Precise Spindle Research Articles

Overcome cancer drug resistance by targeting epigenetic modifications of centrosome.

The centrosome is an organelle that serves as the microtubule- and actin-organizing center of human cells. Although the centrosome is small of size, it is great important on cellular function that regulates cytoskeletal organization and governs precise spindle orientation/positioning ensuring equal distribution of cellular components in cell division. Epigenetic modifications to centrosome proteins can lead to centrosome aberrations, such as disorganized spindles and centrosome amplification causing aneuploidy and genomic instability. Epigenetic disturbances are associated not only with carcinogenesis and cancer progression, but also with drug resistance to chemotherapy. In this review, we discuss mechanisms of epigenetic alteration during the centrosome biogenesis in cancer. We provide an update on the current status of clinical trials that aim to target epigenetic modifications in centrosome aberrations and to thwart drug resistance.

Ground truth construction and parameter tuning for the detection of sleep spindle timing in rodents

BackgroundThe precise detection of cortical sleep spindles is critical to basic research on memory consolidation in rodents. Previous research using automatic spindle detection algorithms often lacks systematic parameter variations and validations. New MethodWe present a method to systematically tune and validate algorithm parameters in automatic spindle detection algorithms using a moderate number of human raters. ResultsComparing a Hilbert transform-based algorithm to a ground truth constructed by six human raters, this method produced a parameter set yielding an F1 score of 0.82 at 10 ms resolution. The algorithm performance fell within the range of human agreement with the ground truth. Both human and algorithm failures arose largely from disagreement in spindle boundaries rather than spindle occurrence. With no additional tuning, the algorithm performed similarly in recordings from different days or rats. Comparison with existing methodsMost spindle detection algorithms do not perform systematic parameter variations and validation using a ground truth. To our knowledge, our study is the first in which rodent spindle data is scored by humans, and in which an automatic spindle detection algorithm is evaluated with respect to this ground truth. The rodent data from this study make it possible to compare our algorithm with others previously tested on human data. ConclusionsWe present a general ground truth based approach for the tuning and validation of spindle extraction algorithms and suggest that algorithms aimed at extracting precise spindle timing in rats should use a systematic approach for parameter tuning.

Plk1 regulates spindle orientation by phosphorylating NuMA in human cells.

Proper orientation of the mitotic spindle defines the correct division plane and is essential for accurate cell division and development. In metazoans, an evolutionarily conserved complex comprising of NuMA/LGN/Gαi regulates proper orientation of the mitotic spindle by orchestrating cortical dynein levels during metaphase. However, the molecular mechanisms that modulate the spatiotemporal dynamics of this complex during mitosis remain elusive. Here, we report that acute inactivation of Polo-like kinase 1 (Plk1) during metaphase enriches cortical levels of dynein/NuMA/LGN and thus influences spindle orientation. We establish that this impact of Plk1 on cortical levels of dynein/NuMA/LGN is through NuMA, but not via dynein/LGN. Moreover, we reveal that Plk1 inhibition alters the dynamic behavior of NuMA at the cell cortex. We further show that Plk1 directly interacts and phosphorylates NuMA. Notably, NuMA-phosphorylation by Plk1 impacts its cortical localization, and this is needed for precise spindle orientation during metaphase. Overall, our finding connects spindle-pole pool of Plk1 with cortical NuMA and answers a long-standing puzzle about how spindle-pole Plk1 gradient dictates proper spindle orientation for error-free mitosis.

