Spindle Forces Research Articles

Noise, vibration, harshness model of a rotating tyre

ABSTRACTThe tyre plays a fundamental role in the generation of acoustically perceptible driving noise and vibrations inside the vehicle. An essential part of these vibrations is induced by the road excitation and transferred via the tyre into the vehicle. There are two basic ways to study noise, vibration, harshness (NVH) behaviour: Simulations in time and frequency domains. Modelling the tyre transfer behaviour in frequency domain requires special attention to the rotation of the tyre. This paper shows the approach taken by the authors to include the transfer behaviour in the frequency range up to 250 Hz from geometric road excitations to resulting spindle forces in frequency domain. This paper validates the derived NVH tyre model by comparison with appropriate transient simulations of the base transient model.

A Balance between Nuclear and Cytoplasmic Volumes Controls Spindle Length.

Proper assembly of the spindle apparatus is crucially important for faithful chromosome segregation during anaphase. Thanks to the effort over the last decades, we have very detailed information about many events leading to spindle assembly and chromosome segregation, however we still do not understand certain aspects, including, for example, spindle length control. When tight regulation of spindle size is lost, chromosome segregation errors emerge. Currently, there are several hypotheses trying to explain the molecular mechanism of spindle length control. The number of kinetochores, activity of molecular rulers, intracellular gradients, cell size, limiting spindle components, and the balance of the spindle forces seem to contribute to spindle size regulation, however some of these mechanisms are likely specific to a particular cell type. In search for a general regulatory mechanism, in our study we focused on the role of cell size and nuclear to cytoplasmic ratio in this process. To this end, we used relatively large cells isolated from 2-cell mouse embryos. Our results showed that the spindle size upper limit is not reached in these cells and suggest that accurate control of spindle length requires balanced ratio between nuclear and cytoplasmic volumes.

Unattached kinetochores rather than intrakinetochore tension arrest mitosis in taxol-treated cells.

Kinetochores attach chromosomes to the spindle microtubules and signal the spindle assembly checkpoint to delay mitotic exit until all chromosomes are attached. Light microscopy approaches aimed to indirectly determine distances between various proteins within the kinetochore (termed Delta) suggest that kinetochores become stretched by spindle forces and compact elastically when the force is suppressed. Low Delta is believed to arrest mitotic progression in taxol-treated cells. However, the structural basis of Delta remains unknown. By integrating same-kinetochore light microscopy and electron microscopy, we demonstrate that the value of Delta is affected by the variability in the shape and size of outer kinetochore domains. The outer kinetochore compacts when spindle forces are maximal during metaphase. When the forces are weakened by taxol treatment, the outer kinetochore expands radially and some kinetochores completely lose microtubule attachment, a condition known to arrest mitotic progression. These observations offer an alternative interpretation of intrakinetochore tension and question whether Delta plays a direct role in the control of mitotic progression.

Local Anchorage of Kinetochore-Fibers to the Mammalian Spindle Provides Mechanical Isolation and Load-Bearing Redundancy

During cell division, kinetochores attach chromosomes to the spindle through microtubule bundles called k-fibers. Forces generated at kinetochores move chromosomes, rather than k-fibers; thus, the latter must be structurally anchored to the spindle. How a dynamic spindle robustly anchors its k-fibers is not understood. Here, we probe where and how the mammalian spindle holds on to its k-fibers to bear the load of chromosome movement. We use laser ablation to sever k-fibers at different locations and detach them from spindle poles, thereby revealing their anchorage within the spindle body. The immediate relaxation response post-ablation indicates that k-fibers are anchored not only at their ends, but also along their lengths within the spindle. Effective anchorage scales with k-fiber length for the first few microns, but then saturates, indicating that k-fibers are effectively locally anchored within the first few microns of their lengths. This anchorage also occurs locally along the spindle's width, as little load is shared between neighboring k-fibers. We find that increasing microtubule crosslinking increases k-fiber anchorage, and that depleting NuMA, known to crosslink microtubules at poles, significantly disrupts local anchorage of k-fibers to the spindle body. In contrast, depletion of microtubule crosslinkers Eg5 and PRC1 does not affect anchorage despite these proteins’ local mechanical functions. Together, the data indicate that NuMA-mediated microtubule crosslinking in the spindle body allows for local anchorage and isolation of k-fibers, and mechanical redundancy in their connections to the spindle. Such mechanical isolation and redundancy are well-suited to ensure robust k-fiber load-bearing and chromosome segregation despite dynamic spindle forces and structures.

