Mps1-dependent phosphorylation of the kinetochore protein Ndc80 is an intrinsic step in spindle checkpoint activation

The kinetochore is a multi-protein complex which supports chromosome segregation in mitosis by mediating the connection between centromeric DNA and the mitotic spindle. In addition, it serves as a docking platform for the spindle checkpoint proteins. The spindle checkpoint is a surveillance mechanism which monitors the proper assembly of the mitotic spindle apparatus. It allows the transition from meta- into anaphase only if all kinetochores are bipolarly attached to the spindle microtubules and if tension across the spindle is applied. The tetrameric Ndc80-complex (Ndc80, Nuf2, Spc24, Spc25) is part of the kinetochore. It is highly conserved and was shown to be required for the spindle checkpoint in S. cerevisiae (Janke et al. 2001). The kinase Mps1 has also been implicated in spindle checkpoint control (Hartwick et al. 1996). In mammalian cells, the kinetochore localisation of Mps1 depends on the presence of the Ndc80-complex (Stucke et al. 2004). Furthermore, endogenous Mps1 was found as a faint Coomassie-stained protein band in a Ndc80-complex pull down from yeast (C. Jaeger, unpublished results). Based on these observations, the goal of the present studies was to elucidate whether components of the S. cerevisiae Ndc80-complex are phosphorylated by Mps1 and if this phosphorylation has an influence on the regulation of the spindle checkpoint. The following observations were made: 1. Mps1 physically interacts with Ndc80 ∑ Mps1 weakly associates with the kinetochore by ChIP-analysis in S. cerevisiae. ∑ Mps1 physically interacts with the Ndc80-complex after spindle checkpoint activation by Mps1-overexpression. ∑ Mps1 specifically interacts with the conserved, globular N-terminus of Ndc80 in vitro. 2. Mps1 phosphorylates Ndc80 ∑ Mps1 phosphorylates the N-terminus of Ndc80 in vitro. ∑ Ndc80-phosphorylation depends on Mps1 in vivo. 3. Ndc80-phosphorylation activates the spindle assembly checkpoint ∑ Ndc80 in its non-phosphorylated state (ndc8014A) is checkpoint deficient. ∑ constitutive pseudo-phosphorylation of Ndc80 (ndc8014D) causes lethality due to a permanently activated spindle checkpoint (cell cycle arrest in mitosis). This checkpoint activation specifically depends on the checkpoint proteins Mad2 and Bub1. fi Ndc80-phosphorylation by Mps1 is an integral step in the spindle checkpoint pathway! 4. The kinetochore of ndc8014D-cells is functional ∑ The expression levels and the kinetochore localisation of the ndc8014D protein are comparable to the wild-type protein. ∑ The ndc8014D-protein is able to compete with the wild-type protein for binding-sites at the kinetochore. ∑ the spindle in ndc8014D-cells is under tension, a state which can only be achieved in the presence of an intact kinetochore. fi the observed checkpoint activation is specifically caused by the introduced point mutations rather than by a general kinetochore defect. 5. Mps1-activity is required downstream of Ndc80 ∑ The ndc8014D mutant is unable to activate the spindle checkpoint in Mps1-depleted cells. fi Mps1 has a second function in spindle checkpoint activation downstream of Ndc80-phosphorylation which might involve the phosphorylation of the checkpoint protein Mad1. The results which were obtained in the current work provide evidence that Mps1 specifically phosphorylates Ndc80 and that Ndc80-phosphorylation is a mechanism by which the spindle checkpoint is activated in the presence of unattached kinetochores in early stages of mitosis. The functional characterisation of Ndc80-phosphorylation allows for a refinement of the current model of the spindle assembly checkpoint (Figure 32)

Magnetic hysteresis effects in superconducting coplanar microwave resonators

We performed transmission spectroscopy experiments on coplanar half wavelength niobium resonators at a temperature T=4.2 K. We observe not only a strong dependence of the quality factor Q and the resonance frequency f_res on an externally applied magnetic field but also on the magnetic history of our resonators, i.e. on the spatial distribution of trapped Abrikosov vortices in the device. We find these results to be valid for a broad range of frequencies and angles between the resonator plane and the magnetic field direction as well as for resonators with and without antidots near the edges of the center conductor and the ground planes. In a detailed analysis we show, that characteristic features of the experimental data can only be reproduced in calculations, if a highly inhomogeneous rf-current density and a flux density gradient with maxima at the edges of the superconductor is assumed. We furthermore demonstrate, that the hysteretic behaviour of the resonator properties can be used to considerably reduce the vortex induced losses and to fine-tune the resonance frequency by the proper way of cycling to a desired magnetic field

ERA - Energy-based reliability analysis - Energiebasierte Zuverlässigkeitsanalyse

