Asymmetric Spindle Research Articles

Time-Lapse Imaging of Asymmetric Spindle Positioning During Endothelial Tip Cell Division in Angiogenesis In Vivo.

The branching of new blood vessels by angiogenesis is critical to the development, growth, and repair of most vertebrate tissues and is frequently dysregulated in disease. At its core, angiogenesis is driven by the collective migration of leading "tip" and follower "stalk" endothelial cells. Recent work reveals that this collective movement is coordinated by asymmetric tip cell divisions that generate daughters of distinct size, signaling capacity and tip-stalk behaviors. Polarized mitotic spindle positioning is critical to generating such asymmetries in daughter cell size. However, the spatiotemporal dynamics of vertebrate spindle movement are often difficult to explore using in vivo systems. Here we describe a method for the sample preparation, live-imaging and data analysis of endothelial cell mitotic spindle positioning in developing zebrafish embryos. This method enables single-cell and population-level spindle dynamics to be monitored and quantified, both in wild-type or genetically/pharmacologically perturbed embryos. Moreover, this approach can be easily adapted for live imaging of spindle dynamics in other zebrafish embryonic tissues that experience similar asymmetric divisions, such as the trunk neural crest.

Caspase-4 has a role in cell division in epithelial cells through actin depolymerization

In addition to its role in pyroptosis and inflammatory cytokine maturation, caspase-4 (CASP4) also contributes to the fusion of phagosomes with lysosomes and cell migration. However, its role in cell division remains elusive. In this study, we demonstrate that CASP4 is indispensable for proper cell division in epithelial cells. Knockout of CASP4 (CASP4 KO) in HepG2 cells led to delayed cell proliferation, increased cell size, and increased multinucleation. In mitosis, CASP4 KO cells showed multipolar spindles, asymmetric spindle positioning, and chromosome segregation errors, ultimately increasing DNA content and chromosome number. We also found that phalloidin, a marker of filamentous actin, increased in CASP4 KO cells owing to suppressed actin depolymerization. Moreover, the levels of actin polymerization-related proteins, including Rho-associated protein kinase1 (ROCK1), LIM kinase1 (LIMK1), and phosphorylated cofilin, significantly increased in CASP4 KO cells. These results suggest that CASP4 contributes to proper cell division through actin depolymerization.

A monoastral mitotic spindle determines lineage fate and position in the mouse embryo.

During mammalian development, the first asymmetric cell divisions segregate cells into inner and outer positions of the embryo to establish the pluripotent and trophectoderm lineages. Typically, polarity components differentially regulate the mitotic spindle via astral microtubule arrays to trigger asymmetric division patterns. However, early mouse embryos lack centrosomes, the microtubule-organizing centres (MTOCs) that usually generate microtubule asters. Thus, it remains unknown whether spindle organization regulates lineage segregation. Here we find that heterogeneities in cell polarity in the early 8-cell-stage mouse embryo trigger the assembly of a highly asymmetric spindle organization. This spindle arises in an unusual modular manner, forming a single microtubule aster from an apically localized, non-centrosomal MTOC, before joining it to the rest of the spindle apparatus. When fully assembled, this 'monoastral' spindle triggers spatially asymmetric division patterns to segregate cells into inner and outer positions. Moreover, the asymmetric inheritance of spindle components causes differential cell polarization to determine pluripotent versus trophectoderm lineage fate.

