS would like to thank Bill Bement for his thoughtful suggestions, advice, and mentorship. JCS also appreciates the contributions to his lab’s general research progress made by many undergraduate students including: A. Boldt, Z. Ding, T. Law, M. Miller, C. Zuo. JCS acknowledges awards made to him by the NIH (NRSA: F32GM090674) and NSF (MRI: DBI-1428384) in support of his research. JCS and KH are indebted to Grinnell College for its financial support of their work through the CSFS and the MAP program. MEL is supported by F30CA189673, R01GM052932 to William Bement, and T32GM008692 to the UW MSTP.


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Abbreviations MyTH4

myosin tail homology 4

Myo10

myosin-10

F-actin

filamentous actin

NEB

nuclear envelope breakdown

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Author Manuscript Author Manuscript Author Manuscript Cytoskeleton (Hoboken). Author manuscript; available in PMC 2017 June 22.

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Figure 1.

Myosin-10 localizes to the nucleus and mitotic spindle. (A) Stage 10 Xenopus laevis embryos were immunostained with antibodies against α-tubulin (microtubules) and the head domain of myosin-10 (Myo10) and propidium iodide, to mark DNA. The left panel shows secondary antibody only (2° Ab) as a negative control for Myo10 staining. (B) Stage 10 embryos were pre-permeabilized in a mild detergent prior to fixation in order to reduce the signal from soluble protein and then immunostained as in ‘A’. (C) Embryos were microinjected with mRNA encoding 3xGFP (0.5 mg/mL) or GFP-Myo10 (0.75–1 mg/mL)


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at the 2-cell stage. At stage 10 of development the embryos were fixed with formaldehyde either directly (no perm) or after pre-permeabilization (perm). Fixed samples were immunostained with antibodies made against α-tubulin, Myo10, or GFP as indicated in the panels. The region boxed in red is magnified 1.5× in order to show the distinct pole localization of GFP-Myo10 (arrow). In all images the scale bar = 10 microns.


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Figure 2.

GFP-myosin-10 localizes dynamically with the mitotic spindle throughout mitosis. Xenopus laevis embryos were microinjected at the 2- or 4-cell stage with mRNAs encoding for mCherry-α-tubulin (0.5 mg/mL needle concentration) and GFP-Myo10 (0.75–1 mg/mL). At stage 10 of development the live embryos were mounted and imaged via time-lapse confocal microscopy at 10 second intervals. Time is indicated in mm:ss. NEB occurs in the left cell prior to filming and in the right cell at 01:40. Scale bar = 20 microns.


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Figure 3.

The MyTH4 domain of Myo10 localizes directly to the spindle. (A, B) Xenopus laevis embryos were left uninjected (control) or microinjected at the 2-cell stage with mRNA encoding GFP-MyTH4 or GFP-MyTH4-DD (0.25–0.5 mg/mL needle concentration) and then allowed to develop at 17°C. At stage 10 of development the embryos were fixed with formaldehyde either directly (A) or after pre-permeabilization (B). Fixed samples were immunostained with antibodies made against α-tubulin (microtubules) and GFP. Bar = 10 microns. (C) Xenopus laevis embryos were microinjected at the 2- or 4-cell stage with


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mRNAs encoding for 3xmChe-EMTB (microtubules, 0.1 mg/mL), GFP-tubulin (0.5 mg/ mL), mChe-H2B (DNA, 0.05 mg/mL), or GFP-MyTH4 (0.5 mg/mL) and developed at 17°C. At stage 9–10 live embryos were mounted and imaged via time-lapse confocal microscopy (see supplemental movie 3). Arrow tracks the same pole of the spindle in order to show rotation of the spindle. Bar = 20 microns.


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Figure 4.

Localization of full length GFP-Myo10 to the spindle is blocked by point mutations in the MyTH4 domain. (A) Xenopus laevis embryos were left uninjected (control) or microinjected at the 2-cell stage with mRNA encoding GFP-Myo10 or GFP-Myo10-DD (5 nL per cell, 2 mg/mL needle concentration) and then allowed to develop at 17°C. At stage 10 of development the embryos were pre-permeabilized before fixation and then immunostained with antibodies made against α-tubulin (microtubules) and GFP. (B) Line scans 10 pixels wide were taken from left to right and through both poles of the spindle using the center image (GFP-Myo10) from panel A. The green line represents the relative fluorescence intensity of the GFP-Myo10 signal. Dashed lines indicate position of the cell cortex and arrows indicate spindle poles. X-axis represents distance in micrometers. (C) Same as B except using the right image (GFP-Myo10-DD) from panel A.

Author Manuscript Cytoskeleton (Hoboken). Author manuscript; available in PMC 2017 June 22.

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Figure 5.

