Optimization of two support spindle shaft on nonlinear elastic supports by rigidity characteristics

One of the main structural elements of metalworking machines is the spindle assembly (spindle), which is used to hold cutting tools or workpieces. The rigidity of the spindle assembly plays a decisive role in ensuring the accuracy and efficiency of the machine as a whole. The assessment of the spindle shaft stiffness is carried out on the basis of the analysis of the static bending of the spindle shaft, which made it possible to formulate and solve the problems of optimizing the spindle shaft according to the stiffness characteristics for two supporting structures on nonlinear elastic supports. To determine the stiffness of roller bearings, the work uses the dependence obtained on the basis of solving the problem of contact interaction of an elastic steel cylinder with curvilinear elastic steel half-spaces. For the considered design scheme, the optimization goals were chosen for the conditions of the smallest displacement of the end section of the spindle shaft console, the achievement of the minimum angle of rotation in this section or the minimum of their normalized superposition, which ensures maximum rigidity in the processing zone. Consideration has also been given to minimizing the swing angle at the front support to maximize bearing life. Mathematically, the problem is presented in the form of minimizing one of the 4 proposed objective functions by changing the variable parameters - the length of the cantilever and the value of the inter-support distance, represented as dimensionless quantities - the cantilever coefficient and the inter-support distance coefficient. Minimum and maximum values ​​of the cantilever length and shaft span were considered as constraints on the variable parameters. Varying the console coefficients and the inter-support distance was carried out by the method of sequential enumeration within the specified constraints, the solution of optimization problems is presented in a graphical form. The solution to the problem of shaft bending was carried out on the basis of the equation of the bent axis of the beam in the framework of the Euler - Bernoulli hypotheses and presented in an analytical form together with analytical dependencies for calculating the radial stiffness of a roller bearing as a function of the supporting force acting on it. The algorithm for solving optimization problems is implemented in the MatLAB package. Optimal solutions have shown that the minimum of the combined functions, consisting of the sum of the relative deflection values ​​at the end of the console and the angles of rotation at the end of the console and on the front support, is achieved at the same variable parameters as the minima of the angles of rotation at the end of the console and on the front support. The proposed approach to the design of the shafts of spindle units of metal-cutting machines, which are optimal in terms of rigidity characteristics, forms a tool for a reasonable choice of bearings and design parameters of spindle shafts.

The APC/C targets the Cep152-Cep63 complex at the centrosome to regulate mitotic spindle assembly

The control of protein abundance is a fundamental regulatory mechanism during mitosis. The anaphase promoting complex/cyclosome (APC/C) is the main protein ubiquitin ligase responsible for the temporal regulation of mitotic progression. It has been proposed that the APC/C might fulfil other functions including assembly of the mitotic spindle. Here, we show that the APC/C localizes to centrosomes, the organizers of the eukaryotic microtubule cytoskeleton, specifically during mitosis. Recruitment of the APC/C to spindle poles requires the centrosomal protein Cep152, and we identified Cep152 as both an APC/C interaction partner and as an APC/C substrate. Previous studies showed that Cep152 forms a complex with Cep57 and Cep63. The APC/C-mediated ubiquitination of Cep152 at the centrosome releases Cep57 from this inhibitory complex and enables its interaction with pericentrin, a critical step in promoting microtubule nucleation. Thus, our study extends the function of the APC/C from being a regulator of mitosis to also acting as a positive governor of spindle assembly. The APC/C thereby integrates control of these two important processes in a temporal manner.

Structure of the RZZ complex and molecular basis of Spindly-driven corona assembly at human kinetochores

In metazoans, a ≍1 megadalton (MDa) super-complex comprising the Dynein-Dynactin adaptor Spindly and the ROD-Zwilch-ZW10 (RZZ) complex is the building block of a fibrous biopolymer, the kinetochore fibrous corona. The corona assembles on mitotic kinetochores to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. We report here a high-resolution cryo-EM structure that captures the essential features of the RZZ complex, including a farnesyl binding site required for Spindly binding. Using a highly predictive in vitro assay, we demonstrate that the SAC kinase MPS1 is necessary and sufficient for corona assembly at supercritical concentrations of the RZZ-Spindly (RZZS) complex, and describe the molecular mechanism of phosphorylation-dependent filament nucleation. We identify several structural requirements for RZZS polymerization in rings and sheets. Finally, we identify determinants of kinetochore localization and corona assembly of Spindly. Our results describe a framework for the long-sought-for molecular basis of corona assembly on metazoan kinetochores.

