Spindle Structure Research Articles

Effect of Change in Spindle Structure on Proliferation Inhibition of Osteosarcoma Cells and Osteoblast under Simulated Microgravity during Incubation in Rotating Bioreactor

In order to study the effect of microgravity on the proliferation of mammalian osteosarcoma cells and osteoblasts, the changes in cell proliferation, spindle structure, expression of MAD2 or BUB1, and effect of MAD2 or BUB1 on the inhibition of cell proliferation is investigated by keeping mammalian osteosarcoma cells and osteoblasts under simulated microgravity in a rotating wall vessel (2D-RWVS) bioreactor. Experimental results indicate that the effect of microgravity on proliferation inhibition, incidence of multipolar spindles, and expression of MAD2 or BUB1 increases with the extension of treatment time. And multipolar cells enter mitosis after MAD2 or BUB1 is knocked down, which leads to the decrease in DNA content, and decrease the accumulation of cells within multipolar spindles. It can therefore be concluded that simulated microgravity can alter the structure of spindle microtubules, and stimulate the formation of multipolar spindles together with multicentrosomes, which causes the overexpression of SAC proteins to block the abnormal cells in metaphase, thereby inhibiting cell proliferation. By clarifying the relationship between cell proliferation inhibition, spindle structure and SAC changes under simulated microgravity, the molecular mechanism and morphology basis of proliferation inhibition induced by microgravity is revealed, which will give experiment and theoretical evidence for the mechanism of space bone loss and some other space medicine problems.

Investigate the spindle errors motions from thermal change for high-precision CNC machining capability

As the demand for high speed and highly accurate machines has significantly increased, error motions from thermal change, which is up to 70 % of the total machining error, is found to be the main hurdles to overcome in improving the accuracy of CNC machine tools. In this research work, the authors installed four eddy current displacement sensors in the spindle structure near to the front bearing to monitor the spindle offset in the bearing level, which is mainly attributable to the thermal error motions of the spindle. In addition, another three capacitance sensors are mounted on the machine table level and aligned with the x-, y-, and z-axis of the machine to monitor the spindle shift in the table level to find out the correlation between temperature change and the thermal error motions of the spindle. To measure the temperature changes, we attached thermal sensors in the machine and cooling system. The estimation of the spindle thermal displacement based on temperature data from these thermal sensors can provide more information for the monitoring of thermal error motions of the spindle.

The Spindle Component Analysis of Inner Cavity Shaping Machine Tool

This paper takes the spindle of inner cavity shaping machine tool as the research object, analyzes the spindle structure of the machine tool and establishes the equations of motion, analyzes the influence that the spindle speed and tool deflection speed have on the tool rotary speed using Matlab and ADAMS, determines the allowed deflection range of the tool, sets up the transfer function of the servo motor current loop and analyzes the stability of spindle servo motor current loop .

A Cryptic TOG Domain with a Distinct Architecture Underlies CLASP-Dependent Bipolar Spindle Formation

CLASP is a key regulator of microtubule (MT) dynamics and bipolar mitotic spindle structure with CLASP mutants displaying a distinctive monopolar spindle phenotype. It has been postulated that cryptic TOG domains underlie CLASP’s ability to regulate MT dynamics. Here, we report the crystal structure of a cryptic TOG domain (TOG2) from human CLASP1, demonstrating the presence of a TOG array in the CLASP family. Strikingly, CLASP1 TOG2 exhibits a convex architecture across the tubulin-binding surface that contrasts with the flat tubulin-binding surface of XMAP215 family TOG domains. Mutations in key conserved TOG2 determinants abrogate the ability of CLASP mutants to rescue bipolar spindle formation in Drosophila cells depleted of endogenous CLASP. These findings highlight the common mechanistic use of TOG domains in XMAP215 and CLASP families to regulate MT dynamics and suggest that differential TOG domain architecture may confer distinct functions to these critical cytoskeletal regulators.

