Studying kinetochore kinases

Mitotic kinetochores are signaling network hubs that regulate chromosome movements, attachment error-correction, and the spindle assembly checkpoint. Key switches in these networks are kinases and phosphatases that enable rapid responses to changing conditions. Describing the mechanisms and dynamics of their localized activation and deactivation is therefore instrumental for understanding the spatiotemporal control of chromosome segregation

Aurora B potentiates Mps1 activation to ensure rapid checkpoint establishment at the onset of mitosis

The mitotic checkpoint prevents mitotic exit until all chromosomes are attached to spindle microtubules. Aurora B kinase indirectly invokes this checkpoint by destabilizing incorrect attachments; however, a more direct role remains controversial. In contrast, activity of the kinase Mps1 is indispensible for the mitotic checkpoint. Here we show that Aurora B and Hec1 are needed for efficient Mps1 recruitment to unattached kinetochores, allowing rapid Mps1 activation at the onset of mitosis. Live monitoring of cyclin B degradation reveals that this is essential to establish the mitotic checkpoint quickly at the start of mitosis. Delayed Mps1 activation and checkpoint establishment upon Aurora B inhibition or Hec1 depletion are rescued by tethering Mps1 to kinetochores, demonstrating that Mps1 recruitment is the primary role of Aurora B and Hec1 in mitotic checkpoint signalling. These data demonstrate a direct role for Aurora B in initiating the mitotic checkpoint rapidly at the onset of mitosis

Conditional targeting of MAD1 to kinetochores is sufficient to reactivate the spindle assembly checkpoint in metaphase

Fidelity of chromosome segregation is monitored by the spindle assembly checkpoint (SAC). Key components of the SAC include MAD1, MAD2, BUB1, BUB3, BUBR1, and MPS1. These proteins accumulate on kinetochores in early prometaphase but are displaced when chromosomes attach to microtubules and/or biorient on the mitotic spindle. As a result, stable attachment of the final chromosome satisfies the SAC, permitting activation of the anaphase promoting complex/cyclosome (APC/C) and subsequent anaphase onset. SAC satisfaction is reversible, however, as addition of taxol during metaphase stops cyclin B1 degradation by the APC/C. We now show that targeting MAD1 to kinetochores during metaphase is sufficient to reestablish SAC activity after initial silencing. Using rapamycin-induced heterodimerization of FKBP-MAD1 to FRB-MIS12 and live monitoring of cyclin B1 degradation, we show that timed relocalization of MAD1 during metaphase can stop cyclin B1 degradation without affecting chromosome-spindle attachments. APC/C inhibition represented true SAC reactivation, as FKBP-MAD1 required an intact MAD2-interaction motif and MPS1 activity to accomplish this. Our data show that MAD1 kinetochore localization dictates SAC activity and imply that SAC regulatory mechanisms downstream of MAD1 remain functional in metaphase. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00412-014-0458-9) contains supplementary material, which is available to authorized users

Quantitative Mass Spectrometry Analysis Reveals Similar Substrate Consensus Motif for Human Mps1 Kinase and Plk1

Background Members of the Mps1 kinase family play an essential and evolutionarily conserved role in the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures accurate chromosome segregation during mitosis. Human Mps1 (hMps1) is highly phosphorylated during mitosis and many phosphorylation sites have been identified. However, the upstream kinases responsible for these phosphorylations are not presently known. Methodology/Principal Findings Here, we identify 29 in vivo phosphorylation sites in hMps1. While in vivo analyses indicate that Aurora B and hMps1 activity are required for mitotic hyper-phosphorylation of hMps1, in vitro kinase assays show that Cdk1, MAPK, Plk1 and hMps1 itself can directly phosphorylate hMps1. Although Aurora B poorly phosphorylates hMps1 in vitro, it positively regulates the localization of Mps1 to kinetochores in vivo. Most importantly, quantitative mass spectrometry analysis demonstrates that at least 12 sites within hMps1 can be attributed to autophosphorylation. Remarkably, these hMps1 autophosphorylation sites closely resemble the consensus motif of Plk1, demonstrating that these two mitotic kinases share a similar substrate consensus. Conclusions/Significance hMps1 kinase is regulated by Aurora B kinase and its autophosphorylation. Analysis on hMps1 autophosphorylation sites demonstrates that hMps1 has a substrate preference similar to Plk1 kinase