MISP regulates the IQGAP1/Cdc42 complex to collectively orchestrate spindle orientation and mitotic progression

Precise mitotic spindle orientation is essential for both cell fate and tissue organization while defects in this process are associated with tumorigenesis and other diseases. In most animal cell types, the dynein motor complex is anchored at the cell cortex and exerts pulling forces on astral microtubules to position the spindle. The actin-binding protein MISP controls spindle orientation and mitotic progression in human cells. However, the exact underlying mechanism remains to be elucidated. Here we report that MISP interacts with the multidomain scaffolding protein IQGAP1. We further show that MISP binds to the active form of Cdc42 through IQGAP1. Depletion of MISP promotes increased accumulation of IQGAP1 at the cell cortex and a decrease in its Cdc42-binding capacity leading to reduced active Cdc42 levels. Interestingly, overexpression of IQGAP1 can rescue mitotic defects caused by MISP downregulation including spindle misorientation, loss of astral microtubules and prolonged mitosis and also restores active Cdc42 levels. Importantly, we find that IQGAP1 acts downsteam of MISP in regulating astral microtubule dynamics and the localization of the dynactin subunit p150glued that is crucial for proper spindle positioning. We propose that MISP regulates IQGAP1 and Cdc42 to ensure proper mitotic progression and correct spindle orientation.

UTX Affects Neural Stem Cell Proliferation and Differentiation through PTEN Signaling.

SummaryNeural stem cell (NSC) proliferation and differentiation in the developing brain is a complex process precisely regulated by intrinsic and extrinsic signals. Although epigenetic modification has been reportedly involved in the regulation of the cerebral cortex, whether UTX, an H3K27me3 demethylase, regulates the development of cerebral cortex during the embryonic period is unclear. In this study, we demonstrate that Utx deficiency by knockdown and conditional knockout increases NSC proliferation and decreases terminal mitosis and neuronal differentiation. Furthermore, we find that impairment of cortical development caused by lack of Utx is less significant in males than in females. In addition, UTX demethylates H3K27me3 at the Pten promoter and promotes Pten expression. P-AKT and P-mTOR levels are increased in the Utx conditional knockout cortices. Finally, Utx or Pten overexpression can rescue the impairment of brain development caused by Utx loss. These findings may provide important clues toward deciphering brain diseases.

Optimization of Electrical Discharge Turning Process using Taguchi-Grey Relational Approach

Electrical discharge turning (EDT) is a unique form of electrical discharge machining (EDM) process, which is being especially developed to generate cylindrical forms and helical profiles on difficult to machine materials at both macro and micro levels. Generally, wire-cut electrical discharge machining is used for turning because it is not concerned with electrode tooling cost. The process variant die-sinking electrical discharge machining can also be effectively used to generate free-form cylindrical geometries on hard to cut materials. In EDT process a precise spindle is incorporated with conventional die-sinking EDM machine to rotate the workpiece. Axially symmetric parts can be manufactured by feeding the shaped conductive strip of copper into the rotating workpiece. Thus, a mirror image of the tool is formed on the circumference of the workpiece. In this way, cylindrical workpiece can be made with small tools. This study presents an experimental investigation of electrical discharge turning of titanium Ti-6Al-4V alloy. The objective was to investigate the influence of machining parameters including peak current, pulse-on time, gap voltage, spindle speed and flushing pressure on performance characteristics at reverse polarity. Taguchi-grey relational approach based on multi objective optimization has been used to optimize material removal and surface roughness simultaneously.

JMJD5 (Jumonji Domain-containing 5) Associates with Spindle Microtubules and Is Required for Proper Mitosis

Precise mitotic spindle assembly is a guarantee of proper chromosome segregation during mitosis. Chromosome instability caused by disturbed mitosis is one of the major features of various types of cancer. JMJD5 has been reported to be involved in epigenetic regulation of gene expression in the nucleus, but little is known about its function in mitotic process. Here we report the unexpected localization and function of JMJD5 in mitotic progression. JMJD5 partially accumulates on mitotic spindles during mitosis, and depletion of JMJD5 results in significant mitotic arrest, spindle assembly defects, and sustained activation of the spindle assembly checkpoint (SAC). Inactivating SAC can efficiently reverse the mitotic arrest caused by JMJD5 depletion. Moreover, JMJD5 is found to interact with tubulin proteins and associate with microtubules during mitosis. JMJD5-depleted cells show a significant reduction of α-tubulin acetylation level on mitotic spindles and fail to generate enough interkinetochore tension to satisfy the SAC. Further, JMJD5 depletion also increases the susceptibility of HeLa cells to the antimicrotubule agent. Taken together, these results suggest that JMJD5 plays an important role in regulating mitotic progression, probably by modulating the stability of spindle microtubules.

