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Title: Mitotic spindle scaling during Xenopus development by kif2a and importin α}
Article:
Abstract:
Early development of many animals is characterized by rapid cleavages that dramatically decrease cell size, but how the mitotic spindle adapts to changing cell dimensions is not understood. To identify mechanisms that scale the spindle during Xenopus laevis embryogenesis, we established an in vitro system using cytoplasmic extracts prepared from embryos that recapitulates in vivo spindle size differences between stage 3( 4 cells, 37 µm) and stage 8( ∼4000 cells, 18 µm). We identified the kinesin-13 kif2a as a driver of developmental spindle scaling whose microtubule-destabilizing activity is inhibited in stage 3 spindles by the transport receptor importin α, and activated in stage 8 when importin α partitions to a membrane pool. Altering spindle size in developing embryos impaired spindle orientation during metaphase, but chromosome segregation remained robust. Thus, spindle size in Xenopus development is coupled to cell size through a ratiometric mechanism controlling microtubule destabilization.
Introduction:
Cell size varies widely among different organisms and cell types, and changes rapidly during early animal development when cell division occurs in the absence of growth. The mitotic spindle, a dynamic bipolar structure composed primarily of microtubules( MTs) and organizing proteins( Walczak and Heald, 2008), must adjust to cell size to segregate chromosomes the proper distance and signal to the cell cortex to specify orientation of the cleavage furrow( Levy and Heald, 2012). However, the mechanisms coordinating cell and spindle size are poorly understood, and the functional consequences of altering these mechanisms during vertebrate development are unknown. Embryogenesis of the frog Xenopus laevis typifies the developmental scaling problem, and both cell and spindle sizes have been measured( Wuhr et al., 2008). Following fertilization, the ∼1. 2-mm-diameter egg synchronously divides 12 times at approximately 30-min intervals, leading to a ∼100-fold decrease in cell volume and at least a 20-fold decrease in cell diameter by the midblastula transition( stage 8. 5), when zygotic transcription starts. Initially, the cortically localized meiosis II spindle( ∼35 µm) segregates chromosomes a short distance, generating a small polar body that preserves egg cytoplasm. During the early cleavages, there is an upper limit to spindle size( ∼60 µm), and astral microtubules mediate movement of chromosomes long distances during anaphase( Wuhr et al., 2008). At these stages, spindle size is small compared to cell size, and cytoplasmic mechanisms likely operate, since in vivo spindle size is maintained in extracts prepared from eggs or two-cell stage embryos( Mitchison et al., 2005; Wuhr et al., 2008; Loughlin et al., 2011). As cell and spindle size converge, a constant ratio of ∼2:1 cell diameter:spindle length is established, and spindle length reduces to ∼20 µm by the end of the synchronous cleavage divisions. To what extent cell size influences spindle size or if changes in cytoplasmic activities contribute to spindle scaling at these later stages is unknown. Experiment and simulation indicate that the balance of MT nucleation, transport, and destabilization are critical for setting spindle size in egg extracts( Loughlin et al., 2010; Brugues et al., 2012). By comparing two closely related Xenopus species, we showed previously that meiotic spindle size is partially controlled by activity of the MT severing protein katanin( Loughlin et al., 2011). However, the genetically encoded differences that underpinned variation in katanin activity cannot play a role during early development in an embryo with a single genetic background and when transcription is repressed. In early Caenorhabditis elegans development, decreasing spindle length correlates with a decrease in centrosome size and a decay gradient of the protein of TPXL-1 along spindle MTs( Greenan et al., 2010). In contrast, spindle assembly in X. laevis egg extracts does not require centrosomes and depends heavily on a chromatin-centered gradient of RanGTP, which locally releases spindle assembly factors from the nucleocytoplasmic transport protein importin β and its adaptor importin α that binds nuclear localization sequence( NLS) –containing cargos( Gruss et al., 2001; Kalab and Heald, 2008). The respective contribution of gradients and centrosomes are expected to alter spindle architecture and size, and could be influenced by cell size through availability of components( Goehring and Hyman, 2012). The gradual transition from meiotic to mitotic spindle architecture observed in mouse embryos supports this view( Courtois et al., 2012). Collectively, these observations suggest that changes in gradients and spindle assembly pathways are likely to accompany cell size changes and could influence spindle size. To address how spindle size is determined during vertebrate embryogenesis, we created an embryo extract system capable of assembling spindles in cytoplasmic extracts at any stage of X. laevis development up to the midblastula transition when cell divisions become asynchronous( Newport and Kirschner, 1982). We compared stage 3 and stage 8, the two extremes of spindle size in our extract system, and found that MT dynamics are modified to reduce spindle size upon release of an inhibitory interaction between the MT depolymerizing kinesin kif2a and importin α. This occurs through redistribution of importin α from the cytoplasm to a membrane fraction. Taking advantage of this mechanism to modify spindle size without functionally perturbing the spindle allowed us to uncover an important role for spindle size in maintaining spindle orientation in the large cells of early Xenopus development. This work suggests that the changing physical features of the cell can drive intracellular scaling through biochemical partitioning and steady-state redistribution of activities.
