Peter De Wulf

The yeast DASH complex forms closed rings on microtubules

The Saccharomyces cerevisiae DASH complex is an essential microtubule-binding component of the kinetochore. We coexpressed all ten subunits of this assembly in Escherichia coli and purified a single complex, a ∼210-kDa heterodecamer with an apparent stoichiometry of one copy of each subunit. The hydrodynamic properties of the recombinant assembly are indistinguishable from those of the native complex in yeast extracts. The structure of DASH alone and bound to microtubules was visualized by EM. The free heterodecamer is relatively globular. In the presence of microtubules, DASH oligomerizes to form rings and paired helices that encircle the microtubules. We discuss potential roles for such collar-like structures in maintaining microtubule attachment and spindle integrity during chromosome segregation.

Probing the ArcA-P Modulon of Escherichia coli by Whole Genome Transcriptional Analysis and Sequence Recognition Profiling

The ArcB/ArcA two-component signal transduction system of Escherichia coli regulates gene expression in response to the redox conditions of growth. Over the years, genetic screens have lead to the identification of about 30 ArcA-P-controlled operons that are involved in redox metabolism. However, the discovery of 3 targets that are not implicated in respiratory metabolism (the tra operon for plasmid conjugation, psi site for Xer-based recombination, and oriC site for chromosome replication) suggests that the Arc modulon may comprise additional operons that are involved in a myriad of functions. To identify these operons, we derived the ArcA-P-dependent transcription profile of E. coli using oligonucleotide-based microarray analysis. The findings indicated that 9% of all open reading frames in E. coli are affected either directly or indirectly by ArcA-P. To identify which operons are under the direct control of ArcA-P, we developed the ArcA-P recognition weight matrix from footprinting data and used it to scan the genome, yielding an ArcA-P sequence affinity map. By overlaying both methods, we identified 55 new Arc-regulated operons that are implicated in energy metabolism, transport, survival, catabolism, and transcriptional regulation. The data also suggest that the Arc response pathway, which translates into a net global downscaling of gene expression, overlaps partly with the FNR regulatory network. A conservative but reasonable assessment is that the Arc pathway recruits 100–150 operons to mediate a role in cellular adaptation that is more extensive than hitherto anticipated.

Protein phosphatases take the mitotic stage

Following the identification of cyclin-dependent kinases in the 1980s, kinases were hailed as the directors of mitosis. Although the action of kinases must necessarily be reversible, only recently has the involvement of specific phosphatases in mitosis become appreciated. Studies are now revealing how the timely execution of mitotic events depends on the delicate interplay between kinases and phosphatases. To date, the best-characterized mitotic phosphatases are Cdc25, that is required for entry into mitosis and Cdc14, that controls exit from mitosis in budding yeast. Recent work has now exposed the conserved serine-threonine phosphatases PP1 and PP2A as key regulators of various mitotic processes.

Roles for the Conserved Spc105p/Kre28p Complex in Kinetochore-Microtubule Binding and the Spindle Assembly Checkpoint

Background Kinetochores attach sister chromatids to microtubules of the mitotic spindle and orchestrate chromosome disjunction at anaphase. Although S. cerevisiae has the simplest known kinetochores, they nonetheless contain ∼70 subunits that assemble on centromeric DNA in a hierarchical manner. Developing an accurate picture of the DNA-binding, linker and microtubule-binding layers of kinetochores, including the functions of individual proteins in these layers, is a key challenge in the field of yeast chromosome segregation. Moreover, comparison of orthologous proteins in yeast and humans promises to extend insight obtained from the study of simple fungal kinetochores to complex animal cell kinetochores. Principal Findings We show that S. cerevisiae Spc105p forms a heterotrimeric complex with Kre28p, the likely orthologue of the metazoan kinetochore protein Zwint-1. Through systematic analysis of interdependencies among kinetochore complexes, focused on Spc105p/Kre28p, we develop a comprehensive picture of the assembly hierarchy of budding yeast kinetochores. We find Spc105p/Kre28p to comprise the third linker complex that, along with the Ndc80 and MIND linker complexes, is responsible for bridging between centromeric heterochromatin and kinetochore MAPs and motors. Like the Ndc80 complex, Spc105p/Kre28p is also essential for kinetochore binding by components of the spindle assembly checkpoint. Moreover, these functions are conserved in human cells. Conclusions/Significance Spc105p/Kre28p is the last of the core linker complexes to be analyzed in yeast and we show it to be required for kinetochore binding by a discrete subset of kMAPs (Bim1p, Bik1p, Slk19p) and motors (Cin8p, Kar3p), all of which are nonessential. Strikingly, dissociation of these proteins from kinetochores prevents bipolar attachment, even though the Ndc80 and DASH complexes, the two best-studied kMAPs, are still present. The failure of Spc105 deficient kinetochores to bind correctly to spindle microtubules and to recruit checkpoint proteins in yeast and human cells explains the observed severity of missegregation phenotypes.