Polar Ejection Forces Promote the Conversion from Lateral to End-on Kinetochore-Microtubule Attachments on Mono-oriented Chromosomes.

SummaryChromosome bi-orientation occurs after conversion of initial lateral attachments between kinetochores and spindle microtubules into stable end-on attachments near the cell equator. After bi-orientation, chromosomes experience tension from spindle forces, which plays a key role in the stabilization of correct kinetochore-microtubule attachments. However, how end-on kinetochore-microtubule attachments are first stabilized in the absence of tension remains a key unanswered question. To address this, we generated Drosophila S2 cells undergoing mitosis with unreplicated genomes (SMUGs). SMUGs retained single condensed chromatids that attached laterally to spindle microtubules. Over time, laterally attached kinetochores converted into end-on attachments and experienced intra-kinetochore stretch/structural deformation, and SMUGs eventually exited a delayed mitosis with mono-oriented chromosomes after satisfying the spindle-assembly checkpoint (SAC). Polar ejection forces (PEFs) generated by Chromokinesins promoted the conversion from lateral to end-on kinetochore-microtubule attachments that satisfied the SAC in SMUGs. Thus, PEFs convert lateral to stable end-on kinetochore-microtubule attachments, independently of chromosome bi-orientation.

Dyskerin, tRNA genes, and condensin tether pericentric chromatin to the spindle axis in mitosis.

Condensin is enriched in the pericentromere of budding yeast chromosomes where it is constrained to the spindle axis in metaphase. Pericentric condensin contributes to chromatin compaction, resistance to microtubule-based spindle forces, and spindle length and variance regulation. Condensin is clustered along the spindle axis in a heterogeneous fashion. We demonstrate that pericentric enrichment of condensin is mediated by interactions with transfer ribonucleic acid (tRNA) genes and their regulatory factors. This recruitment is important for generating axial tension on the pericentromere and coordinating movement between pericentromeres from different chromosomes. The interaction between condensin and tRNA genes in the pericentromere reveals a feature of yeast centromeres that has profound implications for the function and evolution of mitotic segregation mechanisms.

CENP-W plays a role in maintaining bipolar spindle structure.

The CENP-W/T complex was previously reported to be required for mitosis. HeLa cells depleted of CENP-W displayed profound mitotic defects, with mitotic timing delay, disorganized prometaphases and multipolar spindles as major phenotypic consequences. In this study, we examined the process of multipolar spindle formation induced by CENP-W depletion. Depletion of CENP-W in HeLa cells labeled with histone H2B and tubulin fluorescent proteins induced rapid fragmentation of originally bipolar spindles in a high proportion of cells. CENP-W depletion was associated with depletion of Hec1 at kinetochores. The possibility of promiscuous centrosomal duplication was ruled out by immunofluorescent examination of centrioles. However, centrioles were frequently observed to be abnormally split. In addition, a large proportion of the supernumerary poles lacked centrioles, but were positively stained with different centrosomal markers. These observations suggested that perturbation in spindle force distribution caused by defective kinetochores could contribute to a mechanical mechanism for spindle pole disruption. ‘Spindle free’ nocodazole arrested cells did not exhibit pole fragmentation after CENP-W depletion, showing that pole fragmentation is microtubule dependent. Inhibition of centrosome separation by monastrol reduced the incidence of spindle pole fragmentation, indicating that Eg5 plays a role in spindle pole disruption. Surprisingly, CENP-W depletion rescued the monopolar spindle phenotype of monastrol treatment, with an increased frequency of bipolar spindles observed after CENP-W RNAi. We overexpressed the microtubule cross-linking protein TPX2 to create spindle poles stabilized by the microtubule cross-linking activity of TPX2. Spindle pole fragmentation was suppressed in a TPX2-dependent fashion. We propose that CENP-W, by influencing proper kinetochore assembly, particularly microtubule docking sites, can confer spindle pole resistance to traction forces exerted by motor proteins during chromosome congression. Taken together, our findings are consistent with a model in which centrosome integrity is controlled by the pathways regulating kinetochore-microtubule attachment stability.

