CRUK London Research Institute

London, United Kingdom

CRUK London Research Institute

London, United Kingdom
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Vladimirou E.,University of Warwick | Mchedlishvili N.,ETH Zurich | Mchedlishvili N.,University College London | Gasic I.,ETH Zurich | And 7 more authors.
Developmental Cell | Year: 2013

Kinetochores are the central force-generating machines that move chromosomes during cell division. It is generally assumed that kinetochores move in an autonomous manner. However, we reveal here that movements of neighboring sister-kinetochore pairs in metaphase are correlated in a distance-dependent manner. This correlation increases in the absence ofkinetochore oscillations or stable end-on attachments. This suggests that periodic movements of bioriented chromosomes limit the correlated motion of nonsisters. Computer simulations show that these correlated movements can occur when elastic crosslinks are placed between the K-fibers of oscillating kinetochores. Strikingly, inhibition of the microtubule crosslinking motor kinesin-5 Eg5 leads to an increase in nonsister correlation and impairs periodic oscillations. These phenotypes are partially rescued by codepletion of the kinesin-12 Kif15, demonstrating a function for kinesin-5 and kinesin-12 motors in driving chromosome movements, possibly as part of a crosslinking structure that correlates the movements of nonsister kinetochores. © 2013 Elsevier Inc.

Bancroft J.,University of Warwick | Auckland P.,University of Oxford | Samora C.P.,CRUK London Research Institute | McAinsh A.D.,University of Warwick
Journal of Cell Science | Year: 2015

A key step of mitosis is the congression of chromosomes to the spindle equator. Congression is driven by at least two distinct mechanisms: (1) kinetochores slide along the microtubule lattice using the plus-end directed CENP-E motor, and (2) kinetochores biorientating near the pole move to the equator through microtubule depolymerisation-coupled pulling. Here, we show that CENP-Q - a subunit of the CENP-O complex (comprising CENP-O, CENP-P, CENP-Q and CENP-U) that targets polo-like kinase (Plk1) to kinetochores - is also required for the recruitment of CENP-E to kinetochores. We further reveal a CENP-E recruitment-independent role for CENP-Q in depolymerisation-coupled pulling. Both of these functions are abolished by a single point mutation in CENP-Q (S50A) - a residue that is phosphorylated in vivo. Importantly, the S50A mutant does not affect the loading of Plk1 onto kinetochores and leaves the CENP-O complex intact. Thus, the functions of CENP-Q in CENP-E loading and depolymerisation-coupled pulling are independent from its role in Plk1 recruitment and CENP-O complex stabilisation. Taken together, our data provide evidence that phosphoregulation of CENP-Q plays a central function in coordinating chromosome congression mechanisms. © 2015.

Quintana-Bustamante O.,CRUK London Research Institute | Smith S.L.-L.,King's College London | Griessinger E.,CRUK London Research Institute | Reyal Y.,CRUK London Research Institute | And 4 more authors.
Leukemia | Year: 2012

CCAAT/enhancer-binding protein-α (C/EBPα/CEBPA) is mutated in approximately 8% of acute myeloid leukemia (AML) in both familial and sporadic AML and, with FLT3 and NPM1, has received most attention as a predictive marker of outcome in patients with normal karyotype disease. Mutations clustering to either the N-or C-terminal (N-and C-ter) portions of the protein have different consequences on the protein function. In familial cases, the N-ter form is inherited with patients exhibiting long latency period before the onset of overt disease, typically with the acquisition of a C-ter mutation. Despite the essential insights murine models provide the functional consequences of wild-type C/EBPα in human hematopoiesis and how different mutations are involved in AML development have received less attention. Our data underline the critical role of C/EBPα in human hematopoiesis and demonstrate that C/EBPα mutations (alone or in combination) are insufficient to convert normal human hematopoietic stem/progenitor cells into leukemic-initiating cells, although individually each altered normal hematopoiesis. It provides the first insight into the effects of N-and C-ter mutations acting alone and to the combined effects of N/C double mutants. Our results mimicked closely what happens in CEBPA mutated patients. © 2012 Macmillan Publishers Limited.

McGranahan N.,CRUK London Research Institute | McGranahan N.,University College London | Burrell R.A.,CRUK London Research Institute | Endesfelder D.,CRUK London Research Institute | And 4 more authors.
EMBO Reports | Year: 2012

Chromosomal instability (CIN)-which is a high rate of loss or gain of whole or parts of chromosomes-is a characteristic of most human cancers and a cause of tumour aneuploidy and intra-tumour heterogeneity. CIN is associated with poor patient outcome and drug resistance, which could be mediated by evolutionary adaptation fostered by intra-tumour heterogeneity. In this review, we discuss the clinical consequences of CIN and the challenges inherent to its measurement in tumour specimens. The relationship between CIN and prognosis supports assessment of CIN status in the clinical setting and suggests that stratifying tumours according to levels of CIN could facilitate clinical risk assessment. ©2012 European Molecular Biology Organization.