Peroxynitrite affects the cumulus cell defense of metaphase II mouse oocytes leading to disruption of the spindle structure in vitro

To demonstrate the effects of peroxynitrite (ONOO(-)) on metaphase II mouse oocyte spindle structure and chromosomal alignment in presence and absence of cumulus cells. Experimental study. University-based research laboratory. Metaphase II mouse oocytes (n = 440). Metaphase II mouse oocytes, with and without cumulus cells, were exposed to ONOO(-), nitrite/nitrate, the final product of ONOO(-), and nontreated controls for 15 minutes. Oocytes were fixed and subjected to indirect immunofluorescence for detecting changes in the spindle and chromosomal alignment. Viability staining in exposed oocytes with and without cumulus cells was performed using the trypan blue dye exclusion method and compared with controls. Scoring the alterations in spindle and chromosomal alignment using immunofluorescent and confocal microscopy based on a previously validated system. Most oocytes had poor scores for the spindle and chromosomal alignment with exposure to ONOO(-) in a dose-dependent manner compared with controls. Trypan blue staining revealed that most of the cumulus cells failed to survive treatment with ONOO(-) compared with controls. ONOO(-) affects the viability of cumulus cells and the oocyte spindle structure in a dose-dependent manner. Collectively, these effects compromise oocyte quality, which may lead to female infertility.

Mitotic Rounding Alters Cell Geometry to Ensure Efficient Bipolar Spindle Formation

Accurate animal cell division requires precise coordination of changes in the structure of the microtubule-based spindle and the actin-based cell cortex. Here, we use a series of perturbation experiments to dissect the relative roles of actin, cortical mechanics, and cell shape in spindle formation. We find that, whereas the actin cortex is largely dispensable for rounding and timely mitotic progression in isolated cells, it is needed to drive rounding to enable unperturbed spindle morphogenesis under conditions of confinement. Using different methods to limit mitotic cell height, we show that a failure to round up causes defects in spindle assembly, pole splitting, and a delay in mitotic progression. These defects can be rescued by increasing microtubule lengths and therefore appear to be a direct consequence of thelimited reach of mitotic centrosome-nucleated microtubules. These findings help to explain why most animal cells round up as they enter mitosis.

Glaziovianin A Prevents Endosome Maturation via Inhibiting Microtubule Dynamics

Glaziovianin A, an isoflavone isolated from the leaves of Ateleia glazioviana, inhibits the cell cycle progression in M-phase with an abnormal spindle structure, but its inhibitory mechanism has not been revealed. Here, we report that glaziovianin A and its derivatives are microtubule dynamics inhibitors. Glaziovianin A extended the time lag of tubulin polymerization without changing the net amount of polymerized tubulin in vitro and suppressed microtubule dynamics in cells. Furthermore, glaziovianin A inhibited the transport of endosomes containing EGF-stimulated EGFR and prolonged the EGFR activation. Consistent with the prolonged activation of EGFR, glaziovianin A enhanced the EGF-dependent apoptosis in A431 cells. These results strongly suggested that microtubule dynamics is important for endosome transport and maturation, and that glaziovianin A shows cytotoxicity by two pathways, the mitotic arrest and inadequate activation of receptor kinases via the inhibition of endosome maturation.

Aurora B kinase is required for cytokinesis through effecting spindle structure

The spindle, composed of microtubules and associated proteins, serves as major machinery for cell division. Aurora B is a chromosome passenger protein that has important functions in mitosis. Its dynamic distribution at mitosis goes along with spindle structure and dynamics. We used the siRNA technique to knockdown protein expression and immunofluorescence technique to follow Aurora B during mitosis. Aurora B regulates microtubule plus ends as mitosis progresses, including both kinetochore and polar microtubules. Interactions between Aurora B and polar microtubule plus ends lead to failure in cytokinesis and abnormal midbody structure. We think Aurora B may not only play a role as kinase, but regulate microtubule plus ends.