Da die Wechselwirkungen zwischen mechatronischen Komponenten in Systemen eine entscheidende Rolle auf ihre Belastung einnehmen, ist die Betrachtung dieser Wechselwirkungen un- verzichtbar. Zur Identifikation solcher Wechselwirkungen ist eine ergänzende Methode zur den bisher klassischen Systemanalysen von Nöten. Dies wird bei der vorgestellten energiebasierten Zuverlässigkeitsanalyse (engl. Energy-based Reliability Analysis - ERA) berücksichtigt, indem die stationären Energie- beziehungsweise die dynamischen Leistungsflüsse mechatronischer Systemen in Form von Energieflussdiagrammen dargestellt werden. Mit der Modellierung des Energieflusses und damit das Ansetzen des ERA-Verfahrens kann der Nutzer Wirkzusammenhänge und Schwachstellen erkennen, eine exaktere Bestimmung der Zuverlässigkeit durch Berechnung der Belastung erreichen und folglich Komponenten zuverlässigkeitsbasiert auslegen.The interactions between components in mechatronic systems play a crucial role on the components’ load. This indispensably demands the consideration of these interactions regarding a reliability analysis. In order to identify such interactions a method complementing already existing and well established system analyses is needed. The method presented here called "Energy-based Reliability Analysis (ERA)" takes into account the stationary as well as the dynamic power flows in mechatronic systems. Energy flow diagrams illustrate these flows. By means of the modeling of the energy flux and thus of the implementation of the ERA process, the user can recognize cause-effect relationships and vulnerabilities. The individual component’s as well as the entire system’s reliability can therefore be described more accurately based on a more precise load calculation

Non-FG mediated transport of the large pre-ribosomal subunit through the nuclear pore complex by the mRNA export factor Gle2

Multiple export receptors passage bound pre-ribosomes through nuclear pore complexes (NPCs) by transiently interacting with the Phe-Gly (FG) meshwork of their transport channels. Here, we reveal how the non-FG interacting yeast mRNA export factor Gly-Leu-FG lethal 2 (Gle2) functions in the export of the large pre-ribosomal subunit (pre-60S). Structure-guided studies uncovered conserved platforms used by Gle2 to export pre-60S: an uncharacterized basic patch required to bind pre-60S, and a second surface that makes non-FG contacts with the nucleoporin Nup116. A basic patch mutant of Gle2 is able to function in mRNA export, but not pre-60S export. Thus, Gle2 provides a distinct interaction platform to transport pre-60S to the cytoplasm. Notably, Gle2's interaction platforms become crucial for pre-60S export when FG-interacting receptors are either not recruited to pre-60S or are impaired. We propose that large complex cargos rely on non-FG as well as FG-interactions for their efficient translocation through the nuclear pore complex channe

Method for the development of a functional adaptive simulation model for designing robust products

Products have to ensure their function under the inuence of internal and external noise factors in order to remain competitive in the current market. Therefore the step of designing robust products should be integrated in early stages of the Product Development Process (PDP). Robust products are developed using the Robust Design Method SMART (Systematic Method for Axiomatic Robustness-Testing). Thus far, SMART was applied and veri ed based on a simple mechanical machine element. In this paper, the method will be applied to a complex technical system. Additionally, the confict of aiming between the high e orts and the level of detail in the creation of a simulation model are discussed. This confict is brought about owing to the complex functionality of the design. In order to solve the conict, an approach is given for the creation of an adjusted simulation model. Short simulation times are an advantage for the analysis of parameters regarding robustness. The adaptive simulation model discussed in this paper is based on a exible and equation-based model, which is extended with local -structural-mechanical SUB-models for a more detailed analysis. This approach o ers the option of obtaining rst insights about the functionality of the product and the opportunity to complement the simulation model iteratively for the following design phases. This approach complements SMART on the one hand in the simulative design of robust design parameters and, on the other hand, in their reliability prediction in both the Parameter Design and Tolerance Design phase

Lebensdaueranalyse auf Basis von multidimensionaler Zuverlässigkeits- und Robust Design Simulation : integrale Betrachtung der robusten Zuverlässigkeit

Konventionelle Methoden des Robust Design verfolgen größtenteils Ansätze zur Varianzbetrachtung, die potentiell über der Produkteinsatzzeit auftreten können. Daher geben diese Methoden keine genauen Informationen über das zeitlich funktionale Verhalten der sich verändernden Produkteigenschaften und -anforderungen sowie deren Lebensdauer. Um genaue Lebensdauerprognosen bezüglich dieser Funktionserfüllung zu beschreiben, ist eine kontinuierliche Merkmalsänderung in Form von sogenannten Degradationsmodellen von Vorteil. Diese werden im Allgemeinen durch reale Versuche mit einem hohen Grad an zeitlichem und kostenintensivem Aufwand durchgeführt. Für eine effizientere Ermittlung der Modelle sollten bereits in frühen Phasen des Produktentwicklungsprozesses, virtuelle Degradationsmodelle entwickelt werden. Durch die genaue Kennung von Funktionsausfällen über der Zeit können nicht nur Produkte zielgerichtet ausgelegt, sondern auch unnötige Ressourcen eingespart werden