Overexpression of the MyTH4 domain of Myo10 produces spindle phenotypes. (A) Xenopus laevis embryos were left uninjected (control) or microinjected at the 2-cell stage with morpholino against Myo10 (Myo10-MO, 0.5–1 mM needle concentration) or mRNA encoding GFP-MyTH4 or GFP-MyTH4-DD (0.5 mg/mL). At stage 10 of development the embryos were fixed and immunostained with antibodies made against α-tubulin (microtubules) and GFP to verify expression of the expressed proteins (not shown). Bar = 20 microns. (B) Images such as those shown in ‘A’ were counted for total cell number and


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number of cells with mitotic figures (prophase through telophase). Plot shows the mitotic index (MI, cells with mitotic figures divided by total number of cells) as mean + SE. Pvalues determined by ANOVA with Tukey’s post-hoc analysis (sample sizes = 23–24). White bars indicate only bipolar spindles were counted in the mitotic index, while gray bars indicate the mitotic index including multipolar spindles. (C) Immunoblot verifying expression of GFP-MyTH4 and GFP-MyTH4-DD protein at stage 10. Arrow indicates common degradation product of MyTH4 domain. (D) Embryos microinjected with mRNA encoding GFP-PH4F (combined PH-MyTH4-FERM domains) was treated and imaged as in ‘A’. Bar = 10 microns. (E) Diagram showing the various protein domains of myosin-10. Motor, contains the ATPase and actin-binding domains; IQ, light chain binding motifs; CC, coiled-coil; PEST, protease-sensitive region rich in pro-glu-ser-thr repeats; PH, pleckstrin homology; MyTH4, myosin tail homology 4; FERM, Band 4.1/ezrin/radixin/moesin motif. The MyTH4 domain is enlarged and shows the approximate location of two point mutations shown to reduce binding to microtubules, R1638D and R1641D. Numbers indicate amino acid number in frog myosin.

Author Manuscript Author Manuscript Cytoskeleton (Hoboken). Author manuscript; available in PMC 2017 June 22.

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Figure 6.

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Overexpression of the MyTH4 domain of Myo10 induces a cell cycle delay. (A) Xenopus laevis embryos were left uninjected (control) or microinjected at the 2-cell stage with various amounts of mRNA encoding GFP-MyTH4 or GFP-MyTH4-DD (inject 5 nL per cell with needle concentrations ranging from 0.25–0.5 mg/mL) and then allowed to develop at 17°C. Expression can vary between batches of embryos, but ‘low’ expressors always came from embryos injected with 0.25 mg/mL, while ‘medium’ and ‘high’ expressors were found in embryos injected with 0.5 mg/mL. At stage 10 of development the embryos were fixed and immunostained with antibodies made against α-tubulin (microtubules) and GFP to verify expression levels. Bar = 20 microns. (B) Images of control embryos (white) or embryos expressing medium levels of GFP-MyTH4 (gray) or GFP-MyTH4-DD (black) were captured and scored for total cell number and cell cycle stage: G2, prophase (Pro), metaphase (Meta), anaphase/telophase (Post-Meta). Cells with multipolar spindles were counted separately. Bars represent mean + SEM. Sample sizes: a total of 22–24 images from Cytoskeleton (Hoboken). Author manuscript; available in PMC 2017 June 22.

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12–15 embryos from 5 different experiments were counted for each treatment group. (C) Same as B except embryos were expressing low levels of GFP-MyTH4 (gray bars) or GFPMyTH4-DD (black bars).


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Figure 7.

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Low expression of the MyTH4 or MyTH4-DD domains of Myo10 induce prolonged metaphase. Xenopus laevis embryos were microinjected at the 2-cell stage with mRNA encoding mCherry-histone-H2B alone (control, 0.008 mg/mL needle concentration) or in combination with GFP-MyTH4 or GFP-MyTH4-DD (0.25 mg/mL) and then allowed to develop at 16°C. At stage 10 of development the embryos were mounted and imaged via time-lapse confocal microscopy at 5-second intervals. The survival plots indicate the length of metaphase, with the x-axis depicting time in seconds from chromosome alignment at the metaphase plate to the separation of the duplicate chromosomes, and the y-axis indicating the proportion of cells that have not yet entered anaphase. There is a significant difference in the three groups by Log-Rank test (p

ATM controls proper mitotic spindle structure.

Regulation of mitotic progression by the spindle assembly checkpoint.

Mechanisms of Mitotic Spindle Assembly.

Force and counterforce in the mitotic spindle.

Force and the spindle: mechanical cues in mitotic spindle orientation.

Mitotic spindle failure in human cancer.

Membrane-based mechanisms of mitotic spindle assembly.

Nonthermal fluctuations of the mitotic spindle.

Mechanical design principles of a mitotic spindle.

The evolution of the mitotic spindle.

Nuclear Mitotic Apparatus (NuMA) Interacts with and Regulates Astrin at the Mitotic Spindle.

Mitotic spindle and mitotic cell volumes in the root meristem of Zea mays.

KIFC3 promotes mitotic progression and integrity of the central spindle in cytokinesis.

Aurora-A-Dependent Control of TACC3 Influences the Rate of Mitotic Spindle Assembly.

EML4 promotes the loading of NUDC to the spindle for mitotic progression.

Reduced O-GlcNAcase expression promotes mitotic errors and spindle defects.

Mitotic Spindle Assembly in Land Plants: Molecules and Mechanisms.

Centrosomes and the art of mitotic spindle maintenance.

Analysis and modeling of mitotic spindle orientations in three dimensions.

Regulation of spindle integrity and mitotic fidelity by BCCIP.

Scaling, selection, and evolutionary dynamics of the mitotic spindle.

Depletion of pre-mRNA splicing factor Cdc5L inhibits mitotic progression and triggers mitotic catastrophe.

Disruption of Mitotic Progression by Arsenic.

Mitotic spindle orientation: semaphorin-plexin signaling flags the way.