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Smits A.H.,Universiteitsweg 100 | Jansen P.W.T.C.,Universiteitsweg 100 | Poser I.,Max Planck Institute of Molecular Cell Biology and Genetics | Hyman A.A.,Max Planck Institute of Molecular Cell Biology and Genetics | Vermeulen M.,Universiteitsweg 100
Nucleic Acids Research | Year: 2013

Many cellular proteins assemble into macromolecular protein complexes. The identification of protein-protein interactions and quantification of their stoichiometry is therefore crucial to understand the molecular function of protein complexes. Determining the stoichiometry of protein complexes is usually achieved by mass spectrometry-based methods that rely on introducing stable isotope-labeled reference peptides into the sample of interest. However, these approaches are laborious and not suitable for high-throughput screenings. Here, we describe a robust and easy to implement label-free relative quantification approach that combines the detection of high-confidence protein-protein interactions with an accurate determination of the stoichiometry of the identified protein-protein interactions in a single experiment. We applied this method to two chromatin-associated protein complexes for which the stoichiometry thus far remained elusive: the MBD3/NuRD and PRC2 complex. For each of these complexes, we accurately determined the stoichiometry of the core subunits while at the same time identifying novel interactors and their stoichiometry. © 2012 The Author(s). Published by Oxford University Press.


Kuijt T.E.F.,University Utrecht | Omerzu M.,University Utrecht | Saurin A.T.,University Utrecht | Saurin A.T.,University of Dundee | And 2 more authors.
Chromosoma | Year: 2014

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. © 2014 The Author(s).


Szypowska A.A.,University Utrecht | Burgering B.M.T.,University Utrecht | Burgering B.M.T.,Universiteitsweg 100
Antioxidants and Redox Signaling | Year: 2011

Recent compelling data show that reactive oxygen species (ROS) not only are a harmful by-product of aerobic metabolism, but also are used as signaling molecules to regulate various cellular processes. In mammalian cells, ROS are produced transiently in response to many extracellular stimuli, including insulin, and specific inhibition of the ROS suppresses insulin-dependent signaling. Initially, this finding rationalized the concept of ROS acting as insulin mimetics. However, it is becoming evident that ROS are also causal to diabetes, a metabolic disorder characterized by insufficiency of secretion of, or receptor insensitivity to, endogenous insulin. This notion underlines a dual role for ROS in insulin signaling as both deleterious and beneficiary. Moreover, it strongly suggests that a delicate redox balance is required for insulin signaling to remain "healthy" for an organism. © 2011 Mary Ann Liebert, Inc.


Ohl F.,University Utrecht | Ohl F.,Universiteitsweg 100 | van der Staay F.J.,University Utrecht | van der Staay F.J.,Universiteitsweg 100
Veterinary Journal | Year: 2012

The general concept of animal welfare embraces a continuum between negative/bad welfare and positive/good welfare. Early approaches to defining animal welfare were mainly based on the exclusion of negative states, neglecting the fact that during evolution animals optimised their ability to interact with and adapt to their environment(s). An animal's welfare status might best be represented by the adaptive value of the individual's interaction with a given environmental setting but this dynamic welfare concept has significant implications for practical welfare assessments.Animal welfare issues cannot simply be addressed by means of objective biological measurements of an animal's welfare status under certain circumstances. In practice, interpretation of welfare status and its translation into the active management of perceived welfare issues are both strongly influenced by context and, especially, by cultural and societal values. In assessing whether or not a given welfare status is morally acceptable, animal welfare scientists must be aware that scientifically based, operational definitions of animal welfare will necessarily be influenced strongly by a given society's moral understanding. © 2011 Elsevier Ltd.