Mouilleron S.,CRUK London Research Institute | Wiezlak M.,CRUK London Research Institute | O'Reilly N.,CRUK London Research Institute | Treisman R.,CRUK London Research Institute | And 2 more authors.
Structure | Year: 2012

The Phactr family of PP1-binding proteins and the myocardin-related transcription factor family of transcriptional coactivators contain regulatory domains comprising three copies of the RPEL motif, a G-actin binding element. We report the structure of a Phactr1 G-actinṡRPEL domain complex. Three G-actins surround the crank-shaped RPEL domain forming a closed helical assembly. Their spatial relationship is identical to the RPEL-actins within the pentavalent MRTF G-actinṡRPEL domain complex, suggesting that conserved cooperative interactions between actinṡRPEL units organize the assembly. In the trivalent Phactr1 complex, each G-actinṡRPEL unit makes secondary contacts with its downstream actin involving distinct RPEL residues. Similar secondary contacts are seen in G-actinṡRPEL peptide crystals. Loss-of-secondary-contact mutations destabilize the Phactr1 G-actinṡRPEL assembly. Furthermore, actin-mediated inhibition of Phactr1 nuclear import requires secondary contact residues in the Phactr1 N-terminal RPEL-N motif, suggesting that it involves interaction of RPEL-N with the C-terminal assembly. Secondary actin contacts by actin-bound RPEL motifs thus govern formation of multivalent actinṡRPEL assemblies. © 2012 Elsevier Ltd All rights reserved.

Wiezlak M.,CRUK London Research Institute | Diring J.,CRUK London Research Institute | Abella J.,CRUK London Research Institute | Mouilleron S.,CRUK London Research Institute | And 4 more authors.
Journal of Cell Science | Year: 2012

The Phactr family of PP1-binding proteins is implicated in human diseases including Parkinson's, cancer and myocardial infarction. Each Phactr protein contains four G-actin binding RPEL motifs, including an N-terminal motif, abutting a basic element, and a Cterminal triple RPEL repeat, which overlaps a conserved C-terminus required for interaction with PP1. RPEL motifs are also found in the regulatory domains of the MRTF transcriptional coactivators, where they control MRTF subcellular localisation and activity by sensing signal-induced changes in G-actin concentration. However, whether G-actin binding controls Phactr protein function - and its relation to signalling - has not been investigated. Here, we show that Rho-actin signalling induced by serum stimulation promotes the nuclear accumulation of Phactr1, but not other Phactr family members. Actin binding by the three Phactr1 C-terminal RPEL motifs is required for Phactr1 cytoplasmic localisation in resting cells. Phactr1 nuclear accumulation is importin α-β dependent. G-actin and importin α-β bind competitively to nuclear import signals associated with the N- and C-terminal RPEL motifs. All four motifs are required for the inhibition of serum-induced Phactr1 nuclear accumulation when G-actin is elevated. G-actin and PP1 bind competitively to the Phactr1 C-terminal region, and Phactr1 C-terminal RPEL mutants that cannot bind G-actin induce aberrant actomyosin structures dependent on their nuclear accumulation and on PP1 binding. In CHL-1 melanoma cells, Phactr1 exhibits actin-regulated subcellular localisation and is required for stress fibre assembly, motility and invasiveness. These data support a role for Phactr1 in actomyosin assembly and suggest that Phactr1 G-actin sensing allows its coordination with F-actin availability. © 2012. Published by The Company of Biologists Ltd.

Taussig D.C.,Queen Mary, University of London | Taussig D.C.,CRUK London Research Institute | Vargaftig J.,CRUK London Research Institute | Miraki-Moud F.,Queen Mary, University of London | And 10 more authors.
Blood | Year: 2010

Leukemia-initiating cells (LICs) in acute myeloid leukemia (AML) are believed to be restricted to the CD34+ fraction. However, one of the most frequently mutated genes in AML is nucleophosmin (NPM), and this is associated with low CD34 expression. We, therefore, investigated whether NPM-mutated AMLs have LICs restricted to the CD34+ fraction.We transplanted sorted fractions of primary NPM-mutated AML into immunodeficient mice to establish which fractions initiate leukemia. Approximately one-half of cases had LICs exclusively within the CD34- fraction, whereas the CD34+ fraction contained normal multilineage hematopoietic repopulating cells. Most of the remaining cases had LICs in both CD34 + and CD34- fractions. When samples were sorted based on CD34 and CD38 expression, multiple fractions initiated leukemia in primary and secondary recipients. The data indicate that the phenotype of LICs is more heterogeneous than previously realized and can vary even within a single sample. This feature of LICs may make them particularly difficult to eradicate using therapies targeted against surface antigens. © 2010 by The American Society of Hematology.

