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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.

Cells are filled with macromolecules and polymer networks that set scale-dependent viscous and elastic properties to the cytoplasm. Although the role of these parameters in molecular diffusion, reaction kinetics, and cellular biochemistry is being increasingly recognized, their contributions to the motion and positioning of larger organelles, such as mitotic spindles for cell division, remain unknown. Here, using magnetic tweezers to displace and rotate mitotic spindles in living embryos, we uncovered that the cytoplasm can impart viscoelastic reactive forces that move spindles, or passive objects with similar size, back to their original positions. These forces are independent of cytoskeletal force generators yet reach hundreds of piconewtons and scale with cytoplasm crowding. Spindle motion shears and fluidizes the cytoplasm, dissipating elastic energy and limiting spindle recoils with functional implications for asymmetric and oriented divisions. These findings suggest that bulk cytoplasm material properties may constitute important control elements for the regulation of division positioning and cellular organization.

The mitotic spindle must function in cell types that vary greatly in size, and its dimensions scale with the rapid, reductive cell divisions that accompany early stages of development. The mechanism responsible for this scaling is unclear, because uncoupling cell size from a developmental or cellular context has proven experimentally challenging. We combined microfluidic technology with Xenopus egg extracts to characterize spindle assembly within discrete, geometrically defined volumes of cytoplasm. Reductions in cytoplasmic volume, rather than developmental cues or changes in cell shape, were sufficient to recapitulate spindle scaling observed in Xenopus embryos. Thus, mechanisms extrinsic to the spindle, specifically a limiting pool of cytoplasmic component(s), play a major role in determining spindle size.

Key Points The assembly of a bipolar microtubule-based spindle is crucial for the accurate and timely segregation of the chromosomes into the two daughter cells during mitosis. Three microtubule nucleation pathways function in mitotic cells to contribute microtubules to the assembling spindle, namely the centrosome-, chromatin- and microtubule-mediated pathways. Microtubules nucleated from the centrosomes find the chromosomes via 'search and capture' — a process that relies on the dynamic instability of microtubules — which allows them to search space to contact the chromosomes. When a microtubule contacts a kinetochore, it is captured and becomes stabilized to form kinetochore fibres (K-fibres). The speed and efficiency of chromosome capture is promoted by the chromatin-mediated pathway that biases microtubule nucleation and stabilization to the vicinity of the chromosomes. Microtubules are also generated from within the spindle itself via Augmin, which promotes microtubule nucleation from pre-existing microtubules. This nucleation increases the density of microtubules within the spindle and thus contributes to its robustness. Although the nucleation pathways are at least partially redundant, they are integrated to provide an intricate balance of microtubule nucleation that ensures the fidelity of chromosome segregation and the timely completion of mitosis. In the absence of any one of the pathways, spindle assembly still occurs — although with increased use of the remaining pathways — but mitosis takes longer and this can result in genome instability. Three microtubule nucleation pathways — initiated from centrosomes, chromatin and existing spindle microtubules — contribute to the assembly of a functional mitotic spindle in animal cells to ensure accurate chromosome segregation. Recent findings have shed light on their relative contributions to building the spindle and on adaptation of the spindle to variations in cell size and shape. The mitotic spindle has a crucial role in ensuring the accurate segregation of chromosomes into the two daughter cells during cell division, which is paramount for maintaining genome integrity. It is a self-organized and dynamic macromolecular structure that is constructed from microtubules, microtubule-associated proteins and motor proteins. Thirty years of research have led to the identification of centrosome-, chromatin- and microtubule-mediated microtubule nucleation pathways that each contribute to mitotic spindle assembly. Far from being redundant pathways, data are now emerging regarding how they function together to ensure the timely completion of mitosis. We are also beginning to comprehend the multiple mechanisms by which cells regulate spindle scaling. Together, this research has increased our understanding of how cells coordinate hundreds of proteins to assemble the dynamic, precise and robust structure that is the mitotic spindle.

The centrosome acts as the main microtubule-nucleating organelle in animal cells and plays a critical role in mitotic spindle orientation and in genome stability. Yet, despite its central role in cell biology, the centrosome is not present in all multicellular organisms or in all cells of a given organism. The main outcome of centrosome reproduction is the transmission of polarity to daughter cells and, in most animal species, the sperm-donated centrosome defines embryo polarity. Here I will discuss the role of the centrosome in cell polarity, resulting from its ability to position the nucleus at the cell center, and discuss how centrosome innovation might have been critical during metazoan evolution.

The metaphase spindle is a dynamic structure orchestrating chromosome segregation during cell division. Recently, soft matter approaches have shown that the spindle behaves as an active liquid crystal. Still, it remains unclear how active force generation contributes to its characteristic spindle-like shape. Here we combine theory and experiments to show that molecular motor-driven forces shape the structure through a barreling-type instability. We test our physical model by titrating dynein activity in Xenopus egg extract spindles and quantifying the shape and microtubule orientation. We conclude that spindles are shaped by the interplay between surface tension, nematic elasticity, and motor-driven active forces. Our study reveals how motor proteins can mold liquid crystalline droplets and has implications for the design of active soft materials.

