Rebecca Heald

A model for the proposed roles of different microtubule-based motor proteins in establishing spindle bipolarity

BACKGROUND In eukaryotes, assembly of the mitotic spindle requires the interaction of chromosomes with microtubules. During this process, several motor proteins that move along microtubules promote formation of a bipolar microtubule array, but the precise mechanism is unclear. In order to examine the roles of different motor proteins in building a bipolar spindle, we have used a simplified system in which spindles assemble around beads coated with plasmid DNA and incubated in extracts from Xenopus eggs. Using this system, we can study spindle assembly in the absence of paired cues, such as centrosomes and kinetochores, whose microtubule-organizing properties might mask the action of motor proteins. RESULTS We blocked the function of individual motor proteins in the Xenopus extracts using specific antibodies. Inhibition of Xenopus kinesin-like protein 1 (Xklp1) led either to the dissociation of chromatin beads from microtubule arrays, or to collapsed microtubule bundles on beads. Inhibition of Eg5 resulted in monopolar microtubule arrays emanating from chromatin beads. Addition of antibodies against dynein inhibited the focusing of microtubule ends into spindle poles in a dose-dependent manner. Inhibition of Xenopus carboxy-terminal kinesin 2 (XCTK2) affected both pole formation and spindle stability. Co-inhibition of XCTK2 and dynein dramatically increased the severity of spindle pole defects. Inhibition of Xklp2 caused only minor spindle pole defects. CONCLUSIONS Multiple microtubule-based motor activities are required for the bipolar organization of microtubules around chromatin beads, and we propose a model for the roles of the individual motor proteins in this process.

Importin β Is a Mitotic Target of the Small GTPase Ran in Spindle Assembly

Abstract The GTPase Ran has recently been shown to stimulate microtubule polymerization in mitotic extracts, but its mode of action is not understood. Here we show that the mitotic role of Ran is largely mediated by the nuclear transport factor importin β. Importin β inhibits spindle formation in vitro and in vivo and sequesters an aster promoting activity (APA) that consists of multiple, independent factors. One component of APA is the microtubule-associated protein NuMA. NuMA and other APA components are discharged from importin β by RanGTP and induce spindle-like structures in the absence of centrosomes, chromatin, or Ran. We propose that RanGTP functions in mitosis as in interphase by locally releasing cargoes from transport factors. In mitosis, this promotes spindle assembly by organizing microtubules in the vicinity of chromosomes.

Analysis of a RanGTP-regulated gradient in mitotic somatic cells

The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating interactions between cargoes and nuclear transport receptors of the importin-β family. The Ran–importin-β system also functions in mitotic spindle assembly and nuclear pore and nuclear envelope formation. The common principle underlying these diverse functions throughout the cell cycle is thought to be anisotropy of the distribution of RanGTP (the RanGTP gradient), driven by the chromatin-associated guanine nucleotide exchange factor RCC1 (refs 1, 4, 5). However, the existence and function of a RanGTP gradient during mitosis in cells is unclear. Here we examine the Ran–importin-β system in cells by conventional and fluorescence lifetime microscopy using a biosensor, termed Rango, that increases its fluorescence resonance energy transfer signal when released from importin-β by RanGTP. Rango is predominantly free in mitotic cells, but is further liberated around mitotic chromatin. In vitro experiments and modelling show that this localized increase of free cargoes corresponds to changes in RanGTP concentration sufficient to stabilize microtubules in extracts. In cells, the Ran–importin-β–cargo gradient kinetically promotes spindle formation but is largely dispensable once the spindle has been established. Consistent with previous reports, we observe that the Ran system also affects spindle pole formation and chromosome congression in vivo. Our results demonstrate that conserved Ran-regulated pathways are involved in multiple, parallel processes required for spindle function, but that their relative contribution differs in chromatin- versus centrosome/kinetochore-driven spindle assembly systems.

MECHANISMS OF MITOTIC SPINDLE ASSEMBLY AND FUNCTION

The mitotic spindle is the macromolecular machine that segregates chromosomes to two daughter cells during mitosis. The major structural elements of the spindle are microtubule polymers, whose intrinsic polarity and dynamic properties are critical for bipolar spindle organization and function. In most cell types, spindle microtubule nucleation occurs primarily at two centrosomes, which define the spindle poles, but microtubules can also be generated by the chromosomes and within the spindle itself. Many associated factors help organize the spindle, including molecular motors and regulators of microtubule dynamics. The past decade has provided a wealth of information on the molecular players that are critical for spindle assembly as well as a high-resolution view of the intricate movements and dynamics of the spindle microtubules and the chromosomes. In this chapter we provide a historical account of the key observations leading to current models of spindle assembly, as well as an up-to-date status report on this exciting field.

Mechanisms and Molecules of the Mitotic Spindle

In all eukaryotes, morphogenesis of the microtubule cytoskeleton into a bipolar spindle is required for the faithful transmission of the genome to the two daughter cells during division. This process is facilitated by the intrinsic polarity and dynamic properties of microtubules and involves many proteins that modulate microtubule organization and stability. Recent work has begun to uncover the molecular mechanisms behind these dynamic events. Here we describe current models and discuss some of the complex repertoire of factors required for spindle assembly and chromosome segregation.

