CENP-C promotes kinetochore targeting of other constitutive centromere–associated network (CCAN) subunits by directly interacting with the four-subunit CCAN subcomplex CENP-HIKM and spatially organizing the localization of all other CCAN subunits downstream of CENP-A.

Kinetochores, multi-subunit complexes that assemble at the interface with centromeres, bind spindle microtubules to ensure faithful delivery of chromosomes during cell division. The configuration and function of the kinetochore–centromere interface is poorly understood. We report that a protein at this interface, CENP-M, is structurally and evolutionarily related to small GTPases but is incapable of GTP-binding and conformational switching. We show that CENP-M is crucially required for the assembly and stability of a tetramer also comprising CENP-I, CENP-H, and CENP-K, the HIKM complex, which we extensively characterize through a combination of structural, biochemical, and cell biological approaches. A point mutant affecting the CENP-M/CENP-I interaction hampers kinetochore assembly and chromosome alignment and prevents kinetochore recruitment of the CENP-T/W complex, questioning a role of CENP-T/W as founder of an independent axis of kinetochore assembly. Our studies identify a single pathway having CENP-C as founder, and CENP-H/I/K/M and CENP-T/W as CENP-C-dependent followers.DOI: http://dx.doi.org/10.7554/eLife.02978.001

During kinetochore assembly in budding yeast, the key steps of CENP-A recognition and outer kinetochore recruitment are executed through different yeast CCAN subunits, potentially protecting against inappropriate kinetochore assembly.

EB1 (end binding 1) proteins have emerged as central regulators of microtubule (MT) plus ends in all eukaryotes, but molecular mechanisms controlling the activity of these proteins are poorly understood. In this study, we show that the budding yeast EB1 protein Bim1p is regulated by Aurora B/Ipl1p-mediated multisite phosphorylation. Bim1p forms a stable complex with Ipl1p and is phosphorylated on a cluster of six Ser residues in the flexible linker connecting the calponin homology (CH) and EB1 domains. Using reconstitution of plus end tracking in vitro and total internal reflection fluorescence microscopy, we show that dimerization of Bim1p and the presence of the linker domain are both required for efficient tip tracking and that linker phosphorylation removes Bim1p from static and dynamic MTs. Bim1 phosphorylation occurs during anaphase in vivo, and it is required for normal spindle elongation kinetics and an efficient disassembly of the spindle midzone. Our results define a mechanism for the use and regulation of CH domains in an EB1 protein.

The identification of crosslinks by mass spectrometry has recently been established as an integral part of the hybrid structural analysis of protein complexes and networks. The crosslinking analysis determines distance restraints between two covalently linked amino acids which are typically summarized in a table format that precludes the immediate and comprehensive interpretation of the topological data. xVis displays crosslinks in clear schematic representations in form of a circular, bar or network diagram. The interactive graphs indicate the linkage sites and identification scores, depict the spatial proximity of structurally and functionally annotated protein regions and the evolutionary conservation of amino acids and facilitate clustering of proteins into subcomplexes according to the crosslink density. Furthermore, xVis offers two options for the qualitative assessment of the crosslink identifications by filtering crosslinks according to identification scores or false discovery rates and by displaying the corresponding fragment ion spectrum of each crosslink for the manual validation of the mass spectrometric data. Our web server provides an easy-to-use tool for the fast topological and functional interpretation of distance information on protein complex architectures and for the evaluation of crosslink fragment ion spectra. xVis is available under a Creative Commons Attribution-ShareAlike 4.0 International license at http://xvis.genzentrum.lmu.de/.

Partitioning of the genome requires kinetochores, large protein complexes that mediate dynamic attachment of chromosomes to the spindle. Kinetochores contain two supramolecular protein assemblies. The ten-protein KMN network harbors key microtubule-binding sites in the Ndc80 complex and mediates assembly of checkpoint complexes via the KNL-1/Spc105 protein [1, 2]. As KMN does not contact DNA directly, it relies on different centromere-binding proteins for recruitment and cell-cycle-dependent assembly. These proteins are collectively referred to as the CCAN (constitutive centromere-associated network) [2-4]. The molecular mechanisms by which CCAN subunits associate, however, have remained incompletely defined. In particular, it is unclear how CCAN subunits facilitate the assembly of a microtubule-binding interface that contains multiple Ndc80 molecules bound to different receptors [5]. Here, we dissect molecular mechanisms that underlie targeting of the CCAN subunit Cnn1/CENP-T to the sequence-determined point centromeres of budding yeast. Systematic quantitative mass spectrometry experiments reveal association dependencies within the yeast CCAN network. We show that evolutionarily conserved residues in the histone-fold domain of Cnn1 are required for the formation of a stable five-subunit CCAN subassembly with the Ctf3 complex. Cnn1 localizes in a Ctf3-dependent manner to the core of the yeast point centromere, overlapping with the yeast CENP-A protein Cse4. By arranging the N-terminal domains of the CCAN subunits Mcm16, Mcm22, and Cnn1 into close proximity, the Ctf3c-Cnn1-Wip1 complex configures a composite interaction site for two molecules of the Ndc80 complex. Our experiments show how cooperative assembly mechanisms organize the microtubule-binding interface of the kinetochore.