The Architecture of CCAN Proteins Creates a Structural Integrity to Resist Spindle Forces and Achieve Proper Intrakinetochore Stretch

Constitutive centromere-associated network (CCAN) proteins, particularly CENP-C, CENP-T, and the CENP-H/-I complex, mechanically link CENP-A-containing centromeric chromatin within the inner kinetochore to outer kinetochore proteins, such as the Ndc80 complex, that bind kinetochore microtubules. Accuracy of chromosome segregation depends critically upon Aurora B phosphorylation of Ndc80/Hec1. To determine how CCAN protein architecture mechanically constrains intrakinetochore stretch between CENP-A and Ndc80/Hec1 for proper Ndc80/Hec1 phosphorylation, we used super-resolution fluorescence microscopy and selective protein depletion. We found that at bi-oriented chromosomes in late prometaphase cells, CENP-T is stretched ∼16nm to the inner end of Ndc80/Hec1, much less than expected for full-length CENP-T. Depletion of various CCAN linker proteins induced hyper-intrakinetochore stretch (an additional 20-60nm) with corresponding significant decreases inAurora B phosphorylation of Ndc80/Hec1. Thus, proper intrakinetochore stretch is required for normalkinetochore function and depends critically on all the CCAN mechanical linkers to the Ndc80 complex.

Transient dynamic behaviour of finite element tire traversing obstacles with different heights

Tire models used in vehicle dynamics simulation for CAE durability and ride comfort assessment need to be capable of predicting the non-linear deformation and enveloping characteristics which occur when traversing large road obstacles. Normally, transient dynamic characteristics of a rolling tire are determined from tire rig tests, and the tire parameters are transferred into multi-body system for vehicle dynamic analysis. However rig design limitations mean that tests cannot be carried out in the most severe conditions, particularly for traversing high ramp or large obstacles. However, using detailed FE tire models, such tests can be carried out virtually. A FE tire model was developed specifically for this purpose using explicit integration in ABAQUS™. Tire enveloping tests in traversing obstacles of different sizes were then carried out, virtually, using the validated FE tire model. Satisfactory results of transient responses were obtained by comparison with the experimental tests for the tire traversing obstacles with different heights. Tire transient dynamic behaviour was investigated by analysing the influence of tire rolling velocity and height of road obstacle on transient spindle responses, dynamic stiffness, together with tire deformation for the tire impacting obstacles. Finally, the investigation showed that longitudinal dynamic stiffness decreases when the tire traverses a higher obstacle. In addition, with the increase of height of road obstacle, the resonant amplitude of spindle force response as well as the tire deformation becomes larger in both longitudinal and vertical directions, especially for the tire rolling over 25mm×25mm rectangular obstacle. Also, it is found that higher travelling velocity of the tire leads to higher resonant amplitude of spindle forces in the vertical direction.

Centmitor-1, a novel acridinyl-acetohydrazide, possesses similar molecular interaction field and antimitotic cellular phenotype as rigosertib, on 01910.Na.