Comparison of Taguchi Method and Robust Design Optimization (RDO) : by application of a functional adaptive simulation model for the robust product-optimization of an adjuster unit

Current research and development have been trending towards approaches based on simulation and virtual testing. Industrial development processes for complex products employ optimization methods to ensure results are close to reality, simultaneously minimizing required resources. The results of virtual testing are optimized in accordance with requirements using optimization techniques. Robust Design Optimization (RDO) is one established approach to optimization. RDO is based on the identification of an optimal parameter set which includes a small variance of the target value as a constraint. Under most circumstances, this approach does not involve separate optimization of the target value and target variance. However, the basic strategy of the optimization approach developed by Taguchi is to first optimize the parameter sets for the target value and then optimize and minimize the target variance. According to an application example , the benefit of Taguchi's approach (TM) is that it facilitates the identification of an optimal parameter set of nominal values for technical feasibility and possible manufacturing. If an optimal parameter set is determined, the variance can be minimized under consideration of process parameters. This paper examines and discusses the differences between and shared characteristics of the robust optimization methods TM and RDO, and discusses their shortcomings. In order to provide a better illustration, this paper explains and applies both methods using an adjuster unit of a commercial vehicle braking system. A simulation model is developed including an appropriate work ow by applying optiSLang-modules

Robust Design Methodology for the development of commercial vehicle braking systems

Today’s product requirements demand an ever increasing functionality for the same space and usually the same number of components. Thereby, the quality, reliability and robustness of these products should be preserved or even be increased. This target conflict cannot be solved without compromises. The research community between the Institute of Machine Components (IMA), University of Stuttgart, and the Knorr-Bremse Systeme für Nutzfahrzeuge GmbH is seeking for new solutions for these challenges. The new approaches for designing robust and reliable products are being implemented directly in a current development project of an innovative Air Disc Brake (ADB). With “Systematic Method for Axiomatic Robustness-Testing” (SMART), reliability methods and the basic concept of Robust Design methodology are related to the Taguchi Method. SMART is based on three phases: System, Parameter and Tolerance Design; accordingly, the sample phases of VDA (Association of German Automotives) are used as milestones. In the System Design, SMART focuses on the decreasing complexity according to the functional dependences of the DPs, thus precluding early random failures. In the Parameter Design phase, SMART gives the developer an approach for modeling an adaptive simulation model (SIM-SMART). This model also enables the simulation of random and possible fatigue failures in addition to the nominally robust DPs. In the early stage of product development, reliability predictions are possible. In the iterative Tolerance Design phase, the final tolerance limits for robust and reliable products are defined with consideration of compromises in terms of costs, quality and technical feasibility. With the application of SMART, a design concept of a new generation of an ADB with less complexity is created. The extensive functions for flexible function studies are modeled with the objective of SIM-SMART. Accordingly to this model, parameter studies for determination of the nominal adjustment levels can be performed and their random and fatigue failures modeled. In conclusion, more accurate reliability test strategies are recommended using the definition of tolerance limits. The cost aspect and technical feasibility are also taken into account. So far, SMART has not been added to the iterative Tolerance Design phase. With this paper, the method is not only extended to this phase, but also sufficiently validated. In addition, SMART can predict and analyze random failures. With its three coherent and iterative phases, it is an as yet unpublished and unimplemented approach for designing even more robust and reliable products. Robust Design Methodology and reliability methods are fundamental building blocks for products with high quality requirements. SMART presents an approach to support the designing of robust, reliable, highly functional and innovative ADB

Sem1 is a functional component of the nuclear pore complex–associated messenger RNA export machinery

The evolutionarily conserved protein Sem1/Dss1 is a subunit of the regulatory particle (RP) of the proteasome, and, in mammalian cells, binds the tumor suppressor protein BRCA2. Here, we describe a new function for yeast Sem1. We show that sem1 mutants are impaired in messenger RNA (mRNA) export and transcription elongation, and induce strong transcription-associated hyper-recombination phenotypes. Importantly, Sem1, independent of the RP, is functionally linked to the mRNA export pathway. Biochemical analyses revealed that, in addition to the RP, Sem1 coenriches with components of two other multisubunit complexes: the nuclear pore complex (NPC)-associated TREX-2 complex that is required for transcription-coupled mRNA export, and the COP9 signalosome, which is involved in deneddylation. Notably, targeting of Thp1, a TREX-2 component, to the NPC is perturbed in a sem1 mutant. These findings reveal an unexpected nonproteasomal function of Sem1 in mRNA export and in prevention of transcription-associated genome instability. Thus, Sem1 is a versatile protein that might stabilize multiple protein complexes involved in diverse pathways.España, Ministerio de Educación y Ciencia BFU2006-05260 and BFU2007-28647Andalucia, Junta de Andalucia CVI102 and CVI254