Spindle Structure Research Articles

Engineered non-Mendelian inheritance of entire parental genomes in C. elegans.

The ability to rewrite the rules of genetic segregation would open new possibilities in diverse areas of biotechnology ranging from breeding to epigenetics. Here we engineer non-Mendelian inheritance of the entire maternal or paternal genome in Caenorhabditis elegans by changing the structure of the mitotic spindle during the first cell division of the zygote. Using germline-specific overexpression of a single protein, the conserved microtubule force regulator GPR-1, we increase forces that pull on spindle poles to convert the single bipolar mitotic spindle to two monopolar spindles. This generates two-cell embryos in which one cell contains only the maternal chromosomes and the other cell contains only the paternal chromosomes. As the embryo develops, each cell of the animal, including the germ cells, contains the genetic material of only one parent, resulting in hybrid F1 animals. Progeny of these animals (F2) inherit either only F0 maternal or only F0 paternal chromosomes, and thus descend from only either of their grandparents' gametes.

Comparative analysis of traditional jet vortex spinning and self-twist jet vortex spinning on yarn mechanism and yarn properties

Because of the friction between the hollow spindle surface and the fiber end increasing, the fiber end rolls on the hollow spindle surface under the high-speed rotating airflow during the twisting process. The fiber gets self-twist, which will be wound into the yarn body. The self-twist of the fiber will increase the friction and the cohesion between the fibers in the yarn, which will improve the strength of the yarn. This article analyzes traditional jet vortex spinning and self-twist jet vortex spinning comparatively from the three aspects of the hollow spindle structure, the airflow distribution inside the nozzle, and the yarn performance. It is beneficial for exploring the yarn mechanism, the yarn performance, and the development of self-twist jet vortex spinning.

Development of a novel 2D rotary ultrasonic texturing technique for fabricating tailored structures

Microgrooves, microdimples, or hierarchical surfaces comprising these structures are highly required in many industrial fields for their functional performances. In the present research, a novel two-dimensional (2D) rotary ultrasonic texturing (RUT) technique is developed for efficiently fabricating these precision surface structures. A rotary ultrasonic spindle that can generate circular vibration on the horizontal plane perpendicular to the spindle axis was designed and manufactured for carrying out the 2D RUT processes. The spindle structure and the texturing principle are introduced. Surface generation mechanisms are mathematically analyzed and experimentally verified. A diamond tool with geometrically defined cutting edge was designed and manufactured for carrying out the surface texturing experiments. Different types of nearly burr-free textured surfaces composing of periodic grooves from several micrometers to tens of micrometers were successfully fabricated, and their textural features coincide well with the theoretical prediction. Material removal mechanisms, tools design principles, and machinable structures of the new 2D RUT technique are discussed.

Mechanical stability of bipolar spindle assembly

Assembly and stability of mitotic spindle are governed by the interplay of various intra-cellular forces, e.g. the forces generated by motor proteins by sliding overlapping anti-parallel microtubules (MTs) polymerized from the opposite centrosomes, the interaction of kinetochores with MTs, and the interaction of MTs with the chromosome arms. We study the mechanical behavior and stability of spindle assembly within the framework of a minimal model which includes all these effects. For this model, we derive a closed-form analytical expression for the force acting between the centrosomes as a function of their separation distance and we show that an effective potential can be associated with the interactions at play. We obtain the stability diagram of spindle formation in terms of parameters characterizing the strength of motor sliding, repulsive forces generated by polymerizing MTs, and the forces arising out of the interaction of MTs with kinetochores. The stability diagram helps in quantifying the relative effects of the different interactions and elucidates the role of motor proteins in formation and inhibition of spindle structures during mitotic cell division. We also predict a regime of bistability for a certain parameter range, wherein the spindle structure can be stable for two different finite separation distances between centrosomes. This occurrence of bistability also suggests the mechanical versatility of such self-assembled spindle structures.

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

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. © 2016 Wiley Periodicals, Inc.

In Vitro Maturation of Mouse Oocytes Increases the Level of Kif11/Eg5 on Meiosis II Spindles.

