Sandrine Ruchaud

Making the Auroras glow: regulation of Aurora A and B kinase function by interacting proteins.

The conserved Aurora family of protein kinases have emerged as crucial regulators of mitosis and cytokinesis. Despite their high degree of homology, Aurora A and B have very distinctive localisations and functions: Aurora A associates with the spindle poles to regulate entry into mitosis, centrosome maturation and spindle assembly; Aurora B is a member of the Chromosomal Passenger Complex (CPC) that transfers from the inner centromere in early mitosis to the spindle midzone, equatorial cortex and midbody in late mitosis and cytokinesis. Aurora B functions include regulation of chromosome–microtubule interactions, cohesion, spindle stability and cytokinesis. This review will focus on how interacting proteins make this functional diversity possible by targeting the kinases to different subcellular locations and regulating their activity.

Caspase-6 gene disruption reveals a requirement for lamin A cleavage in apoptotic chromatin condensation

To study the role of caspase‐6 during nuclear disassembly, we generated a chicken DT40 cell line in which both alleles of the caspase‐6 gene were disrupted. No obvious morphological differences were observed in the apoptotic process in caspase‐6‐ deficient cells compared with the wild type. However, examination of apoptosis in a cell‐free system revealed a block in chromatin condensation and apoptotic body formation when nuclei from HeLa cells expressing lamin A or lamin A‐transfected Jurkat cells were incubated in caspase‐6‐deficient apoptotic extracts. Transfection of exogenous caspase‐6 into the clone reversed this phenotype. Lamins A and C, which are caspase‐6‐only substrates, were cleaved by the wild‐type and heterozygous apoptotic extracts but not by the extracts lacking caspase‐6. Furthermore, the caspase‐6 inhibitor z‐VEID‐fmk mimicked the effects of caspase‐6 deficiency and prevented the cleavage of lamin A. Taken together, these observations indicate that caspase‐6 activity is essential for lamin A cleavage and that when lamin A is present it must be cleaved in order for the chromosomal DNA to undergo complete condensation during apoptotic execution.

The chromosomal passenger complex: one for all and all for one.

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.

Deconstructing Survivin: comprehensive genetic analysis of Survivin function by conditional knockout in a vertebrate cell line

Survivin is a key cellular protein thought to function in apoptotic regulation, mitotic progression, or possibly both. In this study, we describe the isolation of two conditional knockouts of the survivin gene in chicken DT40 cells. DT40 cells lacking Survivin die in interphase after failing to complete cytokinesis. However, these cells show normal sensitivity to the chemotherapeutic agent etoposide. Expression of Survivin mutants against a null background to reassess the role of several key residues reveals that DT40 cells can grow normally if their sole Survivin is missing a widely studied cyclin-dependent kinase phosphorylation site or sites reportedly essential for binding to Smac or aurora B. Mutations in the nuclear export sequence or dimerization interface render cells temperature sensitive for growth. As an important caveat for other studies in which protein function is studied by transient transfection, three of the Survivin mutants fail to localize in the presence of the wild-type protein but do localize and indeed support life in its absence.

INCENP–aurora B interactions modulate kinase activity and chromosome passenger complex localization

Dynamic localization of the chromosomal passenger complex (CPC) during mitosis is essential for its diverse functions. CPC targeting to centromeres involves interactions between Survivin, Borealin, and the inner centromere protein (CENP [INCENP]) N terminus. In this study, we investigate how interactions between the INCENP C terminus and aurora B set the level of kinase activity. Low levels of kinase activity, seen in INCENP-depleted cells or in cells expressing a mutant INCENP that cannot bind aurora B, are sufficient for a spindle checkpoint response when microtubules are absent but not against low dose taxol. Intermediate kinase activity levels obtained with an INCENP mutant that binds aurora B but cannot fully activate it are sufficient for a robust response against taxol, but cannot trigger CPC transfer from the chromosomes to the anaphase spindle midzone. This transfer requires significantly higher levels of aurora B activity. These experiments reveal that INCENP interactions with aurora B in vivo modulate the level of kinase activity, thus regulating CPC localization and functions during mitosis.