Thermal error detection and compensation technology for spindle of horizontal CNC machine tool with large torque

Titanium alloys and other difficult-to-machine materials are machined using large-torque CNC machine tools, and asymmetric mechanical spindle is employed in its spindle system. The mechanical spindle demonstrates high power and large torque. The asymmetric design makes the spindle structure more compact while reducing assembly requirements. Nevertheless, when processed, the asymmetric spindle shows complex thermal characteristics and makes it difficult to ensure real-time control, which easily results in significant errors for parts. To eliminate the uncontrollable thermal error, the efforts are made as follows. Firstly, the cold state and hot state attributes in thermal error analysis of mechanical spindle are defined, and the method to distribute the measuring position of asymmetric mechanical spindle temperature is proposed. Secondly, a three-dimensional space detection and analysis method for thermal error of six-vector eight-test based on rotation error vector and deviation error vector is suggested. Thirdly, according to the experimental results, the six-vector thermal error of the three-dimensional space is determined, and then a thermal error compensation technology based on Siemens space coordinate transformation parameters is proposed. Finally, the S specimen recommended by ISO 10791-1-annex A was used for machining verification before and after thermal error compensation. By comparing the 0.129 mm deviation before compensation and the 0.085 mm deviation after compensation, the machining accuracy of parts is discovered to improve by 34.1%. Therefore, the above research lays a theoretical foundation for the follow-up thermal error detection and compensation for the asymmetric spindle of large torque CNC machine tools of the same type.

WDR62 is a novel participator in spindle migration and asymmetric cytokinesis during mouse oocyte meiotic maturation

In female meiosis, oocyte meiotic maturation is a form of asymmetric cell division, producing the first polar body and a large oocyte, in which the asymmetry of oocyte meiotic division depends on spindle migration and positioning, and cortical polarization. In this study, we conclude that WDR62 (WD40-repeat protein 62) plays an important role in asymmetric meiotic division during mouse oocyte maturation. Our initial study demonstrated that WDR62 mainly co-localized with chromosomes during mouse oocyte meiotic maturation. Interference of Wdr62 by siRNA microinjection did not affect germinal vesicle breakdown (GVBD) but compromised the first polar body extrusion (PBE) with the large polar bodies generated, which is coupled with a higher incidence of spindle abnormality and chromosome misalignment. Further analysis concluded that loss of WDR62 blocked asymmetric spindle positioning and actin cap formation, which should be responsible for large polar body extrusion. Moreover, WDR62 decline intervened with the Arp2/3 complex, an upstream regulator for the cortical actin. Besides for p-MAPK, a critical regulator for the asymmetric division of oocyte, WDR62-depleted oocytes showed perturbation only in localization pattern but not expression level. In summary, our study defines WDR62 as an essential cytoskeletal regulator of spindle migration and asymmetric division during mouse oocyte meiotic maturation.

Nondisjunction and unequal spindle organization accompany the drive of Aegilops speltoides B chromosomes.

B chromosomes (Bs) are supernumerary chromosomes, which are often preferentially inherited. When transmission rates of chromosomes are higher than 0.5, not obeying the Mendelian law of equal segregation, the resulting transmission advantage is collectively referred to as 'chromosome drive'. Here we analysed the drive mechanism of Aegilops speltoides Bs. The repeat AesTR-183 of A.speltoides Bs, which also can be detected on the Bs of Aegilops mutica and rye, was used to track Bs during pollen development. Nondisjunction of CENH3-positive, tubulin interacting B sister chromatids and an asymmetric spindle during first pollen grain mitosis are key for the accumulation process. A quantitative flow cytometric approach revealed that, independent of the number of Bs present in the mother plant, Bs accumulate in the generative nuclei to >93%. Nine out of 11 tested (peri)centromeric repeats were shared by A and B chromosomes. Our findings provide new insights into the process of chromosome drive. Quantitative flow cytometry is a useful and reliable method to study the drive frequency of Bs. Nondisjunction and unequal spindle organization accompany during first pollen mitosis the drive of A.speltoides Bs. The prerequisites for the drive process seems to be common in Poaceae.

Brefeldin A impairs porcine oocyte meiotic maturation via interruption of organelle dynamics.