Gagrica S.,CRUK London Research Institute | Gagrica S.,University College London | Brookes S.,CRUK London Research Institute | Anderton E.,CRUK London Research Institute | And 2 more authors.
Cancer Research | Year: 2012

The cyclin-dependent kinase (CDK) inhibitors, p18 INK4c and p16 INK4a, both have the credentials of tumor suppressors in human cancers and mouse models. For p16 INK4a, the underlying rationale is its role in senescence, but the selective force for inactivation of p18 INK4c in incipient cancer cells is less clear. Here, we show that in human fibroblasts undergoing replicative or oncogene-induced senescence, there is a marked decline in the levels of p18 INK4c protein and RNA, which mirrors the accumulation of p16 INK4a. Downregulation of INK4c is not dependent on p16 INK4a, and RAS can promote the loss of INK4c without cell-cycle arrest. Downregulation of p18 INK4c correlates with reduced expression of menin and E2F1 but is unaffected by acute cell-cycle arrest or inactivation of the retinoblastoma protein (pRb). Collectively, our data question the idea that p18 INK4c acts as a backup for loss of p16 INK4a and suggest that the apparent activation of p18 INK4c in some settings represents delayed senescence rather than increased expression. We propose that the contrasting behavior of the two very similar INK4 proteins could reflect their respective roles in senescence versus differentiation. ©2011 AACR.

Izumi N.,Max Planck Institute for Molecular Biomedicine | Helker C.,Max Planck Institute for Molecular Biomedicine | Ehling M.,Max Planck Institute for Molecular Biomedicine | Behrens A.,CRUK London Research Institute | And 2 more authors.
PLoS ONE | Year: 2012

Notch signaling controls fundamental aspects of angiogenic blood vessel growth including the selection of sprouting tip cells, endothelial proliferation and arterial differentiation. The E3 ubiquitin ligase Fbxw7 is part of the SCF protein complex responsible for the polyubiquitination and thereby proteasomal degradation of substrates such as Notch, c-Myc and c-Jun. Here, we show that Fbxw7 is a critical regulator of angiogenesis in the mouse retina and the zebrafish embryonic trunk, which we attribute to its role in the degradation of active Notch. Growth of retinal blood vessel was impaired and the Notch ligand Dll4, which is also a Notch target, upregulated in inducible and endothelial cell-specific Fbxw7iECKO mutant mice. The stability of the cleaved and active Notch intracellular domain was increased after siRNA knockdown of the E3 ligase in cultured human endothelial cells. Injection of fbxw7 morpholinos interfered with the sprouting of zebrafish intersegmental vessels (ISVs). Arguing strongly that Notch and not other Fbxw7 substrates are primarily responsible for these phenotypes, the genetic inactivation of Notch pathway components reversed the impaired ISV growth in the zebrafish embryo as well as sprouting and proliferation in the mouse retina. Our findings establish that Fbxw7 is a potent positive regulator of angiogenesis that limits the activity of Notch in the endothelium of the growing vasculature. © 2012 Izumi et al.

Chandler H.,CRUK London Research Institute | Peters G.,CRUK London Research Institute
Current Opinion in Cell Biology | Year: 2013

Senescence represents a permanent exit from the cell cycle and its role in curtailing the proliferation of damaged and potentially oncogenic cells has relevance both as a front-line defense against cancer and as an underlying cause of aging. The retinoblastoma protein (RB) and p53 tumor suppressors are central to the process and the growth arrest is primarily implemented by the cyclin-dependent kinase (CDK) inhibitors, p16INK4a and p21CIP1. In contrast to terminal differentiation, senescence is a general response to a diverse range of cellular stresses and is typically accompanied by a characteristic set of phenotypic changes. Of particular note is a secretory program whose autocrine and paracrine effects can advertize the presence of senescent cells within a tissue and promote their clearance by the immune system. In this short review, we will highlight recent advances in understanding the relationship between senescence and aging and the distinction between senescence and terminal differentiation, from a cell cycle perspective. © 2013 Elsevier Ltd.

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