Activation of ADF/cofilin by phosphorylation-regulated Slingshot phosphatase is required for the meiotic spindle assembly in Xenopus laevis oocytes

We identify Xenopus ADF/cofilin (XAC) and its activator, Slingshot phosphatase (XSSH), as key regulators of actin dynamics essential for spindle microtubule assembly during Xenopus oocyte maturation. Phosphorylation of XSSH at multiple sites within the tail domain occurs just after germinal vesicle breakdown (GVBD) and is accompanied by dephosphorylation of XAC, which was mostly phosphorylated in immature oocytes. This XAC dephosphorylation after GVBD is completely suppressed by latrunculin B, an actin monomer-sequestering drug. On the other hand, jasplakinolide, an F-actin-stabilizing drug, induces dephosphorylation of XAC. Effects of latrunculin B and jasplakinolide are reconstituted in cytostatic factor-arrested extracts (CSF extracts), and XAC dephosphorylation is abolished by depletion of XSSH from CSF extracts, suggesting that XSSH functions as an actin filament sensor to facilitate actin filament dynamics via XAC activation. Injection of anti-XSSH antibody, which blocks full phosphorylation of XSSH after GVBD, inhibits both meiotic spindle formation and XAC dephosphorylation. Coinjection of constitutively active XAC with the antibody suppresses this phenotype. Treatment of oocytes with jasplakinolide also impairs spindle formation. These results strongly suggest that elevation of actin dynamics by XAC activation through XSSH phosphorylation is required for meiotic spindle assembly in Xenopus laevis

Effect analysis of bearing and interface dynamics on tool point FRF for chatter stability in machine tools by using a new analytical model for spindle-tool assemblies

Self-excited vibration of the tool, regenerative chatter, can be predicted and eliminated if the stability lobe diagram of the spindle–holder–tool assembly is known. Regardless of the approach being used, analytically or numerically, forming the stability lobe diagram of an assembly implies knowing the point frequency response function (FRF) in receptance form at the tool tip. In this paper, it is aimed to study the effects of spindle–holder and holder–tool interface dynamics, as well as the effects of individual bearings on the tool point FRF by using an analytical model recently developed by the authors for predicting the tool point FRF of spindle–holder–tool assemblies. It is observed that bearing dynamics control the rigid body modes of the assembly, whereas, spindle–holder interface dynamics mainly affects the first elastic mode, while holder–tool interface dynamics alters the second elastic mode. Individual bearing and interface translational stiffness and damping values control the natural frequency and the peak of their relevant modes, respectively. It is also observed that variations in the values of rotational contact parameters do not affect the resulting FRF considerably, from which it is concluded that rotational contact parameters of both interfaces are not as crucial as the translational ones and therefore average values can successfully be used to represent their effects. These observations are obtained for the bearing and interface parameters taken from recent literature, and will be valid for similar assemblies. Based on the effect analysis carried out, a systematic approach is suggested for identifying bearing and interface contact parameters from experimental measurements

A Modeling approach for analysis and improvement of spindle-holder-tool assembly dynamics

The most important information required for chatter stability analysis is the dynamics of the involved structures, i.e. the frequency response functions (FRFs) which are usually determined experimentally. In this study, the tool point FRF of a spindle-holder-tool assembly is analytically determined by using the receptance coupling and structural modification techniques. Timoshenko’s beam model is used for increased accuracy. The spindle is also modeled analytically with elastic supports representing the bearings. The mathematical model is used to determine the effects of different parameters on the tool point FRF and to identify contact dynamics from experimental measurements. The applications of the model are demonstrated and the predictions are verified experimentally

Anaphase B.

Anaphase B spindle elongation is characterized by the sliding apart of overlapping antiparallel interpolar (ip) microtubules (MTs) as the two opposite spindle poles separate, pulling along disjoined sister chromatids, thereby contributing to chromosome segregation and the propagation of all cellular life. The major biochemical "modules" that cooperate to mediate pole-pole separation include: (i) midzone pushing or (ii) braking by MT crosslinkers, such as kinesin-5 motors, which facilitate or restrict the outward sliding of antiparallel interpolar MTs (ipMTs); (iii) cortical pulling by disassembling astral MTs (aMTs) and/or dynein motors that pull aMTs outwards; (iv) ipMT plus end dynamics, notably net polymerization; and (v) ipMT minus end depolymerization manifest as poleward flux. The differential combination of these modules in different cell types produces diversity in the anaphase B mechanism. Combinations of antagonist modules can create a force balance that maintains the dynamic pre-anaphase B spindle at constant length. Tipping such a force balance at anaphase B onset can initiate and control the rate of spindle elongation. The activities of the basic motor filament components of the anaphase B machinery are controlled by a network of non-motor MT-associated proteins (MAPs), for example the key MT cross-linker, Ase1p/PRC1, and various cell-cycle kinases, phosphatases, and proteases. This review focuses on the molecular mechanisms of anaphase B spindle elongation in eukaryotic cells and briefly mentions bacterial DNA segregation systems that operate by spindle elongation