Cpx Two-Component Signal Transduction in Escherichia coli: Excessive CpxR-P Levels Underlie CpxA* Phenotypes

In Escherichia coli, the CpxA-CpxR two-component signal transduction system and the sigma(E) and sigma(32) response pathways jointly regulate gene expression in adaptation to adverse conditions. These include envelope protein distress, heat shock, oxidative stress, high pH, and entry into stationary phase. Certain mutant versions of the CpxA sensor protein (CpxA* proteins) exhibit an elevated ratio of kinase to phosphatase activity on CpxR, the cognate response regulator. As a result, CpxA* strains display numerous phenotypes, many of which cannot be easily related to currently known functions of the CpxA-CpxR pathway. It is unclear whether CpxA* phenotypes are caused solely by hyperphosphorylation of CpxR. We here report that all of the tested CpxA* phenotypes depend on elevated levels of CpxR-P and not on cross-signalling of CpxA* to noncognate response regulators.

A weight matrix for binding recognition by the redox‐response regulator ArcA‐P of Escherichia coli

An important aspect of microbial pathogenesis relates to the interaction between the invading pathogen and the host at the molecular level. Information on the three-dimensional structures of the component proteins involved can provide valuable clues concerning the mechanisms that underlie these processes. To date, none of the class 5 outer membrane proteins from Neisseria meningitidis has been investigated at the structural level. In order to obtain an insight into the molecular basis for meningococcal adhesion and recognition of Opc antigen by antibody, we have acquired low-resolution structural data of reconstituted Opc by using electron microscopy of two-dimensional crystals combined with crystallographic image analysis. Here, we present new structural data on the integral membrane protein Opc, an adhesin and invasin from the Gramnegative pathogen Neisseria meningitidis. Opc is a member of the class 5 group of integral outer membrane proteins and is expressed at high levels by certain strains of N. meningitidis (Achtman et al., 1988, J Exp Med 168: 507±525). It is highly immunogenic in humans, and antibodies against Opc are bactericidal (Rosenqvist et al., 1993, J Infect Dis 167: 1065±1073). There is also a substantial body of evidence indicating that Opc facilitates the adhesion and invasion of epithelial and endothelial cells by the recognition of speci®c cell surface receptors (Virji et al., 1992, Mol Microbiol 6: 2785±2795;Virji et al., 1994, Mol Microbiol 14: 173±184; de Vries et al., 1998, Mol Microbiol 27: 1203±1212). A topological model has been proposed for Opc (Merker et al., 1997, Mol Microbiol 23: 281±293), consisting of 10 transmembrane b-strands connected by ®ve large surface-exposed loop regions. The model was based on epitope mapping, mutagenesis and insertion of a foreign epitope into the surface loops. It predicts that a substantial proportion of the total mass of Opc would protrude above the membrane, presumably to facilitate interaction with cell surface receptors in the host.

The kinetochore: From molecular discoveries to cancer therapy

Centromeres and Kinetochores: An Historical Perspective.- The Basics of Chromosome Segregation.- The Centromere.- Neocentromeres.- Human Artificial Centromeres: Assembly of Functional Centromeres on Human Artificial Chromosomes.- Kinetochore Composition, Formation, and Organization.- Evolution of Centromeres and Kinetochores: A Two-Part Fugue.- Mitotic Spindle Assembly Mechanisms.- Kinetochore-Microtubule Interactions.- Post-Translational Modifications that Regulate Kinetochore Activity.- The Role of the Kinetochore in Spindle Checkpoint Signaling.- Kinetochore Regulation of Anaphase and Cytokinesis.- Roles of Centromeres and Kinetochores in Meiosis.- The Kinetochore-Cancer Connection.- The Kinetochore as Target for Cancer Drug Development.