Although in vitro maturation (IVM) of oocytes has been used for a relatively long time, during which the culture conditions have improved remarkably, the resulting germ cells are still not fully comparable to the cells obtained from the ovary in many important aspects, namely in fertilization rate and subsequent embryonic development. Some of the differences between IVM and in vivo maturation (IVV) oocytes were already discovered, including variability in spindle assembly and morphology. In this study we focused on a role of molecular motor Kif11 (hereafter referred to as Eg5) in maintaining bipolar spindle structure in IVM and IVV oocytes. Our experiments revealed that in IVM oocytes, Eg5 is abundant on meiosis II spindle, which makes these cells more sensitive to Eg5 inhibition than IVV oocytes. We further demonstrate that this sensitivity is acquired gradually with exposure to the in vitro conditions. This is a remarkable difference in function of spindle apparatus between IVM and IVV oocytes, and we believe our results are important not only for understanding of the chromosome segregation in mammalian oocytes but also because they indicate cells are using alternative pathways to achieve the same function when exposed to different conditions.

Study on the Dynamic Characteristics of the Winding Head

According to the structure and production processing requirements of polyester filament winding head, a finite element dynamic model of the winding system in parameters time-varying was established. By using the method of numerical calculation, the dynamic characteristics of the winding head were analyzed in three processes: rapid starting-up in empty volume, winding filament process and stopping in full volume. By studying the influence of the three processing under large stiffness coupling and soft stiffness coupling in the winding head, the role of the coupling stiffness and spindle structure was revealed in different working process.

The Finite Element Analysis and the Multi-Objective Optimization Design of Spindle Systems of CNC Gantry Machine Tools

It is discovered that the vibration resistance of spindle systems needs to be improved based on the statics analysis, modal analysis and heating-force coupling analysis of spindle systems of CNC gantry machine tools. The design variables of optimization are set according to sensitivity analysis, multi-objective and dynamic optimization design is realized and its designing scheme is gained for spindle structure. The research results show that vibration resistance can be improved without change of the quality and static property of spindle systems of CNC gantry machine tools.

Molecular requirements for the inter-subunit interaction and kinetochore recruitment of SKAP and Astrin.

Accurate chromosome segregation during cell division is crucial for propagating life and protects from cellular transformation. The SKAP:Astrin heterodimer localizes to spindle microtubules and to mature microtubule–kinetochore attachments during mitosis. Depletion of either subunit disrupts spindle structure and destabilizes kinetochore–microtubule attachments. Here, we identify molecular requirements for the inter-subunit interaction of SKAP and Astrin, and discuss requirements for their kinetochore recruitment. We also identify and characterize a microtubule-binding domain in SKAP, distinct from the SXIP motif that mediates end binding (EB) protein binding and plus end tracking, and show that it stimulates the growth-rate of microtubules, possibly through a direct interaction with tubulin. Mutations targeting this microtubule-binding domain impair microtubule plus-end tracking but not kinetochore targeting, and recapitulate many effects observed during depletion of SKAP. Collectively, our studies represent the first thorough mechanistic analysis of SKAP and Astrin, and significantly advance our functional understanding of these important mitotic proteins.

Investigation of the Machining Stability of a Milling Machine with Hybrid Guideway Systems

This study was aimed to investigate the machining stability of a horizontal milling machine with hybrid guideway systems by finite element method. To this purpose, we first created finite element model of the milling machine with the introduction of the contact stiffness defined at the sliding and rolling interfaces, respectively. Also, the motorized built-in spindle model was created and implemented in the whole machine model. Results of finite element simulations reveal that linear guides with different preloads greatly affect the dynamic responses and machining stability of the horizontal milling machine. The critical cutting depth predicted at the vibration mode associated with the machine tool structure is about 10 mm and 25 mm in the X and Y direction, respectively, while the cutting depth predicted at the vibration mode associated with the spindle structure is about 6.0 mm. Also, the machining stability can be increased when the preload of linear roller guides of the feeding mechanism is changed from lower to higher amount.