S131012 高剛性水静圧スピンドルの設計([S131-01]先進工作機械技術(1))

A precision spindle is a key component of the ultra-precision machine tools that are used for machining precision molds for producing various lenses and other precise parts. The machining accuracy required for the parts now reaches several tens of nanometers. In order to achieve the desired machining accuracy, the precise spindle system with high stiffiiess bearings is needed for the ultra-precision machine tools. This paper deals with designed spindle supported by water hydrostatic bearings. An objective of this study is to design of the spindle with high bearing stiffness supported by water hydrostatic bearings of 1000 N/μm. Thus this paper presents the design of water hydrostatic thrust bearings, the structure of spindle supported by water hydrostatic bearing and the theoretical stiffness of designed spindle. The influences of the gap sizes and the supply water pressure on the bearing stiffness are also given. The stiffness of thrust bearing of the developed spindle is investigated theoretically.

Spatial-Temporal Regulation of Spindle Assembly Checkpoint

During mitosis, microtubule spindle organized by the two spindle poles (SP) need to properly capture and align chromosomes at the cell equator to ensure faithful chromosome segregation. Improper chromosome-spindle attachment will activate spindle assembly checkpoint proteins (SAC) emanated from this chromosome. Active SAC prevents cyclin B degradation in the cytoplasm and, consequently halts the progression of mitosis. Only after the last chromosome gets properly attached can chromosome segregation begin. Given the tens of chromosomes and everlasting fluctuations in the cell, it remains murky how the silencing of SAC faithfully couples with the last chromosome attachment. We established a theoretical model that describes the mitotic spindle structure (chromosomes, mitotic spindles, and spindle poles) as a coherent transport system. Depending on whether the chromosome is properly attached, SAC and cyclin B circulate within the transport system via dyneins, and exchange with the cytoplasm in accordance to their well-known biochemical regulations. The basis of such coherent transport is the compartmentalization underlied by direct and/or indirect protein binding affinities with these mitotic structures. Our model results show that such transport system recapitulates the observed spatial-temporal pattern of SAC and cyclin B. More importantly, the transport system establishes a robust and sensitive mechanism for silencing SAC activity in accordance with the status of chromosome-spindle attachments.

Specific removal of TACC3/ch-TOG/clathrin at metaphase deregulates kinetochore fiber tension

Microtubule-associated proteins of the mitotic spindle are thought to be important for the initial assembly and the maintenance of spindle structure and function. However, distinguishing assembly and maintenance roles for a given protein is difficult. Most experimental methods for protein inactivation are slow and therefore affect both assembly and maintenance. Here, we have used 'knocksideways' to rapidly (∼5 minutes) and specifically remove TACC3-ch-TOG-clathrin non-motor complexes from kinetochore fibers (K-fibers). This method allows the complex to be inactivated at defined stages of mitosis. Removal of TACC3-ch-TOG-clathrin after nuclear envelope breakdown caused severe delays in chromosome alignment. Inactivation at metaphase, following a normal prometaphase, significantly delayed progression to anaphase. In these cells, K-fiber tension was reduced and the spindle checkpoint was not satisfied. Surprisingly, there was no significant loss of K-fiber microtubules, even after prolonged removal. TACC3-ch-TOG-clathrin removal during metaphase also resulted in a decrease in spindle length and significant alteration in kinetochore dynamics. Our results indicate that TACC3-ch-TOG-clathrin complexes are important for the maintenance of spindle structure and function as well as for initial spindle assembly.

Physical Aspects of Spindle Assembly

The spindle is a complex assembly of microtubules, motors, and other associated proteins, which segregate chromosomes during cell division.In metaphase, the spindle exists in a steady-state with a constant flux of molecules and energy continuously modifying and maintaining its architecture. While the self-organization of systems of microtubules and motors have been investigated using theory and experiments, there have been few attempts to test if the proposed theories can be used to understand the dynamics and structure of complex biological systems in vivo. Here we use polarized light microscopy, 3D time-lapse spinning disk confocal microscopy, single molecule imaging, second harmonic generation microscopy, and mechanical measurements to test the validity of continuum models of metaphase spindles. Our results show that a simple continuum model can quantitatively explain spindle structure and dynamics, demonstrate that rigorous physical theories can be used to quantitatively describe complex subcellular systems, and provides a framework for understanding the structure of the spindle and its response to physical and molecular perturbations.