Discussion:
Reconstituting spindle formation in embryo extracts enabled us to elucidate a mechanism that coordinates spindle size with cell size during X. laevis development through differential activation of the kinesin-13 kif2a. While a number of perturbations have been shown to alter spindle size, we have identified, out the hundreds of known spindle associated proteins, physiological effectors that operate to scale the spindle during embryogenesis. Considering our recent work identifying the microtubule severing enzyme katanin as an activity that scales spindle sized between related frog species, modulation of MT destabilization has now emerged as a general means of physiologically scaling spindle size in Xenopus( Loughlin et al., 2011). In contrast to interspecies spindle scaling, which relied on a regulatory phosphorylation site, the developmental mechanism described here depends on decreasing cytoplasmic levels of an inhibitor, importin α, which may be directly linked to changes in the ratio of surface membrane to cell volume( Figure 7D). Importantly, this mechanism functions cell autonomously and independent of developmental stage, thereby assuring that the spindle scales correctly over the range of cell sizes present in the embryo at each cleavage division. It is important to note that neither the interspecies nor developmental scaling mechanisms identified fully account for spindle size differences. Future research will tell whether many factors are involved and if there are other common themes. In principle, any pathway containing factors that partition between the cytoplasm and membrane could have altered output with changing cell size during embryogenesis. Similarly, rounds of DNA, centrosome, or other organelle replication in the absence of cell growth could provide exponentially growing sinks for inhibitory factors or drive spatial redistribution of molecules that contribute to intracellular scaling. Structural components may also become limiting as the cytoplasmic volume decreases, or other aspects of changing cell geometry could alter spatial distributions and reaction rates. It is an open question how many of these processes function to modulate the size of the spindle and other organelles, compared to the contribution of zygotic transcription and other regulatory mechanisms that turn on later in development. We have shown that developmental spindle scaling is coincident with a change in spindle assembly pathway from a chromatin-mediated mechanism to a centrosome-dominated mechanism. In egg extracts and large blastomeres, importin α binds to cargo proteins and provides, in combination with importin β, functional regulation downstream of the RanGTP gradient( Nachury et al., 2001; Wiese et al., 2001). Although the mechanisms that regulate spindle assembly through the RanGTP gradient and downstream effectors are poorly understood( Kalab et al., 2006), we show that the switch in spindle assembly to a more centrosome-mediated pathway occurs concomitantly with a decrease in cytoplasmic levels of importin α. This mechanism is consistent with the gradual change from meiotic to mitotic spindle morphology observed during early mouse development and suggests that the physical changes in embryonic cells size can impact biochemical reactions that alter spindle architecture( Courtois et al., 2012). In egg extracts, importin α phosphorylation is thought to liberate it from membrane stores into the cytoplasm, thereby promoting its transport activity( Hachet et al., 2004). Whether the multiple importin α bands in stage 7 and stage 8 embryos represent different phosphorylation states or some other posttranslational modification remains to be determined. Interestingly, nuclear size is also sensitive to importin α levels, and in this way, nuclear and spindle size may be coordinated, positioning importin α as a central molecule in controlling the size of multiple subcellular structures( Levy and Heald, 2010). While the RanGTP gradient is known to act through activation of MT stabilizers and motor proteins, we have identified kif2a as the first MT-destabilizing protein that could be regulated by this pathway( Walczak and Heald, 2008). Consistent with a role for kif2a in spindle size control, either increasing its levels or disrupting its localization by inhibiting dynein can modulate meiotic spindle size in Xenopus egg extracts( Gaetz and Kapoor, 2004; Ohi et al., 2007). In contrast, inhibiting the other kinesin-13 family member MCAK dramatically affects global MT dynamics( Ohi et al., 2007). This difference may be due to high importin α levels limiting the activity of kif2a in egg extract spindles. Whether importin α regulates kif2a in cultured cells, where it is necessary for bipolar