Mitosis is an attractive target for the development of new anticancer drugs. In a search for novel mitotic inhibitors, we virtually screened for low molecular weight compounds that would possess similar steric and electrostatic features, but different chemical structure than rigosertib (ON 01910.Na), a putative inhibitor of phosphoinositide 3-kinase (PI3K) and polo-like kinase 1 (Plk1) pathways. Highest scoring hit compounds were tested in cell-based assays for their ability to induce mitotic arrest. We identified a novel acridinyl-acetohydrazide, here named as Centmitor-1 (Cent-1), that possesses highly similar molecular interaction field as rigosertib. In cells, Cent-1 phenocopied the cellular effects of rigosertib and caused mitotic arrest characterized by chromosome alignment defects, multipolar spindles, centrosome fragmentation, and activated spindle assembly checkpoint. We compared the effects of Cent-1 and rigosertib on microtubules and found that both compounds modulated microtubule plus-ends and reduced microtubule dynamics. Also, mitotic spindle forces were affected by the compounds as tension across sister kinetochores was reduced in mitotic cells. Our results showed that both Cent-1 and rigosertib target processes that occur during mitosis as they had immediate antimitotic effects when added to cells during mitosis. Analysis of Plk1 activity in cells using a Förster resonance energy transfer (FRET)-based assay indicated that neither compound affected the activity of the kinase. Taken together, these findings suggest that Cent-1 and rigosertib elicit their antimitotic effects by targeting mitotic processes without impairment of Plk1 kinase activity.

Probing Forces on Newly Generated Spindle Microtubule Minus-Ends

The mitotic spindle is a dynamic self-organizing machine that coordinates cell division and preserves genomic stability. The ability to focus microtubule minus-ends into poles is crucial to spindle structure and function. However, our understanding of pole-focusing forces has been limited by the challenges of labeling and imaging microtubule minus-ends in established spindles. Here, we used laser ablation to sever kinetochore-fiber microtubules in mammalian cells and probe how the cell detects and organizes newly generated microtubule minus-ends. Within a few seconds of ablation, the cell recognizes new minus-ends and begins pulling them poleward. These pole-focusing forces exist throughout metaphase and anaphase and can move chromosomes rapidly, dominating other spindle forces. Opposing forces on chromosomes from the other half-spindle are able to slow, though not stop, the pole-focusing response, as indicated by faster pole-focusing speeds in monopolar spindles and during anaphase than in metaphase bipolar spindles. Together, our data indicate that microtubule minus-end focusing forces operate broadly and rapidly and are of similar magnitude to other spindle forces. These pole-focusing forces are thus well-suited to robustly maintain spindle structural integrity despite rapid turnover of spindle components and mechanical challenges.

The Architecture of CCAN Proteins Creates a Structural Integrity to Resist Spindle Forces and Achieve Proper Intrakinetochore Stretch

The Architecture of CCAN Proteins Creates a Structural Integrity to Resist Spindle Forces and Achieve Proper Intrakinetochore Stretch

Calculating road input data for vehicle simulation

Multibody system simulation is an important tool in the development process in vehicle engineering. Without much effort, different vehicle variants and designs can be simulated, analyzed, and optimized. This is of particular relevance in an early stage of development, when no physical prototypes are available yet. In order to simulate the vehicle models under realistic conditions, suitable input data is needed for the simulation. We present an approach to derive a virtual road profile based on a tire-surrogate model, measured spindle forces, and a multibody system model of the measurement vehicle. In contrast to the measured spindle forces, the road profile together with the tire-surrogate model can be used to simulate other vehicle variants, for which no measurements are available. The road profile is derived by solving an inverse control problem. We formulate this inverse problem in the context of system simulation and provide a short mathematical analysis. Additionally, we discuss a solution approach, the method of control-constraints, which is also applied in a numerical simulation study to compute a virtual road profile.