Machining of Micro Rotational Parts with Wire EDM Machine

An optimized long aspect radio micro rotary parts machining process was proposed based on common wire EDM machine. By adding a precise rotary spindle to a conventional three axis CNC wire EDM machine, it can be extended to perform the fabrication of small radius and high aspect ratio rotational components. Additionally, a high material removal rate (MRR) and a fine surface could be achieved by using a new combined feeding strategy in this machining process. A single factor experiment was conducted to investigate the influence of the radial infeed on MRR. The experimental results showed that the maximum MRR can reach up to 3.09 mm3/min when the radial infeed was 50 μm. At last, a feedback rod and a group of pin electrodes were machined by applying optimized parameters and following the new feeding strategy, and the achieved maximum aspect ratio was more than 60 among these pin electrodes.

Syndecan defines precise spindle orientation by modulating Wnt signaling in C. elegans

Wnt signals orient mitotic spindles in development, but it remains unclear how Wnt signaling is spatially controlled to achieve precise spindle orientation. Here, we show that C. elegans syndecan (SDN-1) is required for precise orientation of a mitotic spindle in response to a Wnt cue. We find that SDN-1 is the predominant heparan sulfate (HS) proteoglycan in the early C. elegans embryo, and that loss of HS biosynthesis or of the SDN-1 core protein results in misorientation of the spindle of the ABar blastomere. The ABar and EMS spindles both reorient in response to Wnt signals, but only ABar spindle reorientation is dependent on a new cell contact and on HS and SDN-1. SDN-1 transiently accumulates on the ABar surface as it contacts C, and is required for local concentration of Dishevelled (MIG-5) in the ABar cortex adjacent to C. These findings establish a new role for syndecan in Wnt-dependent spindle orientation.

Syndecan defines precise spindle orientation by modulating Wnt signaling in C. elegans.

Wnt signals orient mitotic spindles in development, but it remains unclear how Wnt signaling is spatially controlled to achieve precise spindle orientation. Here, we show that C. elegans syndecan (SDN-1) is required for precise orientation of a mitotic spindle in response to a Wnt cue. We find that SDN-1 is the predominant heparan sulfate (HS) proteoglycan in the early C. elegans embryo, and that loss of HS biosynthesis or of the SDN-1 core protein results in misorientation of the spindle of the ABar blastomere. The ABar and EMS spindles both reorient in response to Wnt signals, but only ABar spindle reorientation is dependent on a new cell contact and on HS and SDN-1. SDN-1 transiently accumulates on the ABar surface as it contacts C, and is required for local concentration of Dishevelled (MIG-5) in the ABar cortex adjacent to C. These findings establish a new role for syndecan in Wnt-dependent spindle orientation.

Feasibility Study on Design of Spindle Supported by High-Stiffness Water Hydrostatic Thrust Bearing

The machining accuracy of ultra-precision machine tools relies on the performance of the spindle and linear table. The machining accuracy of ultra-precision machine tools is now at the level of several tens of nanometers. In order for ultra-precision machine tools to achieve machining accuracy, a precise spindle system is indispensible. High bearing stiffness is particularly important to minimize displacement due to the cutting force. This paper considers a spindle design supported by high-stiffness water hydrostatic thrust bearings. An objective of this study is to design a precision spindle supported by water hydrostatic thrust bearings with 1 kN/µm bearing stiffness. The bearing restrictors are chosen so that the highest stiffness can be obtained for given bearing parameters. The influences of gap sizes and supply water pressure on the bearing stiffness are presented. Based on the feasibility study done on the design of highstiffness water hydrostatic thrust bearings, the spindle is designed and developed. The influences of the water pressure on the spindle deformation and bearing stiffness are also investigated.