Brefeldin A (BFA) is a lactone antibiotic synthesized from palmitic acid by several fungi that could block anterograde transport of proteins from endoplasmic reticulum to Golgi apparatus by reversible disruption of the Golgi complex. Previous investigations have shown that BFA induces the apoptosis of cancer cells in mitosis and impairs asymmetric spindle positioning in meiosis. Here, we document that exposure to BFA in porcine oocytes compromises the meiotic maturation via disrupting both nuclear and cytoplasmic maturation. We found that BFA exposure collapsed the cytoskeleton assembly by showing the aberrant spindle organization with misaligned chromosomes and defective actin dynamics. Furthermore, the distribution of both mitochondria and cortical granules (CGs), two important indexes of cytoplasmic maturation of oocytes, was disturbed following BFA exposure. We finally validated that the localization of ovastacin, a component of CGs that is essential for the postfertilization removal of sperm-binding sites in the zona pellucida, was also perturbed in BFA-exposed oocytes, which might weaken their fertilization capacity. Collectively, these findings indicate that Golgi-mediated protein transport is indispensable for the porcine oocyte meiotic maturation.

EphA2 phosphorylation at Ser897 by the Cdk1/MEK/ERK/RSK pathway regulates M-phase progression via maintenance of cortical rigidity.

Successful cell division is accomplished by the proper formation of the mitotic spindle. Here, we show that EphA2 knockdown causes mitotic errors, including a delay in M-phase progression, asymmetric spindle positioning, multipolar spindles, and cell blebs. It has been known that EphA2 is phosphorylated at Tyr588, which is triggered by the ligand binding, and at Ser897 downstream of growth factor signaling. Upon mitotic entry, EphA2 is phosphorylated at Ser897, accompanied by a reduction in Tyr588 phosphorylation. This EphA2 phosphorylation at Ser897 is inhibited by MEK/ERK and 90 kDa ribosomal S6 kinase (RSK) inhibitors and is induced by the introduction of active cyclin-dependent kinase 1 (Cdk1) and cyclin B1. EphA2 knockdown-induced M-phase delay and cell blebs are rescued by wild type EphA2 expression but not by Ser897Ala mutant. The Ras homolog gene family member G (RhoG) guanine nucleotide exchange factor Ephexin4 interacts with EphA2 in a Ser897 phosphorylation-dependent manner, and its knockdown delays M-phase progression and causes RhoG delocalization. RhoG knockdown delays M-phase progression, and EphA2 knockdown-induced M-phase delay is partially rescued by the constitutively active RhoG mutant. These results suggest that, in EphA2-expressing cells, EphA2 phosphorylation at Ser897 participates in proper M-phase progression downstream of the Cdk1/MEK/ERK/RSK pathway because of its role in maintaining cortical rigidity via Ephexin4 and RhoG and thereby regulating mitotic spindle formation.-Kaibori, Y. Saito, Y., Nakayama, Y. EphA2 phosphorylation at Ser897 by the Cdk1/MEK/ERK/RSK pathway regulates M-phase progression via maintenance of cortical rigidity.

The kinases PIG-1 and PAR-1 act in redundant pathways to regulate asymmetric division in the EMS blastomere of C. elegans

The PAR-1 kinase of C. elegans is localized to the posterior of the one-cell embryo and its mutations affect asymmetric spindle placement and partitioning of cytoplasmic components in the first cell cycle. However, par-1 mutations do not cause failure to restrict the anterior PAR polarity complex to the same extent as mutations in the posteriorly localized PAR-2 protein. Further, it has been difficult to examine the role of PAR-1 in subsequent divisions due to the early defects in par-1 mutant embryos. Here we show that the PIG-1 kinase acts redundantly with PAR-1 to restrict the anterior PAR-3 protein for normal polarity in the one-cell embryo. By using a temperature sensitive allele of par-1, which exhibits enhanced lethality when combined with a pig-1 mutation, we have further explored roles for these genes in subsequent divisions. We find that both PIG-1 and PAR-1 regulate spindle orientation in the EMS blastomere of the four-cell stage embryo to ensure that it undergoes an asymmetric division. In this cell, PIG-1 and PAR-1 act in parallel pathways for spindle positioning, PIG-1 in the MES-1/SRC-1 pathway and PAR-1 in the Wnt pathway.