Mps1Mph1 kinase phosphorylates Mad3 to inhibit Cdc20Slp1-APC/C and maintain spindle checkpoint arrests

<div><p>The spindle checkpoint is a mitotic surveillance system which ensures equal segregation of sister chromatids. It delays anaphase onset by inhibiting the action of the E3 ubiquitin ligase known as the anaphase promoting complex or cyclosome (APC/C). Mad3/BubR1 is a key component of the mitotic checkpoint complex (MCC) which binds and inhibits the APC/C early in mitosis. Mps1^Mph1 kinase is critical for checkpoint signalling and MCC-APC/C inhibition, yet few substrates have been identified. Here we identify Mad3 as a substrate of fission yeast Mps1^Mph1 kinase. We map and mutate phosphorylation sites in Mad3, producing mutants that are targeted to kinetochores and assembled into MCC, yet display reduced APC/C binding and are unable to maintain checkpoint arrests. We show biochemically that Mad3 phospho-mimics are potent APC/C inhibitors <i>in vitro</i>, demonstrating that Mad3p modification can directly influence Cdc20^Slp1-APC/C activity. This genetic dissection of APC/C inhibition demonstrates that Mps1^Mph1 kinase-dependent modifications of Mad3 and Mad2 act in a concerted manner to maintain spindle checkpoint arrests.</p></div

Probing MPS1 function in mitosis

The importance of MPS1 in mitosis has been established for several years. Even though recent years have seen quite some development in the elucidation of the molecular pathways concerning MPS1 signaling, many aspects remained to be uncovered at the onset of the research presented in this thesis. In Chapter 2 we report the development of mutant cell lines that allow specific, highly penetrant and reversible inhibition of MPS1, providing us with the opportunity to study the role of MPS1 in mitosis. One of these functions - the recruitment of MAD1 to unattached kinetochores - is studied in more detail in Chapter 3. We provide evidence that feedback control between MPS1, BUB1 and ZW10 regulates MAD1 kinetochore recruitment and subsequent SAC activity. In this chapter, we furthermore show that the predominant function for RZZ and BUB1 in SAC signaling is ensuring MAD1 localization. Chapter 4 describes the identification of PLK1 as an auxiliary factor in the establishment and maintenance of the SAC. Under conditions of maximal SAC activity, PLK1 is dispensable, but when SAC signaling is suboptimal, PLK1 becomes essential to maintain SAC signaling. Our data suggest that under these conditions PLK1 activity promotes MCC stability. The work presented in Chapter 5 describes the efforts put into identification of direct MPS1 targets and provides useful lessons for future investigation in the search for novel MPS1 targets

Throwing it out there:Grip on multidimensional performance characteristics of judoka – a systematic review

Judo is a dynamic and complex sport, and therefore talent programs may benefit from a multidimensional approach to identify clear and reliable performance characteristics in order to monitor and train athletes. However, from both a practical and theoretical point of view, understanding on this topic is limited. This systematic review therefore aimed to (1) identify which multidimensional performance characteristics can discriminate between different performance levels of judoka and (2) find the gaps in literature. To categorize the findings the Groningen Sport Talent Model (GSTM) was applied. A systematic search of MEDLINE, PsycINFO, PubMed, and Web of Science was performed following the PRISMA guidelines. In total 23 studies with good to excellent methodological quality were included. All performance characteristics of the GSTM (anthropometrical, physiological, technical, tactical, and psychological) were represented in the included studies. There is strong evidence to suggest that a broad set of physiological characteristics is needed to manage the demands of judo combats. Additionally, there is strong evidence that performance characteristics related to grip fighting discriminate between judoka of different performance levels and higher performance level judoka are characterized by the ability to throw their opponent and to variate in technique. The findings of this review can be used as a basis for talent development and identification in judo, and to optimize training programs. Future research should focus on increasing the understanding of psychological characteristics of judoka and the multidimensional talent trajectory by conducting longitudinal studies.</p

Data accompanying the paper: Is Io's volcanic distribution controlled by a convective layer?

Two sets of Io's volcanic features locations adopted from previous publications, fitted and filtered for the comparision with interior convection dynamics. Set-1 ('active'): Based on active hot-spots (de Kleer et al. 2019, Mura et al. 2020, Davies et al., 2015) Set-2 ('hamilton'): Based on data set 1 and 2 in supplementary material of Hamilton et al, 2013