spindle assembly and poleward flux of MTs( Ganem and Compton, 2004; Ganem et al., 2005), is unknown. Kif2a phosphorylation by aurora and polo kinases modulates its activity, which is also regulated through an interaction with inner centromere Kin-I stimulator( ICIS)( Jang et al., 2009; Knowlton et al., 2009). A comprehensive evaluation of kif2a's many interactions will help determine how layers of regulation are integrated to control developmental spindle scaling. By identifying a physiological mechanism that controls spindle size and then altering it, we have identified a role for spindle size in maintaining spindle orientation in very large cells of the early X. laevis embryo. However, chromosome segregation was robust to decreases in spindle size indicating that spindle architecture remained viable and a large spindle is not essential for division of large cells. Previous work has suggested that in contrast to the canonical spindle positioning pathways, astral MTs cannot reach the cortex of large cells to provide position cues and forces at metaphase( Mitchison et al., 2012). Instead, centrosomes are likely positioned prior to spindle assembly through interactions with telophase MT asters of the previous division( Wuhr et al., 2010, 2011), a mechanism that does not appear disrupted under conditions that reduce spindle size in our experiments. The randomized orientation of smaller spindles specifically at metaphase in stage 3 embryos suggests that in addition to initial positioning of centrosomes to define spindle orientation, there is a mechanism that maintains spindle orientation until anaphase onset, after which large anaphase asters may again provide orientation cues. Considering the spindle as a prolate ellipsoid in a viscous medium, the frictional resistance to rotation is nonlinearly related to length. A larger spindle may therefore resist rotation, especially given the highly viscous cytoplasm and the relatively short metaphase duration in blastomeres. While identifying the mechanisms that control spindle size revealed a cellular role in spindle orientation, in the future, it will be interesting to examine how changing spindle size affects the developmental program of the embryo. Overall, our embryo extract system, in combination with biochemical validation of regulatory interactions and experimental manipulation in developing embryos, allows the identification of physiologically relevant molecular mechanisms controlling spindle architecture and cell division.
In the earliest stages of development , animal cells undergo multiple rounds of division without growth via a process known as mitosis . Over the course of just 12 rounds of cell division , a single fertilized egg is transformed into more than 4000 smaller cells . Before dividing , the cell must first replicate its chromosomes . A structure called the mitotic spindle then separates the members of each chromosome pair and distributes them evenly between the two daughter cells . Given that the daughter cells become smaller with each round of division , the spindle must also become smaller to ensure that the chromosomes are pulled apart an appropriate distance . However , it has been unclear how the cell achieves this . Now , Wilbur and Heald report insights into the mechanism by which spindle size is coordinated with cell size , using the model organism Xenopus laevis—a frog that produces large embryos that are easy to manipulate . They began by preparing extracts of cytoplasm from X . laevis embryos at two different developmental stages: one set of embryos contained 4 cells each and the other set contained ∼4000 cells . They found that the spindle , which is composed largely of microtubules—hollow filaments that can become longer or shorter through the addition or removal of tubulin building blocks—was almost twice as large in the four-cell embryos as in the more developed embryos . Moreover , spindle size was determined by the actions of two proteins: kif2a and importin-α . Binding of kif2a destabilized microtubules and caused them to shorten; importin-α blocked this process by binding to kif2a and preventing it from interacting with microtubules . Wilbur and Heald found that over the course of development , importin-α became increasingly localized to the cell membrane , meaning that there was less available to bind to kif2a in the cytoplasm . This freed up kif2a to interact with and destabilize microtubules , and led ultimately to a reduction in spindle size . Given that the overall ratio of cell surface membrane to cytoplasm increases as cells undergo division without growth , interaction between kif2a and importin-α could be the long-sought mechanism by which spindle and cell sizes are coordinated early in development .