Pericentric chromatin loops function as a nonlinear spring in mitotic force balance

The mechanisms by which sister chromatids maintain biorientation on the metaphase spindle are critical to the fidelity of chromosome segregation. Active force interplay exists between predominantly extensional microtubule-based spindle forces and restoring forces from chromatin. These forces regulate tension at the kinetochore that silences the spindle assembly checkpoint to ensure faithful chromosome segregation. Depletion of pericentric cohesin or condensin has been shown to increase the mean and variance of spindle length, which have been attributed to a softening of the linear chromatin spring. Models of the spindle apparatus with linear chromatin springs that match spindle dynamics fail to predict the behavior of pericentromeric chromatin in wild-type and mutant spindles. We demonstrate that a nonlinear spring with a threshold extension to switch between spring states predicts asymmetric chromatin stretching observed in vivo. The addition of cross-links between adjacent springs recapitulates coordination between pericentromeres of neighboring chromosomes.

Topoisomerase II– and Condensin-Dependent Breakage of MEC1ATR-Sensitive Fragile Sites Occurs Independently of Spindle Tension, Anaphase, or Cytokinesis

Fragile sites are loci of recurrent chromosome breakage in the genome. They are found in organisms ranging from bacteria to humans and are implicated in genome instability, evolution, and cancer. In budding yeast, inactivation of Mec1, a homolog of mammalian ATR, leads to chromosome breakage at fragile sites referred to as replication slow zones (RSZs). RSZs are proposed to be homologous to mammalian common fragile sites (CFSs) whose stability is regulated by ATR. Perturbation during S phase, leading to elevated levels of stalled replication forks, is necessary but not sufficient for chromosome breakage at RSZs or CFSs. To address the nature of additional event(s) required for the break formation, we examined involvement of the currently known or implicated mechanisms of endogenous chromosome breakage, including errors in replication fork restart, premature mitotic chromosome condensation, spindle tension, anaphase, and cytokinesis. Results revealed that chromosome breakage at RSZs is independent of the RAD52 epistasis group genes and of TOP3, SGS1, SRS2, MMS4, or MUS81, indicating that homologous recombination and other recombination-related processes associated with replication fork restart are unlikely to be involved. We also found spindle force, anaphase, or cytokinesis to be dispensable. RSZ breakage, however, required genes encoding condensin subunits (YCG1, YSC4) and topoisomerase II (TOP2). We propose that chromosome break formation at RSZs following Mec1 inactivation, a model for mammalian fragile site breakage, is mediated by internal chromosomal stress generated during mitotic chromosome condensation.

DNA catenation maintains structure of human metaphase chromosomes

Mitotic chromosome structure is pivotal to cell division but difficult to observe in fine detail using conventional methods. DNA catenation has been implicated in both sister chromatid cohesion and chromosome condensation, but has never been observed directly. We have used a lab-on-a-chip microfluidic device and fluorescence microscopy, coupled with a simple image analysis pipeline, to digest chromosomal proteins and examine the structure of the remaining DNA, which maintains the canonical ‘X’ shape. By directly staining DNA, we observe that DNA catenation between sister chromatids (separated by fluid flow) is composed of distinct fibres of DNA concentrated at the centromeres. Disrupting the catenation of the chromosomes with Topoisomerase IIα significantly alters overall chromosome shape, suggesting that DNA catenation must be simultaneously maintained for correct chromosome condensation, and destroyed to complete sister chromatid disjunction. In addition to demonstrating the value of microfluidics as a tool for examining chromosome structure, these results lend support to certain models of DNA catenation organization and regulation: in particular, we conclude from our observation of centromere-concentrated catenation that spindle forces could play a driving role in decatenation and that Topoisomerase IIα is differentially regulated at the centromeres, perhaps in conjunction with cohesin.

크리트 가진법을 이용한 타이어특성에 따른 로드노이즈 예측 연구

It is important for identification of noise and vibration problem of tire to consider influence of interaction between road and tire. A quantification of road noise is a challenging issue in vehicle NVH due to extremely complicated transfer paths of road noise as well as the difficulty in an experimental identification of input force from tire-road interaction. A noise caused by tire is divided into road noise(structure-borne noise) and pattern noise(air-borne noise). Pattern noise is caused by pattern shape of tire, which has larger than 500 Hz, but road noise is generated by the interactions between a tire and a vehicle body. In this paper, we define the quantitative analysis for road noise caused by interactions between tire and road parameters. For the identification of road noise, the chassis dynamometer that is equipped $10mm{\times}10mm $ square cleat in the semi-anechoic chamber is used, and the tire spindle forces are measured by load cell. The vibro-acoustic transfer function between ear position and wheel center was measured by the vibro-acoustic reciprocity method. In this study three tires with different type of mechanical are used for the experiment work.