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.

The numerical analysis of the radial sleeve bearing with combined surface slip

In order to improve the carrying capacity and reduce the temperature rise in high speed and precise spindle systems, a combined surface radial sleeve bearing using the interfacial slip technique was discussed. An extended Reynolds equation was derived based on the modified slip length model considering the limiting shear stress. By means of the finite differential methods, the characteristic analysis and optimization of the slip region of the combined surface sleeve bearing were carried out, and it has been proved that there is still a considerable large load support in a parallel sliding gap. Comparing with the general journal bearing, the load capacity and end leakage rate of the combined surface sleeve bearing can be increased greatly and the load capacity can be increased by 1.75 times. The attitude angle, friction drag, temperature rise of the combined bearing can be decreased distinctly and the temperature rise can be decreased by 92.4%.

Automatic Thermal Expansion Compensation of the Precise Grinding Machine AMB Spindle

Thermal expansion of the magnetic bearings in the process of precise grinding will lead to the posture drift of the grinding head, which can seriously affect the precision of the grinding. In this paper, the module of the relationship between temperature rise and the spindle posture is established, and the relationship between the controller’s five reference inputs and the spindle posture is ascertained. The controller’s five reference inputs are corrected according to the sample value of the temperatures in the system, so the inline adjustment of the grinding head posture and the automatic thermal expansion compensation of the system can be realized. Experiment proved that this algorithm could successfully compensate the thermal expansion of the AMB spindle.

MCPH1 regulates the neuroprogenitor division mode by coupling the centrosomal cycle with mitotic entry through the Chk1–Cdc25 pathway

Primary microcephaly 1 is a neurodevelopmental disorder caused by mutations in the MCPH1 gene, whose product MCPH1 (also known as microcephalin and BRIT1) regulates DNA-damage response. Here we show that Mcph1 disruption in mice results in primary microcephaly, mimicking human MCPH1 symptoms, owing to a premature switching of neuroprogenitors from symmetric to asymmetric division. MCPH1-deficiency abrogates the localization of Chk1 to centrosomes, causing premature Cdk1 activation and early mitotic entry, which uncouples mitosis and the centrosome cycle. This misorients the mitotic spindle alignment and shifts the division plane of neuroprogenitors, to bias neurogenic cell fate. Silencing Cdc25b, a centrosome substrate of Chk1, corrects MCPH1-deficiency-induced spindle misalignment and rescues the premature neurogenic production in Mcph1-knockout neocortex. Thus, MCPH1, through its function in the Chk1-Cdc25-Cdk1 pathway to couple the centrosome cycle with mitosis, is required for precise mitotic spindle orientation and thereby regulates the progenitor division mode to maintain brain size.

Identification of an Aurora-A/PinsLINKER/ Dlg Spindle Orientation Pathway using Induced Cell Polarity in S2 Cells

Asymmetric cell division is intensely studied because it can generate cellular diversity as well as maintain stem cell populations. Asymmetric cell division requires mitotic spindle alignment with intrinsic or extrinsic polarity cues, but mechanistic detail of this process is lacking. Here, we develop a method to construct cortical polarity in a normally unpolarized cell line and use this method to characterize Partner of Inscuteable (Pins; LGN/AGS3 in mammals) -dependent spindle orientation. We identify a previously unrecognized evolutionarily conserved Pins domain (Pins(LINKER)) that requires Aurora-A phosphorylation to recruit Discs large (Dlg; PSD-95/hDlg in mammals) and promote partial spindle orientation. The well-characterized Pins(TPR) domain has no function alone, but placing the Pins(TPR) in cis to the Pins(LINKER) gives dynein-dependent precise spindle orientation. This "induced cortical polarity" assay is suitable for rapid identification of the proteins, domains, and amino acids regulating spindle orientation or cell polarity.

