Marie Curie Research Institute

Oxted, United Kingdom

Marie Curie Research Institute

Oxted, United Kingdom
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Jaqaman K.,National Oceanic and Atmospheric Administration | Jaqaman K.,Scripps Research Institute | King E.M.,National Oceanic and Atmospheric Administration | King E.M.,University of Dundee | And 26 more authors.
Journal of Cell Biology | Year: 2010

During mitosis in most eukaryotic cells, chromosomes align and form a metaphase plate halfway between the spindle poles, about which they exhibit oscillatory movement. These movements are accompanied by changes in the distance between sister kinetochores, commonly referred to as breathing. We developed a live cell imaging assay combined with computational image analysis to quantify the properties and dynamics of sister kinetochores in three dimensions. We show that baseline oscillation and breathing speeds in late prometaphase and metaphase are set by microtubule depolymerases, whereas oscillation and breathing periods depend on the stiffness of the mechanical linkage between sisters. Metaphase plates become thinner as cells progress toward anaphase as a result of reduced oscillation speed at a relatively constant oscillation period. The progressive slowdown of oscillation speed and its coupling to plate thickness depend nonlinearly on the stiffness of the mechanical linkage between sisters. We propose that metaphase plate formation and thinning require tight control of the state of the mechanical linkage between sisters mediated by centromeric chromatin and cohesion. © 2010 Jaqaman et al.

Amaro A.C.,ETH Zurich | Samora C.P.,Marie Curie Research Institute | Samora C.P.,University of Warwick | Holtackers R.,ETH Zurich | And 8 more authors.
Nature Cell Biology | Year: 2010

Chromosome segregation in metazoans requires the alignment of sister kinetochores on the metaphase plate. During chromosome alignment, bioriented kinetochores move chromosomes by regulating the plus-end dynamics of the attached microtubules. The bundles of kinetochore-bound microtubules alternate between growth and shrinkage, leading to regular oscillations along the spindle axis. However, the molecular mechanisms that coordinate microtubule plus-end dynamics remain unknown. Here we show that centromere protein (CENP)-H, a subunit of the CENP-A nucleosome-associated and CENP-A distal complexes (CENP-A NAC/CAD), is essential for this coordination, because kinetochores lacking CENP-H establish bioriented attachments but fail to generate regular oscillations, as a result of an uncontrolled rate of microtubule plus-end turnover. These alterations lead to rapid erratic movements that disrupt metaphase plate organization. We also show that the abundance of the CENP-A NAC/CAD subunits CENP-H and CENP-I dynamically change on individual sister kinetochores in vivo, because they preferentially bind the sister kinetochore attached to growing microtubules, and that one other subunit, CENP-Q, binds microtubules in vitro. We therefore propose that CENP-A NAC/CAD is a direct regulator of kinetochore-microtubule dynamics, which physically links centromeric DNA to microtubule plus ends. © 2010 Macmillan Publishers Limited. All rights reserved.

Llarena M.,Marie Curie Research Institute | Bailey D.,Marie Curie Research Institute | Bailey D.,Public Health England | Curtis H.,Marie Curie Research Institute | And 2 more authors.
Traffic | Year: 2010

CREB-H and activating transcription factor 6 (ATF6) are transmembrane transcription factors that, in response to endoplasmic reticulum (ER) stress, traffic to the Golgi where they are cleaved by specific proteases, producing the N-terminal domains that effect appropriate transcriptional responses. We show that unlike in ATF6 whose lumenal tail binds BiP and contains determinants for stress sensing and Golgi transport, in CREB-H the lumenal tail is not involved in ER retention, not required for Golgi transport and does not bind BiP. The main determinant for CREB-H ER retention resides in a membrane-proximal cytoplasmic determinant that is conserved in related members of the CREB-H family, but lacking in ATF6. We refine requirements within the ER-retention motif (ERM) and show that ERM-ve variants exhibited constitutive Golgi localization and constitutive cleavage by the Golgi protease, S1P. The ERM also conferred ER retention on a heterologous protein. Furthermore, deletion of the lumenal tail of CREB-H had no effect on ER retention of parental CREB-H or Golgi localization of ERM-ve variants. Importantly, when the lumenal tail of ATF6 was transferred into an ERM-ve variant, the chimera was now retained in the ER. Together, these data demonstrate novel and qualitatively distinct mechanisms of trafficking and stress signalling in CREB-H compared to ATF6. © 2009 John Wiley & Sons A/S.