Bacterial chromosomes (though not Escherichia coli and some other c-proteobacterial chromosomes) contain parS sequences and parAB genes encoding partitioning proteins, i.e. ParA (ATPase) and ParB (DNA-binding proteins) that are components of the segregation machinery. Here, mycobacterial parABS elements were characterized for the first time. parAB genes are not essential in Mycobacterium smegmatis; however, elimination or overexpression of ParB protein causes growth inhibition. Deletion of parB also leads to a rather severe chromosome segregation defect: up to 10 % of the cells were anucleate. Mycobacterial ParB protein uses three oriC-proximal parS sequences as targets to organize the origin region into a compact nucleoprotein complex. Formation of such a complex involves ParB-ParB interactions and is assisted by ParA protein. INTRODUCTIONTuberculosis (TB) is the world's most common disease caused by an infectious organism, Mycobacterium tuberculosis (DeAngelis & Flanagin, 2005; and see http:// www.who.int/topics/tuberculosis/en/). Due to the spread of multi-drug-resistant strains of M. tuberculosis and a synergy with HIV, the TB epidemic is growing and becoming more dangerous in both developing and industrialized countries. A unique feature of M. tuberculosis is its ability to maintain a dormant, non-replicating state for extended periods of time under unfavourable conditions. The mechanism (or mechanisms) by which this bacterium shifts to a dormant state and reverts to active growth is not well understood (Hampshire et al., 2004;Gó mez & Smith, 2000;Wayne & Hayes, 1996: Wayne & Sohaskey, 2001. Knowledge regarding the steps of the mycobacterial cell cycle (replication, chromosome segregation and cell division) seems to be critical for understanding the mechanisms that are responsible for the transition from an active to a non-replicative persistent state (and vice versa) of pathogenic mycobacteria, particularly M. tuberculosis. While initiation of chromosome replication Qin et al., 1999;Rajagopalan et al., 1995;Zawilak et al., 2004) and cell division (FtsZ ring formation) (Chauhan et al., 2006; Dziadek et al., 2002Dziadek et al., , 2003Huang et al., 2007;Rajagopalan et al., 2005) are relatively well studied in mycobacteria, nothing is known about the segregation of chromosomes in these bacteria.Bacterial chromosome segregation has been recently found to be an active and complex process closely coupled with replication (see Bartosik & Jagura-Burdzy, 2005;Errington et al., 2005;Hayes & Barilla, 2006; Leonard et al., 2005 for reviews). In bacteria studied to date, the newly synthesized origin (oriC) regions undergo a symmetric or asymmetric segregation process; two copies of the duplicated oriC regions migrate from the cell centre toward opposite cell poles, i.e. to the 1/4 and 3/4 positions (Escherichia coli, Bacillus subtilis, Vibro cholerae chromosome II), or one copy of the newly synthesized origins remains at the pole while the other copy migrates to the opposite pole Abbreviations: EMSA, electrophoretic mo...

The bacterial type VI secretion system is a multicomponent molecular machine directed against eukaryotic host cells and competing bacteria. An intracellular contractile tubular structure that bears functional homology with bacteriophage tails is pivotal for ejection of pathogenic effectors. Here, we present the 6 Å cryoelectron microscopy structure of the contracted Vibrio cholerae tubule consisting of the proteins VipA and VipB. We localized VipA and VipB in the protomer and identified structural homology between the C-terminal segment of VipB and the tail-sheath protein of T4 phages. We propose that homologous segments in VipB and T4 phages mediate tubule contraction. We show that in type VI secretion, contraction leads to exposure of the ClpV recognition motif, which is embedded in the type VI-specific four-helix-bundle N-domain of VipB. Disaggregation of the tubules by the AAA+ protein ClpV and recycling of the VipA/B subunits are thereby limited to the contracted state.