Papers by Sandrine Ruchaud
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The Journal of Cell Biology, 2008
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Journal of Cell Science
Aurora kinases are central regulators of mitotic-spindle assembly, chromosome segregation and cyt... more Aurora kinases are central regulators of mitotic-spindle assembly, chromosome segregation and cytokinesis. Aurora B is a member of the chromosomal passenger complex (CPC) with crucial functions in regulation of the attachment of kinetochores to microtubules and in cytokinesis. We report here that Aurora B contains a conserved SUMO modification motif within its kinase domain. Aurora B can bind SUMO peptides in vitro when bound to the IN-box domain of its CPC partner INCENP. Mutation of Lys207 to arginine (Aurora B(K207R)) impairs the formation of conjugates of Aurora B and SUMO in vivo. Expression of the SUMO-null form of Aurora B results in abnormal chromosome segregation and cytokinesis failure and it is not able to rescue mitotic defects in Aurora-B-knockout cells. These defects are accompanied by increased levels of the CPC on chromosome arms and defective centromeric function, as detected by decreased phosphorylation of the Aurora-B substrate CENP-A. The Aurora-B(K207R) mutant d...
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Reviews and Protocols in DT40 Research, 2006
Chicken DT40 cells are a very favourable system to use for the study of apoptosis. These cells un... more Chicken DT40 cells are a very favourable system to use for the study of apoptosis. These cells undergo apoptosis readily in response to a variety of physiological and experimental stimuli. Their response to chemotherapeutic agents such as etoposide is rapid and efficient, facilitating biochemical analysis of apoptotic execution. Because many of the genes involved in apoptotic execution are not essential for cellular life, DT40 cells also provide a ready system for the genetic analysis of apoptosis, in which true null mutations may be isolated. Here we describe standard procedures for the induction and analysis of apoptosis in DT40 cells. We also describe a few of the conclusions from our studies in which several components of the apoptotic execution pathway have been knocked out.
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Journal of autoimmune diseases, 2007
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Trends in Cell Biology, 2002
In the 'post-genomic&... more In the 'post-genomic' era, cDNA and genomic sequences are now available that encode huge numbers of proteins. Assigning functions to these proteins is a daunting task. Cell biologists have traditionally approached this problem by disrupting protein function with dominant-negative or structural mutants. Here, we describe several alternative approaches whereby cells or cell lines lacking particular gene products can be generated from genomic sequences for use in functional studies. These include gene targeting in mouse, human and chicken DT40 cells, and recent advances in double-stranded RNA-mediated interference (RNAi).
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The Journal of Cell Biology, 2009
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The Journal of Cell Biology, 2004
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The EMBO Journal, 2002
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Proceedings of the National Academy of Sciences, 2008
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Proceedings of the National Academy of Sciences, 1994
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Oncogene, 1997
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Nature Reviews Molecular Cell Biology, 2007
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Molecular Biology of the Cell, 2007
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Methods, 2008
Caspases are aspartate-directed cysteine proteases that cleave a diverse group of intracellular s... more Caspases are aspartate-directed cysteine proteases that cleave a diverse group of intracellular substrates to contribute to various manifestations of apoptosis. These proteases are synthesized as inactive precursors and are activated as a consequence of signaling induced by a wide range of physiological and pathological stimuli. Caspase activation can be detected by measurement of catalytic activity, immunoblotting for cleavage of their substrates, immunolabeling using conformation-sensitive antibodies or affinity labeling followed by flow cytometry or ligand blotting. Here we describe methods for each of these assays, identify recent improvements in these assays and outline the strengths and limitations of each approach.
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Leukemia, 1998
cAMP-dependent signal transduction co-operates with retinoids to induce acute promyelocytic leuka... more cAMP-dependent signal transduction co-operates with retinoids to induce acute promyelocytic leukaemia (APL) cell maturation. The rationale of this work was to determine whether signal cross-talk could be used to decrease the pharmacological doses of retinoids in the treatment of APL. When only the basal level of adenylate-cyclase (AC) activity is present in NB4 cells, up to 1 microM concentration of all-trans retinoic acid (RA) is required for full maturation (100%). In these conditions, with only 10 nM RA less than 20% of cells will differentiate. Although the use of membrane receptor agonists to activate AC has been proved to synergize with RA treatment, these agents were never as potent as cell permeant cAMP analogues. Analogues have disadvantages since cleavage by serum and cellular phosphodiesterases generates metabolites which interfere in cellular response. In the present study, we observed cell maturation by engrafting an autonomous Bordetella pertussis AC which steadily delivers natural cAMP into the cell. The enzyme alone had no effect on cell maturation. Importantly, cell maturation was increased in a dose-dependent manner when the bacterial AC (1 ng/ml to 1 microg/ml) was used to potentiate the effects of low doses RA (10 nM). More than 50% of cells matured with only 10 nM of RA and 200 ng/ml of B. pertussis AC. The maturation response was significantly increased when lower amounts of enzyme were repetitively added to the culture to compensate for enzymatic decay. These results indicate that a sustained AC activity enhanced cell maturation. We were able to reduce to 3 nM the RA requirement, provided that a minimal amount (20 ng/ml) of B. pertussis AC was added every 12 h in culture. Membrane signalling maintaining high the level of cAMP substantially improved the efficacy of APL cell maturation by retinoids. Therefore, therapeutic benefits are expected by lowering the concentration of RA towards physiological (nanomolar) levels, thus reducing the side-effects of the drug. cAMP-elevating drugs that act on a post-cyclase target (cyclic-nucleotide phosphodiesterases) or cell-targeted drug carriers (cAMP and RA loaded liposomes) should be evaluated as maturation therapies combining the activation of multiple signalling pathways.
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Journal of Cell Science, 2010
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Cell Death and Differentiation, 2000
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Cell, 2007
The chromosomal passenger complex-composed of Aurora B kinase and its regulatory subunits INCENP,... more The chromosomal passenger complex-composed of Aurora B kinase and its regulatory subunits INCENP, Survivin, and Borealin-modulates multiple events during mitosis. In this issue, Jeyaprakash et al. (2007) report the crystal structure of the regulatory subunit complex and reveal how interactions between these proteins promote the targeting and function of the chromosomal passenger complex during mitosis.
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Biochemistry, 2009
The chromosomal passenger complex (CPC) comprises at least four protein components and functions ... more The chromosomal passenger complex (CPC) comprises at least four protein components and functions at various cellular localizations during different mitotic stages to ensure correct chromosome segregation and completion of cytokinesis. Borealin, the most recently identified member of the CPC, is an intrinsically unstructured protein of low solubility and stability. Recent reports have demonstrated the formation of binary or ternary CPC subcomplexes incorporating short Borealin fragments in vitro. Using isothermal titration calorimetry, we show that full-length Borealin, instead of a Borealin fragment possessing the complete Survivin and INCENP recognition sequence, is required for the composition of a Borealin-Survivin complex competent to interact with INCENP. In addition, we show evidence that full-length Borealin, which forms high-order oligomers in its isolated form, is a monomer in the Borealin-Survivin CPC subcomplex.
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Papers by Sandrine Ruchaud