Basten S.G.,Universiteitsweg 100 | Basten S.G.,University Utrecht | Giles R.H.,University Utrecht
Cilia | Year: 2013

Dysfunctional cilia underlie a broad range of cellular and tissue phenotypes and can eventually result in the development of ciliopathies: pathologically diverse diseases that range from clinically mild to highly complex and severe multi-organ failure syndromes incompatible with neonatal life. Given that virtually all cells of the human body have the capacity to generate cilia, it is likely that clinical manifestations attributed to ciliary dysfunction will increase in the years to come. Disputed but nevertheless enigmatic is the notion that at least a subset of tumor phenotypes fit within the ciliopathy disease spectrum and that cilia loss may be required for tumor progression. Contending for the centrosome renders ciliation and cell division mutually exclusive; a regulated tipping of balance promotes either process. The mechanisms involved, however, are complex. If the hypothesis that tumorigenesis results from dysfunctional cilia is true, then why do the classic ciliopathies only show limited hyperplasia at best? Although disassembly of the cilium is a prerequisite for cell proliferation, it does not intrinsically drive tumorigenesis per se. Alternatively, we will explore the emerging evidence suggesting that some tumors depend on ciliary signaling. After reviewing the structure, genesis and signaling of cilia, the various ciliopathy syndromes and their genetics, we discuss the current debate of tumorigenesis as a ciliopathy spectrum defect, and describe recent advances in this fascinating field. © 2013 Basten and Giles; licensee BioMed Central Ltd.


Van Kouwenhove M.,Netherlands Cancer Institute | Kedde M.,Netherlands Cancer Institute | Agami R.,Netherlands Cancer Institute | Agami R.,Universiteitsweg 100
Nature Reviews Cancer | Year: 2011

Non-protein-coding transcripts have been conserved throughout evolution, indicating that crucial functions exist for these RNAs. For example, microRNAs (miRNAs) have been found to modulate most cellular processes. The protein classes of RNA-binding proteins include essential regulators of miRNA biogenesis, turnover and activity. RNA-RNA and protein-RNA interactions are essential for post-transcriptional regulation in normal development and may be deregulated in disease. In reviewing emerging concepts of the interplay between miRNAs and RNA-binding proteins, we highlight the implications of these complex layers of regulation in cancer initiation and progression. © 2011 Macmillan Publishers Limited. All rights reserved.


Elkon R.,Netherlands Cancer Institute | Drost J.,Netherlands Cancer Institute | van Haaften G.,Netherlands Cancer Institute | Jenal M.,Netherlands Cancer Institute | And 4 more authors.
Genome Biology | Year: 2012

Background: The majority of mammalian genes contain multiple poly(A) sites in their 3' UTRs. Alternative cleavage and polyadenylation are emerging as an important layer of gene regulation as they generate transcript isoforms that differ in their 3' UTRs, thereby modulating genes' response to 3' UTR-mediated regulation. Enhanced cleavage at 3' UTR proximal poly(A) sites resulting in global 3' UTR shortening was recently linked to proliferation and cancer. However, mechanisms that regulate this enhanced alternative polyadenylation are unknown.Results: Here, we explored, on a transcriptome-wide scale, alternative polyadenylation events associated with cellular proliferation and neoplastic transformation. We applied a deep-sequencing technique for identification and quantification of poly(A) sites to two human cellular models, each examined under proliferative, arrested and transformed states. In both cell systems we observed global 3' UTR shortening associated with proliferation, a link that was markedly stronger than the association with transformation. Furthermore, we found that proliferation is also associated with enhanced cleavage at intronic poly(A) sites. Last, we found that the expression level of the set of genes that encode for 3'-end processing proteins is globally elevated in proliferation, and that E2F transcription factors contribute to this regulation.Conclusions: Our results comprehensively identify alternative polyadenylation events associated with cellular proliferation and transformation, and demonstrate that the enhanced alternative polyadenylation in proliferative conditions results not only in global 3' UTR shortening but also in enhanced premature cleavage in introns. Our results also indicate that E2F-mediated co-transcriptional regulation of 3'-end processing genes is one of the mechanisms that links enhanced alternative polyadenylation to proliferation. © 2012 Elkon et al.; licensee BioMed Central Ltd.