Ion specific effects in bundling and depolymerization of taxol-stabilized microtubules

Microtubules (MTs) are nanometer scale hollow cylindrical biological polyelectrolytes. They are assembled from alpha/beta-tubulin dimers, which stack to form protofilaments (PFs) with lateral interactions between PFs resulting in the curved MT. In cells, MTs and their assemblies are critical components in a range of functions from providing tracks for the transport of cargo to forming the spindle structure during mitosis. Previous studies have, shown that while cations with valence equal to or larger than 3+ tend to assemble tight 3D bundles of taxol-stabilized MTs, certain divalent cations induce relatively loose 2D bundles of different symmetry (D. J. Needleman et al., Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 16099). Similarly, divalent cations form 2D bundles of DNA adsorbed on cationic membranes (I. Koltover et al., Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 14046). The bundling behavior for these biological polyelectrolyte systems is qualitatively in agreement with current theory. Here, we present results which show that, unlike the case for DNA adsorbed on cationic membranes, bundling of taxol-stabilized MTs occurs only for certain divalent cations above a critical ion concentration (e.g. Ca2+, Sr2+, Ba2+). Instead, many divalent cations pre-empt the bundling transition and depolymerize taxol-stabilized MTs at a lower counterion concentration. Although previous cryogenic TEM has shown that, in the absence of taxol, Ca2+ depolymerizes MTs assembling in buffers containing GTP (guanosine triphosphate), our finding is surprising given the know stabilizing effects of taxol on GDP (guanosine diphosphate)-MTs. The ion concentration required for MT depolymerization decreases with increasing atomic number for the divalents Mg2+, Mn2+, Co2+, and Zn2+. GdCl3 (3+) is found to be extremely efficient at MT depolymerization requiring ion concentrations of about 1 mM, while oligolysine(2+), is observed not to depolymerize MTs at concentrations as high as 144 mM. The surprising MT depolymerization results are discussed in the context of divalents either disrupting lateral interactions between PFs (which are strengthened for taxol containing beta-tubulin) or interfering with taxol's ability to induce flexibility at the interface between two tubulin dimers in the same PF (which has been recently suggested as a mechanism by which taxol stabilizes MTs post-hydrolysis with the induced flexibility counteracting the kink between GDP-tublin dimers in a PF).

COORDINATION ANALYSIS ON URBANIZATION LEVEL AND LAND USE EFFICIENCY OF XINJIANG BASED ON ESDA

This paper analyzes spatial effect of urbanization level, land use efficiency and their coordinative development degree of Xinjiang from 1995 to 2010 based on Exploratory Spatial Data Analysis and coordinative development degree model. It was found that the urbanization level and land use efficiency take on apparent spatial agglomeration. The hot spots concentrate mainly in the north slope of Tianshan Mountain and the corridor along South Xinjiang railway, and the cold spots distribute mainly in the south edge of Tarim Basin. Coordinative development degree shows a spindle structure in a whole and different types of counties take on different development tracks. Counties in Xinjiang can be divided into five types, which are synchronization coordination type, lagging urbanization type, lagging land benefit type, gradually fitting type, and low level coordination type, among which, the counties of synchronization coordination type are few, the quantity of other types is relatively uniform.

In situ atom-resolved tracing of element diffusion in NiAu nanospindles

The structure evolution behaviors of NiAu nanospindles in oxidizing/vacuum conditions were studied by in situ scanning transmission electron microscopy. The spindle structure transforms into Ni@Au@NiO multi-shell structure in the oxidizing atmosphere following an oxidation-driving multilayer reconstruction mechanism. In the vacuum condition, the NiAu nanospindles evolve along four distinct stages: (i) polyhedron recrystallization of Ni matrix; (ii) facets-selected segregation and recrystallization of Au component; (iii) single crystallization of Ni matrix; (iv) wrapping diffusion of Au component. The investigation here provides a practical insight into the structural stability of NiAu bimetallic catalysts through a thermal processes.