Asymmetric Tyrosination of Spindle Microtubules Facilitates Selfish Inheritance.

Meiotic drive is an enigmatic process that results from biased segregation of selfish genetic elements that enhance their own transmission and drive evolution. During asymmetric female meiotic divisions, selfish elements segregate preferentially towards the egg rather than polar bodies. Recent findings demonstrate that asymmetric spindle tyrosination helps selfish elements to cheat.

Meeting the meiotic challenge: Specializations in mammalian oocyte spindle formation.

Oocytes uniquely accumulate cytoplasmic constituents to support early embryogenesis. This unique specialization is accompanied by acquisition of a large size and by execution of asymmetric meiotic divisions that preserve precious ooplasm through the expulsion of minimal size polar bodies. While often taken for granted, these basic features of oogenesis necessitate unique specializations of the meiotic apparatus. These include a chromatin-sourced RanGTP gradient that restricts spindle size by defining a spatial domain where meiotic spindles form, acentriolar centrosomes that rely on microtubule organizing centers to form spindle poles, and an actin-based mechanism for asymmetric spindle positioning. Additionally, localized protein synthesis to support spindle formation is achieved in the spindle forming region, whilst protein synthesis is reduced elsewhere in the ooplasm. This is achieved through enrichment of spindle-related mRNAs in the spindle forming region combined with local PLK1-mediated phosphorylation and inactivation of the translational repressor EIF4EBP1. This allows PLK1 to function as an important regulatory nexus through which endogenous and exogenous signals can impact spindle formation and function, and highlights the important role that PLK1 may have in maintaining oocyte quality and fertility.

Actin Disassembly: How to Contract without Motors?

Contractile actin networks take on various functions in cells. How disordered actin networks contract is still poorly understood. A recent study proposes a contractile mechanism that is driven by actin disassembly and required to prevent chromosome losses in starfish oocytes.

Evolution of mitotic spindle behavior during the first asymmetric embryonic division of nematodes.

Asymmetric cell division is essential to generate cellular diversity. In many animal cells, the cleavage plane lies perpendicular to the mitotic spindle, and it is the spindle positioning that dictates the size of the daughter cells. Although some properties of spindle positioning are conserved between distantly related model species and different cell types, little is known of the evolutionary robustness of the mechanisms underlying this event. We recorded the first embryonic division of 42 species of nematodes closely related to Caenorhabditis elegans, which is an excellent model system to study the biophysical properties of asymmetric spindle positioning. Our recordings, corresponding to 128 strains from 27 Caenorhabditis and 15 non-Caenorhabditis species (accessible at http://www.ens-lyon.fr/LBMC/NematodeCell/videos/), constitute a powerful collection of subcellular phenotypes to study the evolution of various cellular processes across species. In the present work, we analyzed our collection to the study of asymmetric spindle positioning. Although all the strains underwent an asymmetric first cell division, they exhibited large intra- and inter-species variations in the degree of cell asymmetry and in several parameters controlling spindle movement, including spindle oscillation, elongation, and displacement. Notably, these parameters changed frequently during evolution with no apparent directionality in the species phylogeny, with the exception of spindle transverse oscillations, which were an evolutionary innovation at the base of the Caenorhabditis genus. These changes were also unrelated to evolutionary variations in embryo size. Importantly, spindle elongation, displacement, and oscillation each evolved independently. This finding contrasts starkly with expectations based on C. elegans studies and reveals previously unrecognized evolutionary changes in spindle mechanics. Collectively, these data demonstrate that, while the essential process of asymmetric cell division has been conserved over the course of nematode evolution, the underlying spindle movement parameters can combine in various ways. Like other developmental processes, asymmetric cell division is subject to system drift.