Spindle Micro-Fluctuations of Length Reveal its Dynamics Over Cell Division

The mitotic spindle ensures correct segregation of sister chromatids and correct partitioning in daughter cells. It comprises dynamical microtubules (alternating polymerizing and depolymerizing), a variety of molecular motors and their regulators. Although spindle structure is well known, the link to its functions remains elusive, calling for including the dynamics of its components and their interactions. This question was mostly investigated by in silico or in vitro approaches. But a detailed characterizing of spindle mechanics, in physiological conditions, is missing. We propose an image processing based, non invasive, method combined to an heuristic model to measure mechanical parameters along time.We tracked fluorescently labeled spindle pole at high temporal and spatial resolution and measured the micro-fluctuations of spindle length, in vivo. We computed its power density spectrum using short time Fourier transform (sliding window) — a blueprint of spindle mechanics. Such a spectrum is then fitted with a Kelvin Voigt model with inertia (a spring, a damper, an inertial element in parallel) convolved with the window. We validated this method by recovering the mechanical parameters over time from simulated data. Then we measured them, in vivo, in two model organisms, nematode C. elegans one-cell embryo and fission yeast S. pombe.In control C. elegans embryos, metaphase appeared dominated by damping element, consistent with the slow spindle elongation observed. At anaphase onset, all three parameters collapsed, before increasing about 50s later to reach a regime where damping and inertia dominated, suggesting a dramatic re-arrangement of the spindle. Using a gene candidate approach, we could relate these mechanical behaviors to known structures at work, overlapping and kinetochore microtubules during metaphase and centralspindle at anaphase.With such a tool, we will propose a model of the spindle accounting for the dynamics of its component.

TCTP regulates spindle microtubule dynamics by stabilizing polar microtubules during mouse oocyte meiosis

Dynamic changes in spindle structure and function are essential for maintaining genomic integrity during the cell cycle. Spindle dynamics are highly dependent on several microtubule-associated proteins that coordinate the dynamic behavior of microtubules, including microtubule assembly, stability and organization. Here, we show that translationally controlled tumor protein (TCTP) is a novel microtubule-associated protein that regulates spindle dynamics during meiotic maturation. TCTP was expressed and widely distributed in the cytoplasm with strong enrichment at the spindle microtubules during meiosis. TCTP was found to be phosphorylated during meiotic maturation, and was exclusively localized to the spindle poles. Knockdown of TCTP impaired spindle organization without affecting chromosome alignment. These spindle defects were mostly due to the destabilization of the polar microtubules. However, the stability of kinetochore microtubules attached to chromosomes was not affected by TCTP knockdown. Overexpression of a nonphosphorylable mutant of TCTP disturbed meiotic maturation, stabilizing the spindle microtubules. In addition, Plk1 was decreased by TCTP knockdown. Taken together, our results demonstrate that TCTP is a microtubule-associating protein required to regulate spindle microtubule dynamics during meiotic maturation in mouse oocytes.

Oocyte Meiotic Spindle Assembly and Function.

Gametogenesis in animal oocytes reduces the diploid genome content of germline precursors to a haploid state in gametes by discarding ¾ of the duplicated chromosomes through a sequence of two meiotic cell divisions called meiosis I and II. The assembly of the microtubule-based spindle structure that mediates this reduction in genome content remains poorly understood compared to our knowledge of mitotic spindle assembly and function. In this review, we consider the diversity of oocyte meiotic spindle assembly and structure across animal phylogeny, review recent advances in our understanding of how animal oocytes assemble spindles in the absence of the centriole-based microtubule-organizing centers that dominate mitotic spindle assembly, and discuss different models for how chromosomes are captured and moved to achieve chromosome segregation during oocyte meiotic cell division.

A Magnetic Suspension Spindle System for Micro EDM

As an important manufacturing technology, micro EDM is widely applied in many industry fields. However, the slow response speed of servo system often leads to short circuit and arc discharge. To deal this problem, a magnetic suspension spindle system is used to improve the response frequency. This paper introduces the structure and principle of the magnetic suspension spindle device used for micro EDM, hardware and software solutions of the control system are illuminated by schematic diagram or flow chart. Using incremental PID algorithm, the axial (Z) response frequency reaches 150Hz, the radial and axial position accuracy is achieved with 5μm and 2μm. And the micro EDM is realized through the magnetic suspension spindle in 1.3mm stroke.