A Role for Metaphase Spindle Elongation Forces in Correction of Merotelic Kinetochore Attachments

During mitosis, equal segregation of chromosomes depends on proper kinetochore-microtubule attachments. Merotelic kinetochore orientation, in which a single kinetochore binds microtubules from both spindle poles [1], is a major cause of chromosome instability [2], which is commonly observed in solid tumors [3, 4]. Using the fission yeast Schizosaccharomyces pombe, we show that a proper force balance between kinesin motors on interpolar spindle microtubules is critical for correcting merotelic attachments. Inhibition of the plus-end-directed spindle elongation motors kinesin-5 (Cut7) and kinesin-6 (Klp9) reduces spindle length, tension at kinetochores, and the frequency of merotelic attachments. In contrast, merotely is increased by deletion of the minus-end-directed kinesin-14 (Klp2) or overexpression of Klp9. Also, Cdk1 regulates spindle elongation forces to promote merotelic correction by phosphorylating and inhibiting Klp9. The role of spindle elongation motors in merotelic correction is conserved, because partial inhibition of the human kinesin-5 homolog Eg5 using the drug monastrol reduces spindle length and lagging chromosome frequency in both normal (RPE-1) and tumor (CaCo-2) cells. These findings reveal unexpected links between spindle forces and correction of merotelic attachments and show that pharmacological manipulation of spindle elongation forces might be used to reduce chromosome instability in cancer cells.

Directionality of Tangential Force Variation in High Speed Uniformity

Abstract Tire nonuniformity may affect vehicle ride comfort quality especially while vehicles are running at highway speeds. Spindle force variations in the radial, tangential, and lateral directions are normally used as parameters evaluating tire high speed uniformity performance. An interesting physical phenomenon—directionality of tangential force variation has been observed for many tires. The directionality of tangential force variation means that tangential force variation is different when a tire is measured in the clockwise and counter-clockwise directions under the same test conditions. In this paper, a theory is developed to explain the interesting physical phenomenon. Simulation and analysis are conducted using a dynamic tire model. It is found that the directionality of tangential force variation is caused by the interaction of different types of nonuniformities in tires such as mass and geometry variations.

Aurora-A inactivation causes mitotic spindle pole fragmentation by unbalancing microtubule-generated forces

BackgroundAurora-A is an oncogenic kinase playing well-documented roles in mitotic spindle organisation. We previously found that Aurora-A inactivation yields the formation of spindles with fragmented poles that can drive chromosome mis-segregation. Here we have addressed the mechanism through which Aurora-A activity regulates the structure and cohesion of spindle poles.ResultsWe inactivated Aurora-A in human U2OS osteosarcoma cells either by RNA-interference-mediated silencing or treating cultures with the specific inhibitor MLN8237. We show that mitotic spindle pole fragmentation induced by Aurora-A inactivation is associated with microtubule hyperstabilisation. Silencing of the microtubule-stabilising factor ch-TOG prevents spindle pole fragmentation caused by inactivation of Aurora-A alone and concomitantly reduces the hyperstabilisation of microtubules. Furthermore, decreasing pole-directed spindle forces by inhibition of the Eg5 kinesin, or by destabilisation of microtubule-kinetochore attachments, also prevents pole fragmentation in Aurora-A-inactivated mitoses.ConclusionsOur findings indicate that microtubule-generated forces are imbalanced in Aurora-A-defective cells and exert abnormal pressure at the level of spindle poles, ultimately causing their fragmentation. This study therefore highlights a novel role of the Aurora-A kinase in regulating the balance between microtubule forces during bipolar spindle assembly.