Kif2 localizes to a subdomain of cortical endoplasmic reticulum that drives asymmetric spindle position

Asymmetric positioning of the mitotic spindle is a fundamental process responsible for creating sibling cell size asymmetry; however, how the cortex causes the depolymerization of astral microtubules during asymmetric spindle positioning has remained elusive. Early ascidian embryos possess a large cortical subdomain of endoplasmic reticulum (ER) that causes asymmetric spindle positioning driving unequal cell division. Here we show that the microtubule depolymerase Kif2 localizes to this subdomain of cortical ER. Rapid live-cell imaging reveals that microtubules are less abundant in the subdomain of cortical ER. Inhibition of Kif2 function prevents the development of mitotic aster asymmetry and spindle pole movement towards the subdomain of cortical ER, whereas locally increasing microtubule depolymerization causes exaggerated asymmetric spindle positioning. This study shows that the microtubule depolymerase Kif2 is localized to a cortical subdomain of endoplasmic reticulum that is involved in asymmetric spindle positioning during unequal cell division.

Molecular Programs Underlying Asymmetric Stem Cell Division and Their Disruption in Malignancy.

Asymmetric division of stem cells is a highly conserved and tightly regulated process by which a single stem cell produces two unequal daughter cells. One retains its stem cell identity while the other becomes specialized through a differentiation program and loses stem cell properties. Coordinating these events requires control over numerous intra- and extracellular biological processes and signaling networks. In the initial stages, critical events include the compartmentalization of fate determining proteins within the mother cell and their subsequent passage to the appropriate daughter cell in order to direct their destiny. Disturbance of these events results in an altered dynamic of self-renewing and differentiation within the cell population, which is highly relevant to the growth and progression of cancer. Other critical events include proper asymmetric spindle assembly, extrinsic regulation through micro-environmental cues, and non-canonical signaling networks that impact cell division and fate determination. In this review, we discuss mechanisms that maintain the delicate balance of asymmetric cell division in normal tissues and describe the current understanding how some of these mechanisms are deregulated in cancer.

Klp10A, a stem cell centrosome-enriched kinesin, balances asymmetries in Drosophila male germline stem cell division.

Asymmetric stem cell division is often accompanied by stereotypical inheritance of the mother and daughter centrosomes. However, it remains unknown whether and how stem cell centrosomes are uniquely regulated and how this regulation may contribute to stem cell fate. Here we identify Klp10A, a microtubule-depolymerizing kinesin of the kinesin-13 family, as the first protein enriched in the stem cell centrosome in Drosophila male germline stem cells (GSCs). Depletion of klp10A results in abnormal elongation of the mother centrosomes in GSCs, suggesting the existence of a stem cell-specific centrosome regulation program. Concomitant with mother centrosome elongation, GSCs form asymmetric spindle, wherein the elongated mother centrosome organizes considerably larger half spindle than the other. This leads to asymmetric cell size, yielding a smaller differentiating daughter cell. We propose that klp10A functions to counteract undesirable asymmetries that may result as a by-product of achieving asymmetries essential for successful stem cell divisions.

Tropomodulin-3 is essential in asymmetric division during mouse oocyte maturation.

The dynamic polymerization and depolymerization of actin filaments is essential for various cellular processes such as cell migration, rotation, cytokinesis, and mammalian oocyte maturation. Tropomodulin 3 (Tmod3) binds to the slow-growing (pointed) ends of the actin filament, thereby protecting the filament from depolymerization. However, the roles of Tmod3 in mammalian oocyte maturation remain elusive. Tmod3 mRNA and protein is present at all stages of mouse oocyte maturation. Tmod3 protein is mainly localized in the cytoplasm and appears enriched near the chromosome during maturation. By knocking down or ectopically overexpressing Tmod3, we confirmed that Tmod3 regulate the level of the intracytoplasmic actin mesh and asymmetric spindle migration. Expression of N-terminal Tmod3 (correspond to 1–155 amino acids), which contains the tropomyosin-binding site, results in decreased density of the actin mesh, thereby demonstrating the importance of the interaction between tropomyosin and tropomodulin for the maintenance of the actin mesh. Taken together, these findings indicate that Tmod3 plays crucial roles in oocyte maturation, presumably by protecting the actin filament from depolymerization and thereby controlling the density of the cytoplasmic actin mesh.