Authors’ response: polarization microscopy and rescue ICSI

1 h We thank Dr Coticchio for his commentary on our article ‘Spindle examination in unfertilized eggs using the polarization microscope can assist rescue ICS’. His view on some key points in our study harmonize with ideas and issues we have discussed and debated over and over. Therefore, we are pleased to be able to highlight answers for issues he has raised. First, we know the issue of whether to perform rescue intracytoplasmic sperm injection (ICSI) or to cancel the current cycle is still under debate. The goal of assisted reproductive technology is to help infertile couples have a healthy baby, not increase the number of transferable embryos of compromised quality. The biological and clinical implications of oocyte ageing are well reported in numerous articles, including cytogenetic abnormalities in the resulting embryo following rescue ICSI (Morton et al., 1997; Pehlivan et al., 2004) as well as congenital abnormalities (Tarin et al., 2002). However, we should also consider that routine ICSI and IVF might also cause an increased risk of major congenital anomalies compared with natural conception (Wen et al., 2012). To our knowledge, there have been reports of successful births following rescue ICSI (Yuzpe et al., 2000; Shalom-PAZ et al., 2011). As previously shown from data in our centre (Shalom-PAZ et al., 2011), out of 20 pregnancies, only one was diagnosed with trisomy 21 and was terminated, while the other children were found to be

104 3-D VISUALIZATION OF THE FIRST CLEAVAGE AND ABERRATIONS OF BOVINE ZYGOTES BY CONFOCAL LASER SCANNING MICROSCOPY

A high proportion of bovine oocytes fertilized in vitro fail to develop beyond the first 4 cleavage cycles. The first mitotic division of the zygote and proper segregation of chromosomes and cytoplasmic components seems to be a particularly delicate task. Notably, zygotes cleaving with a delay of only a few hours seem to have a very low chance of developing to the blastocyst stage. But what exactly goes wrong, how often, and why? To answer such questions we have to visualize in greater detail basic structures and processes such as the sperm aster, DNA replication, migration and apposition of the 2 pronuclei, synchronous chromosome condensation and breakdown of the nuclear envelopes, assembly of the first mitotic spindle and chromosome congression, anaphase, and cytokinesis. Oocytes fertilized in vitro were fixed at different time points around the first cleavage and stained for DNA, Ser10-phosphorylated histone H3, microtubules, and microfilaments. Zygotes were imaged in toto by recording confocal serial sections at 1-µm intervals using a 40× objective (NA = 1.3). Details were recorded with high spatial sampling densities (pixel size 50 × 50 nm, z-step size of 200 nm) close to the Nyquist criterion and restored by maximum likelihood estimation deconvolution using the real point spread function. We present a series of 3-D confocal images captured at different stages of the first cleavage. The images reveal new insights into the formation, structure, and function of the first mitotic spindle and the occurrence of spindle aberrations, irregular chromosome segregation, and abnormal cytokinesis. The microscopic findings guide us to candidate proteins for localization analyses and functional studies based on 3-D fluorescence live-cell imaging of zygotes and early embryos. This work is supported by the Deutsche Forschungsgemeinschaft (DFG FOR 1041).

Motors or dynamics: What really moves chromosomes?

Motors or dynamics: What really moves chromosomes?

Peroxynitrite Affects the Cumulus Cell Defense of Metaphase-II Mouse Oocytes Leading to Disruption of the Spindle Structure in Vitro

Peroxynitrite Affects the Cumulus Cell Defense of Metaphase-II Mouse Oocytes Leading to Disruption of the Spindle Structure in Vitro

Dynamic Characteristic Analysis of the Spindle of Planer Drilling Machine Based on ANSYS

This paper elaborated the practical application of NC Planer Drilling Machine and the important consequence of main shaft structure to working qualities and working accuracy. Then it introduced the design proposal for the spindle structure of drilling machine. The static analysis, modal analysis and harmonic response analysis were done by using ANSYS. It lays a foundation for optimal design and remanufacturing.