Malecova B.,Marie Curie Research Institute | Malecova B.,Sanford Burnham Institute for Medical Research | Caputo V.S.,Marie Curie Research Institute | Caputo V.S.,Imperial College London | And 5 more authors.
Transcription | Year: 2015

TFIIA is an important positive regulator of TFIID, the primary promoter recognition factor of the basal RNA polymerase II transcription machinery. TFIIA antagonises negative TFIID regulators such as negative cofactor 2 (NC2), promotes specific binding of the TBP subunit of TFIID to TATA core promoter sequence elements and stimulates the interaction of TBP-associated factors (TAFs) in the TFIID complex with core promoter elements located downstream of TATA, such as the initiator element (INR). Metazoan TFIIA consists of 3 subunits, TFIIAα (35 kDa), β (19 kDa) and γ (12 kDa). TFIIAα and β subunits are encoded by a single gene and result from site-specific cleavage of a 55 kDa TFIIA(α/β) precursor protein by the protease Taspase1. Metazoan cells have been shown to contain variable amounts of TFIIA (55/12 kDa) and Taspase1-processed TFIIA (35/19/12 kDa) depending on cell type, suggesting distinct gene-specific roles of unprocessed and Taspase1-processed TFIIA. How precisely Taspase1 processing affects TFIIA functions is not understood. Here we report that Taspase1 processing alters TFIIA interactions with TFIID and the conformation of TFIID/TFIIA promoter complexes. We further show that Taspase1 processing induces increased sensitivity of TFIID/TFIIA complexes to the repressor NC2, which is counteracted by the presence of an INR core promoter element. Our results provide first evidence that Taspase1 processing affects TFIIA regulation of TFIID and suggest that Taspase1 processing of TFIIA is required to establish INR-selective core promoter activity in the presence of NC2. © 2015 Taylor & Francis Group, LLC.

Grazini U.,Italian National Cancer Institute | Grazini U.,Marie Curie Research Institute | Grazini U.,University of Oxford | Zanardi F.,Instituto Firc Of Oncologia Molecolare | And 5 more authors.
Molecular Cell | Year: 2010

The RAG1 and RAG2 proteins are the only lymphoid-specific factors required to perform the first step of V(D)J recombination, DNA cleavage. While the catalytic domain of RAG1, the core region, has been well characterized, the role of the noncore region in modulating chromosomal V(D)J recombination efficiency remains ill defined. Recent studies have highlighted the role of chromatin structure in regulation of V(D)J recombination. Here we show that RAG1 itself, through a RING domain within its N-terminal noncore region, preferentially interacts directly with and promotes monoubiquitylation of histone H3. Mutations affecting the RAG1 RING domain reduce histone H3 monoubiquitylation activity, decrease V(D)J recombination activity in vivo, reduce formation of both signal-joint and coding-joint products on episomal substrates, and decrease efficiency of V(D)J recombination at the endogenous IgH locus in lymphoid cells. The results reveal that RAG1-mediated histone monoubiquitylation activity plays a role in regulating the joining phase of chromosomal V(D)J recombination. © 2010 Elsevier Inc. All rights reserved.

Primot A.,French National Center for Scientific Research | Mogha A.,French National Center for Scientific Research | Corre S.,French National Center for Scientific Research | Roberts K.,Marie Curie Research Institute | And 10 more authors.
Pigment Cell and Melanoma Research | Year: 2010

Summary The master regulator of the melanocyte lineage Mitf is intimately involved in development as well as melanoma, controlling cell survival, differentiation, proliferation and metastasis/migration. Consistent with its central role, Mitf expression and Mitf post-translational modifications are tightly regulated. An additional potential level of regulation is afforded by differential splicing of Mitf exon-6 leading to the generation of two isoforms that differ by the presence of six amino-acids in the Mitf (+) isoform and which have differential effects on cell cycle progression. However, whether the ratio of the two isoforms is regulated and whether their expression correlates with melanoma progression is not known. Here, we show that the differential expression of the Mitf 6a/b isoforms is dependent on the MAPKinase signalling, being linked to the activation of MEK1-ERK2, but not to N-RAS/B-RAF mutation status. In addition, quantification of Mitf 6a/b splicing forms in 86 melanoma samples revealed substantially increased levels of the Mitf (-) form in a subset of metastatic melanomas. The results suggest that differential expression of the Mitf 6a/b isoforms may represent an additional mechanism for regulating Mitf function and melanoma biology. © 2009 John Wiley & Sons A/S.

Kimata Y.,University College London | Kimata Y.,Marie Curie Research Institute | Kitamura K.,Hiroshima University | Fenner N.,Marie Curie Research Institute | And 2 more authors.
Molecular Biology of the Cell | Year: 2011

Meiosis is a specialized form of cell division generating haploid gametes and is dependent upon protein ubiquitylation by the anaphase-promoting complex/cyclosome (APC/C). Accurate control of the APC/C during meiosis is important in all eukaryotic cells and is in part regulated by the association of coactivators and inhibitors. We previously showed that the fission yeast meiosis-specific protein Mes1 binds to a coactivator and inhibits APC/C; however, regulation of the Mes1-mediated APC/C inhibition remains elusive. Here we show how Mes1 distinctively regulates different forms of the APC/C. We study all the coactivators present in the yeast genome and find that only Slp1/Cdc20 is essential for meiosis I progression. However, Fzr1/Mfr1 is a critical target for Mes1 inhibition because fzr1Δ completely rescues the defect on the meiosis II entry in mes1Δ cells. Furthermore, cell-free studies suggest that Mes1 behaves as a pseudosubstrate for Fzr1/Mfr1 but works as a competitive substrate for Slp1. Intriguingly, mutations in the D-box or KEN-box of Mes1 increase its recognition as a substrate by Fzr1, but not by Slp1. Thus Mes1 interacts with two coactivators in a different way to control the activity of the APC/C required for the meiosis I/meiosis II transition. © 2011 Kimata et al.

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