Polarized endosome dynamics by spindle asymmetry during asymmetric cell division.

During asymmetric division, fate determinants at the cell cortex segregate unequally into the two daughter cells. It has recently been shown that Sara (Smad anchor for receptor activation) signalling endosomes in the cytoplasm also segregate asymmetrically during asymmetric division. Biased dispatch of Sara endosomes mediates asymmetric Notch/Delta signalling during the asymmetric division of sensory organ precursors in Drosophila. In flies, this has been generalized to stem cells in the gut and the central nervous system, and, in zebrafish, to neural precursors of the spinal cord. However, the mechanism of asymmetric endosome segregation is not understood. Here we show that the plus-end kinesin motor Klp98A targets Sara endosomes to the central spindle, where they move bidirectionally on an antiparallel array of microtubules. The microtubule depolymerizing kinesin Klp10A and its antagonist Patronin generate central spindle asymmetry. This asymmetric spindle, in turn, polarizes endosome motility, ultimately causing asymmetric endosome dispatch into one daughter cell. We demonstrate this mechanism by inverting the polarity of the central spindle by polar targeting of Patronin using nanobodies (single-domain antibodies). This spindle inversion targets the endosomes to the wrong cell. Our data uncover the molecular and physical mechanism by which organelles localized away from the cellular cortex can be dispatched asymmetrically during asymmetric division.

A narrow window of cortical tension guides asymmetric spindle positioning in the mouse oocyte.

Cell mechanics control the outcome of cell division. In mitosis, external forces applied on a stiff cortex direct spindle orientation and morphogenesis. During oocyte meiosis on the contrary, spindle positioning depends on cortex softening. How changes in cortical organization induce cortex softening has not yet been addressed. Furthermore, the range of tension that allows spindle migration remains unknown. Here, using artificial manipulation of mouse oocyte cortex as well as theoretical modelling, we show that cortical tension has to be tightly regulated to allow off-center spindle positioning: a too low or too high cortical tension both lead to unsuccessful spindle migration. We demonstrate that the decrease in cortical tension required for spindle positioning is fine-tuned by a branched F-actin network that triggers the delocalization of myosin-II from the cortex, which sheds new light on the interplay between actin network architecture and cortex tension.

Actin-capping proteins play essential roles in the asymmetric division of maturing mouse oocytes

Actin polymerization is essential for various stages of mammalian oocyte maturation, including spindle migration, actin cap formation, polar body extrusion and cytokinesis. The heterodimeric actin-capping protein is an essential element of the actin cytoskeleton. It binds to the fast-growing (barbed) ends of actin filaments and plays essential roles in various actin-mediated cellular processes. However, the roles of capping protein in mammalian oocyte maturation are poorly understood. We investigated the roles of capping protein in mouse oocytes and found that it is essential for correct asymmetric spindle migration and polar body extrusion. Capping protein mainly localized in the cytoplasm during maturation. By knocking down or ectopically overexpressing this protein, we revealed that it is crucial for efficient spindle migration and maintenance of the cytoplasmic actin mesh density. Expression of the capping-protein-binding region of CARMIL (also known as LRRC16A) impaired spindle migration and polar body extrusion during oocyte maturation and decreased the density of the cytoplasmic actin mesh. Taken together, these findings show that capping protein is an essential component of the actin cytoskeleton machinery that plays crucial roles in oocyte maturation, presumably by controlling the cytoplasmic actin mesh density.