The effects of voluntary exercise on oocyte quality in a diet-induced obese murine model.

Obesity negatively affects many aspects of the human body including reproductive function. In females, the root of the decline in fertility is linked to problems in the oocyte. Problems seen in oocytes that positively correlate with increasing BMI include changes to the metabolism, lipid accumulation, meiosis, and metaphase II (MII) spindle structure. Studies in mice indicate that dietary interventions fail to reverse these problems. How exercise affects the oocytes has not been addressed. Therefore, we hypothesized an exercise intervention would improve oocyte quality. Here we show that in a mouse model of an exercise, intervention can improve lipid metabolism in germinal vesicle (GV) stage oocytes. Oocytes significantly increased activity and transcription of the β-oxidation enzyme hydroxyacyl-coenzyme A dehydrogenase in response to exercise training only if the mice had been fed a high-fat diet (HFD). An exercise intervention also reversed the lipid accumulation seen in GV stage oocytes of HFD females. However, delays in meiosis and disorganized MII spindles remained present. Therefore, exercise is able to improve, but not reverse, damage imparted on oocytes as a result of an HFD and obesity. By utilizing an exercise intervention on an HFD, we determined only lipid content, and lipid metabolism is changed in GV oocytes. Moving forward, interventions to improve oocyte quality may need to be more targeted to the oocyte specifically. Because of the HFD-induced deficiency in β-oxidation, dietary supplementation with substrates to improve lipid utilization may be more beneficial.

Multiphysics-based design and analysis of the high-speed aerostatic spindle with application to micro-milling

High-speed aerostatic spindles are essential for ultra-precision micro-milling machine tools. The distinguishing functional characteristics of the spindle system are achieved through innovative design and integration of thermal, fluidic, and electromagnetic physics on the spindle structure and system. These physics, together with the shaft dynamics, interact dynamically and collectively to determine the spindle’s performance. It is thus required to have a comprehensive analysis and scientific understanding on the multiple physics interactions within the spindle, which is essential for design of the spindle working at much higher speeds and accuracy under various increasingly stringent engineering conditions. This paper presents an integrated multiphysics simulation approach to design and analysis of high-speed aerostatic spindles. Based on the proposed approach, an integrated multiphysics simulation platform is developed with application to micro-milling machines. Experiments have been designed and carried out to validate the integrated multiphysics simulation method and evaluate the simulation results, which show that the integrated multiphysics model is able to predict and analyze the performance characteristics of the high-speed aerostatic spindle. The integrated multiphysics model and the corresponding virtual spindle simulation can be used as a powerful design aid for supporting the design and analysis of next generation high-speed aerostatic spindles at nanometric precision.

Discovery of AZ0108, an orally bioavailable phthalazinone PARP inhibitor that blocks centrosome clustering.

The propensity for cancer cells to accumulate additional centrosomes relative to normal cells could be exploited for therapeutic benefit in oncology. Following literature reports that suggested TNKS1 (tankyrase 1) and PARP16 may be involved with spindle structure and function and may play a role in suppressing multi-polar spindle formation in cells with supernumerary centrosomes, we initiated a phenotypic screen to look for small molecule poly (ADP-ribose) polymerase (PARP) enzyme family inhibitors that could produce a multi-polar spindle phenotype via declustering of centrosomes. Screening of AstraZeneca's collection of phthalazinone PARP inhibitors in HeLa cells using high-content screening techniques identified several compounds that produced a multi-polar spindle phenotype at low nanomolar concentrations. Characterization of these compounds across a broad panel of PARP family enzyme assays indicated that they had activity against several PARP family enzymes, including PARP1, 2, 3, 5a, 5b, and 6. Further optimization of these initial hits for improved declustering potency, solubility, permeability, and oral bioavailability resulted in AZ0108, a PARP1, 2, 6 inhibitor that potently inhibits centrosome clustering and is suitable for in vivo efficacy and tolerability studies.