Mutations in a tubulin gene caused infertility due to oocyte arrest in about a third of families tested. The investigators found that the mutant tubulins wreak havoc on microtubule assembly in the oocyte. Successful human reproduction starts when a metaphase II oocyte fuses with a sperm cell to form a fertilized egg. In human oocytes, the meiotic cell cycle begins in the neonatal ovary and pauses at prophase I of meiosis until puberty, when a surge of luteinizing hormone stimulates the resumption of meiosis and ovulation. This leads to progression of the oocyte from metaphase I to metaphase II. 1 – 3 Oocytes arrested in prophase I have an intact nucleus, termed the germinal vesicle, whereas oocytes that have resumed meiosis are characterized by the breakdown of the germinal vesicle. After germinal-vesicle breakdown, metaphase I . . .

Chromosome segregation typically requires centrosomes, which generate the microtubule spindle. However, mammalian eggs build a spindle and segregate chromosomes without centrosomes. How acentrosomal spindles are organized has remained elusive. So et al. show that centrosomal and microtubule-associated proteins are repurposed into a large “liquid-like meiotic spindle domain” (LISD) in eggs. The domains localized to spindle poles and also extended to the spindle fibers that connect to kinetochores. LISDs formed by phase separation and were required for spindle assembly, serving as reservoirs that locally sequester and mobilize spindle assembly factors within the large egg cytoplasm. Science , this issue p. eaat9557 Phase separation of microtubule regulatory factors promotes acentrosomal spindle assembly in mammalian oocytes. Mammalian oocytes segregate chromosomes with a microtubule spindle that lacks centrosomes, but the mechanisms by which acentrosomal spindles are organized and function are largely unclear. In this study, we identify a conserved subcellular structure in mammalian oocytes that forms by phase separation. This structure, which we term the liquid-like meiotic spindle domain (LISD), permeates the spindle poles and forms dynamic protrusions that extend well beyond the spindle. The LISD selectively concentrates multiple microtubule regulatory factors and allows them to diffuse rapidly within the spindle volume. Disruption of the LISD via different means disperses these factors and leads to severe spindle assembly defects. Our data suggest a model whereby the LISD promotes meiotic spindle assembly by serving as a reservoir that sequesters and mobilizes microtubule regulatory factors in proximity to spindle microtubules.

Polo-like kinase 1 (PLK1), the prototypical member of the polo-like family of serine/threonine kinases, is a pivotal regulator of mitosis and cytokinesis in eukaryotes. Many layers of regulation have evolved to target PLK1 to different subcellular structures and to its various mitotic substrates in line with its numerous functions during mitosis. Collective work is starting to illuminate an important set of substrates for PLK1: the mitotic kinases that together ensure the fidelity of the cell division process. Amongst these, recent developments argue that PLK1 regulates the activity of the histone kinases Aurora B and Haspin to define centromere identity, of MPS1 to initiate spindle checkpoint signaling, and of BUB1 and its pseudokinase paralog BUBR1 to coordinate spindle checkpoint activation and inactivation. Here, we review the recent work describing the regulation of these kinases by PLK1. We highlight common themes throughout and argue that a major mitotic function of PLK1 is as a master regulator of these key kinases.

Key Points The chromosomal passenger complex (CPC) is a 'master controller' of cell division that is formed by a kinase module (Aurora B kinase) and a localization module (the scaffolding protein inner centromere protein (INCENP), survivin and borealin). Multiple post-translational modifications of CPC components contribute to the appropriate localization and regulation of Aurora B activity. Full activation of Aurora B kinase is a complex multistage process that is mediated by the other CPC components and other cell cycle kinases. In early mitosis, CPC recruitment to the inner centromere is mediated by post-translational modifications of two histones: phosphorylation of histone H3 (by haspin kinase) and of histone H2A (by Bub1 kinase). The baculovirus IAP repeat (BIR) domain of survivin recognizes H3 phosphorylated at Thr3. Further enrichment of the CPC at the inner centromere is mediated by Aurora B-dependent regulatory feedback loops. Roles of the CPC in early mitosis include the regulation of chromosome structure, kinetochore–microtubule attachments and the spindle assembly checkpoint. The CPC relocalizes to central spindle microtubules at the onset of anaphase in a highly regulated process that is mediated by a decrease of cyclin-dependent kinase 1 (Cdk1) activity, interaction with the kinesin mitotic kinesin-like protein 2 (Mklp2) and under the control of several phosphatases and Aurora B kinase itself. Functions of the CPC in late mitosis include the formation and stabilization of the spindle midzone in anaphase and the regulation of the contractile ring formation. The CPC has further roles later on in cytokinesis, in which it regulates furrow ingression and the abscission checkpoint. The chromosomal passenger complex (CPC), which is formed by inner centromere protein (INCENP), borealin, survivin and Aurora B kinase, targets to different locations at different times during mitosis. As it regulates key events at each of these locations, the CPC can be considered as a master regulator of mitosis. Successful cell division requires the precise and timely coordination of chromosomal, cytoskeletal and membrane trafficking events. These processes are regulated by the competing actions of protein kinases and phosphatases. Aurora B is one of the most intensively studied kinases. In conjunction with inner centromere protein (INCENP), borealin (also known as Dasra) and survivin it forms the chromosomal passenger complex (CPC). This complex targets to different locations at differing times during mitosis, where it regulates key mitotic events: correction of chromosome–microtubule attachment errors; activation of the spindle assembly checkpoint; and construction and regulation of the contractile apparatus that drives cytokinesis. Our growing understanding of the CPC has seen it develop from a mere passenger riding on the chromosomes to one of the main controllers of mitosis.