Adherens junction domains are split by asymmetric division of embryonic neural stem cells

Investigating the mechanisms controlling the asymmetric division of neocortical progenitors that generate neurones in the mammalian brain is crucial for understanding the abnormalities of cortical development. Partitioning of fate determinants is a key instructive step and components of the apical junctional complex (adherens junctions), including the polarity proteins PAR3 and aPKC as well as adhesion molecules such as N-cadherin, have been proposed to be candidate determinants. In this study, however, we found no correlation between the partitioning of N-cadherin and fate determination. Rather, we show that adherens junctions comprise three membrane domains, and that during asymmetrical division these are split such that both daughters retain the adhesive proteins that control cell position, but only one daughter inherits the polarity proteins along with the apical membrane. This provides a molecular explanation as to how both daughters remain anchored to the ventricular surface after mitosis, while adopting different fates.

LIM Kinase-mediated Cofilin Phosphorylation during Mitosis Is Required for Precise Spindle Positioning

The interaction of astral microtubules with cortical actin networks is essential for the correct orientation of the mitotic spindle; however, little is known about how the cortical actin organization is regulated during mitosis. LIM kinase-1 (LIMK1) regulates actin dynamics by phosphorylating and inactivating cofilin, an actin-depolymerizing protein. LIMK1 activity increases during mitosis. Here we show that mitotic LIMK1 activation is critical for accurate spindle orientation in mammalian cells. Knockdown of LIMK1 suppressed a mitosis-specific increase in cofilin phosphorylation and caused unusual cofilin localization in the cell cortex in metaphase, instability of cortical actin organization and astral microtubules, irregular rotation and misorientation of the spindle, and a delay in anaphase onset. Similar results were obtained by treating the cells with a LIMK1 in hibitor peptide or latrunculin A or by overexpressing a non-phosphorylatable cofilin(S3A) mutant. Furthermore, localization of LGN (a protein containing the repetitive Leu-Gly-Asn tripeptide motifs), an important regulator of spindle orientation, in the crescent-shaped cortical regions was perturbed in LIMK1 knockdown cells. Our results suggest that LIMK1-mediated cofilin phosphorylation is required for accurate spindle orientation by stabilizing cortical actin networks during mitosis.

MINITYPE MACHINING SYSTEM FOR DIAMOND LAPPING & POLISHING BY USING BRUSHLESS DIRECT CURRENT MOTOR AS PRECISE SPINDLE

A minitype precise spindle system which can machine precisely and stably in the process of diamond lapping and polishing is designed.In such minitypc spindle system,the brushless DC spindle motor is used to drive the lapping finish table,which is built with fluid dynamic bearings. Some measures have been taken to make the lapping system dynamic balance,and a serve controller which can adjust the speed of motor from 1200 r/rain to 5400 r/min is designed.Experiments show that the spindle system is reliable and stable for diamond polishing,and the detection results by atomic force microscope(AFM) show that the surfaces of diamond edge′s R= is 6.725 nm and whole diamond average R,is 3.25 nm.

Statistical analysis of wire electrical discharge turning on material removal rate

The application of wire electrical discharge machining (WEDM) for machining of precise cylindrical forms on hard and difficult-to-machine materials is presented. At first, the design of a precise, flexible and corrosion-resistant rotary spindle submerged is introduced. The spindle has been mounted on a five-axis wire EDM machine to rotate the workpiece in order to generate free form cylindrical geometries. The material removal rate (MRR) is an important indicator of the efficiency and cost-effectiveness of the process. Several experiments are conducted to consider effects of power, time-off, voltage, servo, wire speed, wire tension, and rotational speed (factors) on the MRR (response). An L 18 (2 1 × 3 7) Taguchi standard orthogonal array is chosen for the design of experiments. Analyses of variance (ANOVA) as well as regression analysis are performed on experimental data. The signal-to-noise (S/N) ratio analysis is employed to find the optimal condition.