The γ-tubulin-specific inhibitor gatastatin reveals temporal requirements of microtubule nucleation during the cell cycle.

Inhibitors of microtubule (MT) assembly or dynamics that target α/β-tubulin are widely exploited in cancer therapy and biological research. However, specific inhibitors of the MT nucleator γ-tubulin that would allow testing temporal functions of γ-tubulin during the cell cycle are yet to be identified. By evolving β-tubulin-binding drugs we now find that the glaziovianin A derivative gatastatin is a γ-tubulin-specific inhibitor. Gatastatin decreased interphase MT dynamics of human cells without affecting MT number. Gatastatin inhibited assembly of the mitotic spindle in prometaphase. Addition of gatastatin to preformed metaphase spindles altered MT dynamics, reduced the number of growing MTs and shortened spindle length. Furthermore, gatastatin prolonged anaphase duration by affecting anaphase spindle structure, indicating the continuous requirement of MT nucleation during mitosis. Thus, gatastatin facilitates the dissection of the role of γ-tubulin during the cell cycle and reveals the sustained role of γ-tubulin.

Lack of Cytosolic Carboxypeptidase 1 Leads to Subfertility due to the Reduced Number of Antral Follicles in pcd3J-/- Females.

Females homozygous for the Purkinje cell degeneration mutation (pcd) are fertile, although the success rate is much lower than in the wild type. We performed detailed analysis of reproductive abnormalities of pcd females. The number of oocytes produced following exogenous gonadotropin treatment was much lower in pcd 3J-/- females than in pcd 3J+/+ females. Furthermore, the estrous cyclicity of pcd 3J-/- females according to the appearance of the vagina was almost undetectable comparing to that of the wild type. Histological analyses and follicle counting of 4- and 8-week-old pcd 3J-/- ovaries showed an increase in the number of secondary follicles and a decrease in the number of antral follicles, indicating that AGTPBP1/ CCP1 plays an important role in the development of secondary follicles into antral follicles. Consistent with a previous analysis of the pcd cerebellum, pcd 3J-/- ovaries also showed a clear increase in the level of polyglutamylation. Gene expression analysis showed that both oocytes and cumulus cells express CCP1. However, Ccp4 and CCP6, which can compensate the function of CCP1, were not expressed in mouse ovaries. Failure of microtubule deglutamylation did not affect the structure and function of the meiotic spindle in properly aligning chromosomes in the center of the nucleus during meiosis in pcd 3J-/- females. We also showed that the pituitary-derived growth and reproduction-related endocrine system functions normally in pcd 3J-/- mice. The results of this study provide insight into additional functions of CCP1, which cannot be fully explained by the side chain deglutamylation of microtubules alone.

Discovery of Novel Biologically Active Compounds of Natural Origin, with a Focus on Anti-tumor Activity

Numerous clinically valuable medicines, including anticancer drugs, have been developed from biologically active natural compounds and their structurally related derivatives. This review discusses novel natural compounds with promising biological activities and those with novel chemical structures. Glaziovianin A, an isoflavone isolated from the leaves of Ateleia glazioviana (Legminosae), inhibited cell cycle progression at the M-phase with an abnormal spindle structure. AU-1 and YG-1, 5β-steroidal glycosides isolated from the whole plants of Agave utahensis and the underground parts of Yucca glauca (Agavaceae), induced apoptosis of HL-60 cells via caspase-3 activation. Lycolicidinol, an alkaloid isolated from the bulbs of Lycoris albiflora (Amaryllidaceae), induced transient autophagy and morphological changes in mitochondria in the early stage of the apoptotic cell death process in HSC-2 cells. Taccasterosides isolated from the rhizomes of Tacca chantrieri (Taccaceae) and stryphnosides isolated from the pericarps of Stryphnodendron fissuratum (Legminosae) are steroidal and triterpene glycosides with unique chemical structures having novel sugar sequences.