Complementation of fission yeast kinesin-5/Cut7 with human Eg5 provides a versatile platform for screening of anti-cancer compounds

Kinesin-5 family proteins are essential for bipolar spindle assembly to ensure mitotic fidelity. Here we demonstrate evolutionary functional conservation of kinesin-5 between human and fission yeast. Human Eg5 expressed in the nucleus replaces fission yeast counterpart Cut7. Intriguingly, Eg5 overproduction results in cytotoxicity. This phenotype provides a useful platform for the development of novel kinesin-5 inhibitors as anti-cancer drugs.

Inhibitors of the Bub1 spindle assembly checkpoint kinase: synthesis of BAY-320 and comparison with 2OH-BNPP1

Bub1 is a serine/threonine kinase proposed to function centrally in mitotic chromosome alignment and the spindle assembly checkpoint (SAC); however, its role remains controversial. Although it is well documented that Bub1 phosphorylation of Histone 2A at T120 (H2ApT120) recruits Sgo1/2 to kinetochores, the requirement of its kinase activity for chromosome alignment and the SAC is debated. As small-molecule inhibitors are invaluable tools for investigating kinase function, we evaluated two potential Bub1 inhibitors: 2OH-BNPPI and BAY-320. After confirming that both inhibit Bub1 in vitro , we developed a cell-based assay for Bub1 inhibition. We overexpressed a fusion of Histone 2B and Bub1 kinase region, tethering it in proximity to H2A to generate a strong ectopic H2ApT120 signal along chromosome arms. Ectopic signal was effectively inhibited by BAY-320, but not 2OH-BNPP1 at concentrations tested. In addition, only BAY-320 was able to inhibit endogenous Bub1-mediated Sgo1 localization. Preliminary experiments using BAY-320 suggest a minor role for Bub1 kinase activity in chromosome alignment and the SAC; however, BAY-320 may exhibit off-target effects at the concentration required. Thus, 2OH-BNPP1 may not be an effective Bub1 inhibitor in cellulo , and while BAY-320 can inhibit Bub1 in cells, off-target effects highlight the need for improved Bub1 inhibitors.

NuMA regulates mitotic spindle assembly, structural dynamics and function via phase separation

A functional mitotic spindle is essential for accurate chromosome congression and segregation during cell proliferation; however, the underlying mechanisms of its assembly remain unclear. Here we show that NuMA regulates this assembly process via phase separation regulated by Aurora A. NuMA undergoes liquid-liquid phase separation during mitotic entry and KifC1 facilitates NuMA condensates concentrating on spindle poles. Phase separation of NuMA is mediated by its C-terminus, whereas its dynein-dynactin binding motif also facilitates this process. Phase-separated NuMA droplets concentrate tubulins, bind microtubules, and enrich crucial regulators, including Kif2A, at the spindle poles, which then depolymerizes spindle microtubules and promotes poleward spindle microtubule flux for spindle assembly and structural dynamics. In this work, we show that NuMA orchestrates mitotic spindle assembly, structural dynamics and function via liquid-liquid phase separation regulated by Aurora A phosphorylation.

Pak2 reduction induces a failure of early embryonic development in mice

Background The quality of the early embryo is vital to embryonic development and implantation. As a highly conserved serine/threonine kinase, p21-activated kinase 2 (Pak2) participates in diverse biologic processes, especially in cytoskeleton remodeling and cell apoptosis. In mice, Pak2 knock out and endothelial depletion of Pak2 showed embryonic lethality. However, the role of Pak2 in preimplantation embryos remains unelucidated. Methods In the present work, Pak2 was reduced using a specific small interfering RNA in early mouse embryos, validating the unique roles of Pak2 in spindle assembly and DNA repair during mice early embryonic development. We also employed immunoblotting, immunostaining, in vitro fertilization (IVF) and image quantification analyses to test the Pak2 knockdown on the embryonic development progression, spindle assembly, chromosome alignment, oxidative stress, DNA lesions and blastocyst cell apoptosis. Areas in chromatin with γH2AX were detected by immunofluorescence microscopy and serve as a biomarker of DNA damages. Results We found that Pak2 knockdown significantly reduced blastocyst formation of early embryos. In addition, Pak2 reduction led to dramatically increased abnormal spindle assembly and chromosomal aberrations in the embryos. We noted the overproduction of reactive oxygen species (ROS) with Pak2 knockdown in embryos. In response to DNA double strand breaks (DSBs), the histone protein H2AX is specifically phosphorylated at serine139 to generate γH2AX, which is used to quantitative DSBs. In this research, Pak2 knockdown also resulted in the accumulation of phosphorylated γH2AX, indicative of increased embryonic DNA damage. Commensurate with this, a significantly augmented rate of blastocyst cell apoptosis was detected in Pak2-KD embryos compared to their controls. Conclusions Collectively, our data suggest that Pak2 may serve as an important regulator of spindle assembly and DNA repair, and thus participate in the development of early mouse embryos.