Nijenhuis W.,Universiteitsweg 100 | Vallardi G.,University of Dundee | Teixeira A.,Universiteitsweg 100 | Kops G.J.P.L.,Universiteitsweg 100 | Saurin A.T.,University of Dundee
Nature Cell Biology | Year: 2014

Kinetochores are specialized multi-protein complexes that play a crucial role in maintaining genome stability. They bridge attachments between chromosomes and microtubules during mitosis and they activate the spindle assembly checkpoint (SAC) to arrest division until all chromosomes are attached. Kinetochores are able to efficiently integrate these two processes because they can rapidly respond to changes in microtubule occupancy by switching localized SAC signalling ON or OFF. We show that this responsiveness arises because the SAC primes kinetochore phosphatases to induce negative feedback and silence its own signal. Active SAC signalling recruits PP2A-B56 to kinetochores where it antagonizes Aurora B to promote PP1 recruitment. PP1 in turn silences the SAC and delocalizes PP2A-B56. Preventing or bypassing key regulatory steps demonstrates that this spatiotemporal control of phosphatase feedback underlies rapid signal switching at the kinetochore by: allowing the SAC to quickly transition to the ON state in the absence of antagonizing phosphatase activity; and ensuring phosphatases are then primed to rapidly switch the SAC signal OFF when kinetochore kinase activities are diminished by force-producing microtubule attachments. © 2014 Macmillan Publishers Limited. All rights reserved.


Nijenhuis W.,Universiteitsweg 100
Nature Cell Biology | Year: 2014

Kinetochores are specialized multi-protein complexes that play a crucial role in maintaining genome stability. They bridge attachments between chromosomes and microtubules during mitosis and they activate the spindle assembly checkpoint (SAC) to arrest division until all chromosomes are attached. Kinetochores are able to efficiently integrate these two processes because they can rapidly respond to changes in microtubule occupancy by switching localized SAC signalling ON or OFF. We show that this responsiveness arises because the SAC primes kinetochore phosphatases to induce negative feedback and silence its own signal. Active SAC signalling recruits PP2A-B56 to kinetochores where it antagonizes Aurora B to promote PP1 recruitment. PP1 in turn silences the SAC and delocalizes PP2A-B56. Preventing or bypassing key regulatory steps demonstrates that this spatiotemporal control of phosphatase feedback underlies rapid signal switching at the kinetochore by: allowing the SAC to quickly transition to the ON state in the absence of antagonizing phosphatase activity; and ensuring phosphatases are then primed to rapidly switch the SAC signal OFF when kinetochore kinase activities are diminished by force-producing microtubule attachments. © 2014 Nature Publishing Group


Suijkerbuijk S.,Universiteitsweg 100 | Vleugel M.,Universiteitsweg 100 | Teixeira A.,Universiteitsweg 100 | Kops G.,Universiteitsweg 100
Developmental Cell | Year: 2012

Maintenance of chromosomal stability depends on error-free chromosome segregation. The pseudokinase BUBR1 is essential for this, because it is a core component of the mitotic checkpoint and is required for formation of stable kinetochore-microtubule attachments. We have identified a conserved and highly phosphorylated domain (KARD) in BUBR1 that is crucial for formation of kinetochore-microtubule attachments. Deletion of this domain or prevention of its phosphorylation abolishes formation of kinetochore microtubules, which can be reverted by inhibiting Aurora B activity. Phosphorylation of KARD by PLK1 promotes direct interaction of BUBR1 with the PP2A-B56α phosphatase that counters excessive Aurora B activity at kinetochores. As a result, removal of BUBR1 from mitotic cells or inhibition of PLK1 reduces PP2A-B56α kinetochore binding and elevates phosphorylation of Aurora B substrates on the outer kinetochore. We propose that PLK1 and BUBR1 cooperate to stabilize kinetochore-microtubule interactions by regulating PP2A-B56α-mediated dephosphorylation of Aurora B substrates at the kinetochore-microtubule interface. Error-free chromosome segregation relies on stabilization of kinetochore-microtubule attachments by the pseudokinase BUBR1. Suijkerbuijk et al. show that PLK1 promotes interaction of BUBR1 with PP2A-B56, leading to dampening of the microtubule-destabilizing activity of Aurora B kinase at kinetochores. BUBR1 thus promotes stable kinetochore-microtubule attachments by integrating PLK1 and PP2A signaling. © 2012 Elsevier Inc.

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