Length-dependent anisotropic scaling of spindle shape

Spindle length varies dramatically across species and during early development to segregate chromosomes optimally. Both intrinsic factors, such as regulatory molecules, and extrinsic factors, such as cytoplasmic volume, determine spindle length scaling. However, the properties that govern spindle shape and whether these features can be modulated remain unknown. Here, we analyzed quantitatively how the molecular players which regulate microtubule dynamics control the kinetics of spindle formation and shape. We find that, in absence of Clasp1 and Clasp2, spindle assembly is biphasic due to unopposed inward pulling forces from the kinetochore-fibers and that kinetochore-fibers also alter spindle geometry. We demonstrate that spindle shape scaling is independent of the nature of the molecules that regulate dynamic microtubule properties, but is dependent on the steady-state metaphase spindle length. The shape of the spindle scales anisotropically with increasing length. Our results suggest that intrinsic mechanisms control the shape of the spindle to ensure the efficient capture and alignment of chromosomes independently of spindle length

Influence of bearing kinematics hypotheses on ball bearing heat generation

Spindle dynamics is a key issue in machining. Thermo-mechanical models of spindle need to be developed to understand and predict the complex behavior of spindles at high speed. Accurate bearing stiffness model is required, since it is a boundary condition of the shaft. Besides, bearings also play an important role in heat generation in the spindle. Bearing models rely on kinematics hypotheses at the ball-race contacts. The aim of the paper is to study the influence of these kinematics hypotheses on bearing heat generation. The different kinematics hypotheses that can be found in literature are presented and implemented into the bearing friction model. Simulations of bearing dynamics are performed and contact force and torque at ball-race contacts are compared. Behavior of the bearing models with different kinematic models is studied using a coupled thermo-mechanical model of a specialized test bed. Loss torque and temperatures simulated are compared and validated by experiments

Regulation of Op18 during Spindle Assembly in Xenopus Egg Extracts

Oncoprotein 18 (Op18) is a microtubule-destabilizing protein that is negatively regulated by phosphorylation. To evaluate the role of the three Op18 phosphorylation sites in Xenopus (Ser 16, 25, and 39), we added wild-type Op18, a nonphosphorylatable triple Ser to Ala mutant (Op18-AAA), and to mimic phosphorylation, a triple Ser to Glu mutant (Op18-EEE) to egg extracts and monitored spindle assembly. Op18-AAA dramatically decreased microtubule length and density, while Op18-EEE did not significantly affect spindle microtubules. Affinity chromatography with these proteins revealed that the microtubule-destabilizing activity correlated with the ability of Op18 to bind tubulin. Since hyperphosphorylation of Op18 is observed upon addition of mitotic chromatin to extracts, we reasoned that chromatin-associated proteins might play a role in Op18 regulation. We have performed a preliminary characterization of the chromatin proteins recruited to DNA beads, and identified the Xenopus polo-like kinase Plx1 as a chromatin-associated kinase that regulates Op18 phosphorylation. Depletion of Plx1 inhibits chromatin-induced Op18 hyperphosphorylation and spindle assembly in extracts. Therefore, Plx1 may promote microtubule stabilization and spindle assembly by inhibiting Op18

On the stability of high-speed milling with spindle speed variation

Spindle speed variation is a well-known technique to suppress regenerative machine tool vibrations, but it is usually considered to be effective only for low spindle speeds. In this paper, the effect of spindle speed variation is analyzed in the high-speed domain for spindle speeds corresponding to the first flip (period doubling) and to the first Hopf lobes. The optimal amplitudes and frequencies of the speed modulations are computed using the semidiscre- tization method. It is shown that period doubling chatter can effectively be suppressed by spindle speed variation, although, the technique is not effective for the quasiperiodic chatter above the Hopf lobe. The results are verified by cutting tests. Some special cases are also discussed where the practical behavior of the system differs from the predicted one in some ways. For these cases, it is pointed out that the concept of stability is understood on the scale of the principal period of the system—that is, the speed modulation period for variable spindle speed machining and the tooth passing period for constant spindle speed machining