Rio1 promotes rDNA stability and downregulates RNA polymerase I to ensure rDNA segregation

The conserved protein kinase Rio1 localizes to the cytoplasm and nucleus of eukaryotic cells. While the roles of Rio1 in the cytoplasm are well characterized, its nuclear function remains unknown. Here we show that nuclear Rio1 promotes rDNA array stability and segregation in Saccharomyces cerevisiae. During rDNA replication in S phase, Rio1 downregulates RNA polymerase I (PolI) and recruits the histone deacetylase Sir2. Both interventions ensure rDNA copy-number homeostasis and prevent the formation of extrachromosomal rDNA circles, which are linked to accelerated ageing in yeast. During anaphase, Rio1 downregulates PolI by targeting its subunit Rpa43, causing PolI to dissociate from the rDNA. By stimulating the processing of PolI-generated transcripts at the rDNA, Rio1 allows for rDNA condensation and segregation in late anaphase. These events finalize the genome transmission process. We identify Rio1 as an essential nucleolar housekeeper that integrates rDNA replication and segregation with ribosome biogenesis.

A Screen for Kinetochore-Microtubule Interaction Inhibitors Identifies Novel Antitubulin Compounds

Background Protein assemblies named kinetochores bind sister chromatids to the mitotic spindle and orchestrate sister chromatid segregation. Interference with kinetochore activity triggers a spindle checkpoint mediated arrest in mitosis, which frequently ends in cell death. We set out to identify small compounds that inhibit kinetochore-microtubule binding for use in kinetochore-spindle interaction studies and to develop them into novel anticancer drugs. Methodology/Principal Findings A fluorescence microscopy-based in vitro assay was developed to screen compound libraries for molecules that prevented the binding of a recombinant human Ndc80 kinetochore complex to taxol-stabilized microtubules. An active compound was identified that acted at the microtubule level. More specifically, by localizing to the colchicine-binding site in αβ-tubulin the hit compound prevented the Ndc80 complex from binding to the microtubule surface. Next, structure-activity analyses distinguished active regions in the compound and led to the identification of highly potent analogs that killed cancer cells with an efficacy equaling that of established spindle drugs. Conclusions/Significance The compound identified in our screen and its subsequently identified analogs represent new antitubulin chemotypes that can be synthetically developed into a novel class of antimitotic spindle drugs. In addition, they are stereochemically unique as their R- and S-isomers mimic binding of colchicine and podophyllotoxin, respectively, two antitubulin drugs that interact differently with the tubulin interface. Model-driven manipulation of our compounds promises to advance insight into how antitubulin drugs act upon tubulin. These advances in turn may lead to tailor-made colchicine site agents which would be valuable new assets to fight a variety of tumors, including those that have become resistant to the (antispindle) drugs used today.

Evading Pgp activity in drug-resistant cancer cells: a structural and functional study of antitubulin furan metotica compounds.

Tumor resistance to antitubulin drugs resulting from P-glycoprotein (Pgp) drug-efflux activity, increased expression of the βIII tubulin isotype, and alterations in the drug-binding sites are major obstacles in cancer therapy. Consequently, novel antitubulin drugs that overcome these challenges are of substantial interest. Here, we study a novel chemotype named furan metotica that localizes to the colchicine-binding site in β-tubulin, inhibits tubulin polymerization, and is not antagonized by Pgp. To elucidate the structure–activity properties of this chiral chemotype, the enantiomers of its most potent member were separated and their absolute configurations determined by X-ray crystallography. Both isomers were active and inhibited all 60 primary cancer cell lines tested at the U.S. National Cancer Institute. They also efficiently killed drug-resistant cancer cells that overexpressed the Pgp drug-efflux pump 106-fold. In vitro, the R-isomer inhibited tubulin polymerization at least 4-fold more potently than the S-isomer, whereas in human cells the difference was 30-fold. Molecular modeling showed that the two isomers bind to β-tubulin in distinct manners: the R-isomer binds in a colchicine-like mode and the S-isomer in a podophyllotoxin-like fashion. In addition, the dynamic binding trajectory and occupancy state of the R-isomer were energetically more favorable then those of the S-isomer, explaining the observed differences in biologic activities. The ability of a racemic drug to assume the binding modes of two prototypical colchicine-site binders represents a novel mechanistic basis for antitubulin activity and paves the way toward a comprehensive design of novel anticancer agents. Mol Cancer Ther; 11(5); 1103–11. ©2012 AACR.