A Rae1-Containing Ribonucleoprotein Complex Is Required for Mitotic Spindle Assembly

Centrosome-independent microtubule polymerization around chromosomes has been shown to require a local gradient of RanGTP, which discharges mitotic cargoes from the nuclear import receptor importin beta. Here, we have used an activity-based assay in Xenopus egg extracts to purify the mRNA export protein Rae1 as a spindle assembly factor regulated by this pathway. Rae1 is a microtubule-associated protein that binds directly to importin beta. Depletion of Rae1 from extracts or cells severely inhibits mitotic spindle assembly. A purified Rae1 complex stabilizes microtubules in egg extracts in a RanGTP/importin beta-regulated manner. Interestingly, Rae1 exists in a large ribonucleoprotein complex, which requires RNA for its activity to control microtubule dynamics in vitro. Furthermore, we provide evidence that RNA associates with the mitotic spindle and that it plays a direct, translation-independent role in spindle assembly. Our studies reveal an unexpected function for RNA in spindle morphogenesis.

Genome evolution in the allotetraploid frog Xenopus laevis

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of ‘fossil’ transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17–18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

Motor Function in the Mitotic Spindle Minireview

Motor function is integral to spindle morphogenesis. Whereas the structural cues provided by centrosomes and kinetochores previously dominated our understanding of mitosis, the identification and characterization of spindle motors has illuminated less obvious mechanisms, which have only been appreciated since advancing technology has allowed a wider range of manipulations and visualization of spindle dynamics at high resolution. The more we learn, the more we realize that multiple overlapping and redundant forces contribute to the high fidelity of chromosome segregation, which is not surprising considering the devastating effects of errors in this process.Studies in many different eukaryotic systems have led to models for the function of individual spindle motors based on their molecular features and the effects of their disruption, but have not always led to a consensus for the role of a particular motor. The lesson here is that different systems emphasize different motors and mechanisms. For example, while all eukaryotes require one or more bipolar KIN N kinesin for spindle assembly, yeast appear to lack chromokinesins as well as poleward microtubule flux (Mallavarapu et al. 1999xMallavarapu, A, Sawin, K, and Mitchison, T. Curr. Biol. 1999; 9: 1423–1426Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesMallavarapu et al. 1999). Is poleward microtubule flux an important component of spindle function in larger eukaryotic cells? Or simply a by-product of the many forces exerted on spindle microtubules? More complex reconstitution experiments using pure motors and dynamic microtubules may help address this question.The next big step in understanding how spindle motors function requires studying their interacting proteins, their regulation and the integration of their activities. Some spindle proteins that play important roles in conjunction with motors have already been identified. For example, NuMA is transported to microtubule minus ends by dynein/dynactin where it plays a key role in spindle pole cohesion (Merdes et al. 2000xMerdes, A, Heald, R, Samejima, K, Earnshaw, W.C, and Cleveland, D.W. J. Cell Biol. 2000; 149: 851–862Crossref | PubMed | Scopus (213)See all ReferencesMerdes et al. 2000). Interactions with motor accessory proteins, motility itself, and motor stability are likely to be further regulated by posttranslational modifications such as phosphorylation. As we have seen, changing the balance of motor activities can drive changes in spindle structure or chromosome movement, yet the integration of motor activities is poorly understood. Altogether, the many directions in this field promise to engage spindle enthusiasts for years to come.*E-mail: heald@socrates.berkeley.edu

Genome-wide analysis demonstrates conserved localization of messenger RNAs to mitotic microtubules.

RNA localization is of critical importance in many fundamental cell biological and developmental processes by regulating the spatial control of gene expression. To investigate how spindle-localized RNAs might influence mitosis, we comprehensively surveyed all messenger RNAs (mRNAs) that bound to microtubules during metaphase in both Xenopus laevis egg extracts and mitotic human cell extracts. We identify conserved classes of mRNAs that are enriched on microtubules in both human and X. laevis. Active mitotic translation occurs on X. laevis meiotic spindles, and a subset of microtubule-bound mRNAs (MT-mRNAs) associate with polyribosomes. Although many MT-mRNAs associate with polyribosomes, we find that active translation is not required for mRNA localization to mitotic microtubules. Our results represent the first genome-wide survey of mRNAs localized to a specific cytoskeletal component and suggest that microtubule localization of specific mRNAs is likely to function in mitotic regulation and mRNA segregation during cell division.

Cytoplasmic Volume Modulates Spindle Size During Embryogenesis

Scaling Spindle Size The difficulty of modulating cell size in vivo has made it hard to test hypotheses for organelle size scaling during development. To this end, Hazel et al. (p. 853) and Good et al. (p. 856) developed microfluidic systems in which cytoplasmic extracts are encapsulated in compartments with definable size. The size of mitotic spindles assembled within cell-free extracts scaled with the volume of the compartment within which the spindle assembled. The findings suggest that the diminished availability of cytoplasmic components, notably tubulin, concomitant with cell size reduction, prescribes a smaller spindle size. Microfluidic techniques reveal how mitotic spindle size is regulated by titratable cytosolic factors. Rapid and reductive cell divisions during embryogenesis require that intracellular structures adapt to a wide range of cell sizes. The mitotic spindle presents a central example of this flexibility, scaling with the dimensions of the cell to mediate accurate chromosome segregation. To determine whether spindle size regulation is achieved through a developmental program or is intrinsically specified by cell size or shape, we developed a system to encapsulate cytoplasm from Xenopus eggs and embryos inside cell-like compartments of defined sizes. Spindle size was observed to shrink with decreasing compartment size, similar to what occurs during early embryogenesis, and this scaling trend depended on compartment volume rather than shape. Thus, the amount of cytoplasmic material provides a mechanism for regulating the size of intracellular structures.