Apoptosis-associated caspase activation assays

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.

SUMOylation modulates the function of Aurora-B kinase

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 BK207R) 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-BK207R mutant does not display reduced kinase activity, suggesting that functional defects are probably a consequence of the altered localization, rather than decreased intrinsic kinase activity. These data suggest that SUMOylation of Aurora B modulates its function, possibly by mediating the extraction of CPC complexes from chromosome arms during prometaphase.

The transcriptional repressor ICER and cAMP-induced programmed cell death

The cAMP pathway plays a central role in the response to hormonal signals for cell proliferation, differentiation and apoptosis. In IPC-81 leukaemia cells, activation of the cAMP pathway by prostaglandin E1 treatment, or other cAMP-elevating agents, induces apoptosis within 4 – 6 h. Inhibition of mRNA or protein synthesis during the first 2 h of cAMP induction protects cells from apoptosis, suggesting a requirement for early gene expression. cAMP-dependent protein kinase phosphorylates a class of nuclear factors and thereby regulates the transcription of a specific set of genes. Here we show that CREM (cAMP Responsive Element Modulator) expression is induced rapidly upon prostaglandin E1 treatment of IPC-81 cells. The induced transcripts correspond to the early product ICER (Inducible cAMP Early Repressor). ICER expression remains elevated until the burst of cell death. Protein synthesis inhibitors which prevent cAMP-induced apoptosis also block de novo ICER synthesis. Transfected IPC-81 cell lines, constitutively expressing high level of ICER are resistant to cAMP-induced cell death. In these transfected cells, cAMP fails to upregulate the ICER transcripts demonstrating that ICER exerts strongly its repressor function on CRE-containing genes. That an early expression of ICER blocks apoptosis, suggests that gene repression by endogenous ICER in IPC-81 is unsufficient or occurs too late to protect cells against death. ICER transfected cells rescued from cAMP-induced apoptosis are growth arrested. It shows for the first time that CREM activation directly participates to the decision of the cell to die. ICER, by sequentially repressing distinct sets of CRE-containing genes could modulate cell fate.

Synthesis and biological evaluation of 2,3-diarylimidazo[1,2-a]pyridines as antileishmanial agents

A novel series of 2,3-diarylimidazo[1,2-a]pyridines was synthesized and evaluated for their antileishmanial activities. Four derivatives exhibited good activity against the promastigote and intracellular amastigote stages of Leishmania major, coupled with a low cytotoxicity against the HeLa human cell line. The impact of compound lipophilicity on antiparasitic activities was investigated by Log D comparison. Although LmCK1 could be the parasitic target for three compounds (13, 18, 21), the inhibition of another target is under study to explain the antileishmanial effect of the most promising compounds.

Bioprospecting marine plankton.

The ocean dominates the surface of our planet and plays a major role in regulating the biosphere. For example, the microscopic photosynthetic organisms living within provide 50% of the oxygen we breathe, and much of our food and mineral resources are extracted from the ocean. In a time of ecological crisis and major changes in our society, it is essential to turn our attention towards the sea to find additional solutions for a sustainable future. Remarkably, while we are overexploiting many marine resources, particularly the fisheries, the planktonic compartment composed of zooplankton, phytoplankton, bacteria and viruses, represents 95% of marine biomass and yet the extent of its diversity remains largely unknown and underexploited. Consequently, the potential of plankton as a bioresource for humanity is largely untapped. Due to their diverse evolutionary backgrounds, planktonic organisms offer immense opportunities: new resources for medicine, cosmetics and food, renewable energy, and long-term solutions to mitigate climate change. Research programs aiming to exploit culture collections of marine micro-organisms as well as to prospect the huge resources of marine planktonic biodiversity in the oceans are now underway, and several bioactive extracts and purified compounds have already been identified. This review will survey and assess the current state-of-the-art and will propose methodologies to better exploit the potential of marine plankton for drug discovery and for dermocosmetics.