Molecular insights into the function and regulation of the budding yeast EB1 homolog Bim1p

Proteine der EB1 (End Binding 1) - Familie sind konservierte Regulatoren der Mikrotubuli (MT) -Dynamik in allen Eukaryoten. Sie binden spezifisch an die Plus-Enden der Mikrotubuli, transportieren assoziierte Faktoren und regulieren so die Interaktion von Mikrotubuli mit zellulä-ren Strukuren, beispielsweise mit Kinetochoren oder mit dem Zellkortex. Trotz vielfältiger Funk-tionen in der Zelle sind die molekularen Mechanismen, die der Aktivität dieser Proteine zugrunde liegen, großteils unbekannt. In dieser Arbeit zeige ich, dass das Hefe EB1 Protein Bim1 von der konservierten Kinase Aurora B/Ipl1 reguliert wird und zwar durch Phosphorylierung an der Linkerdomaene. Detaillierte biochemische Studien haben gezeigt, dass Bim1 physisch mit der Ipl1 Kinase interagiert und dass diese, nach vorangehender Aktivierung durch Sli15, Bim1 an sechs direkt benachbarten Stellen in der flexiblen Linkerdomäne phosphoryliert. Bim1 wird im Laufe des Zellzyklus in der Anaphase phosphoryliert und kontrolliert so die Kinetik der Spindelelongation und spielt eine wichtige Rolle im effizienten Abbau ausgehend vom Zentrum der Spindel. Rekombinant hergestelltes Bim1 welches vom Ipl1-Sli15 Komplex phosphoryliert wurde oder auch eine Mutante die konstitutive Phosphorylierung imitiert zeigen verminderte Affinität für Taxol- stabilisierte Mikrotubuli. Meine Experimente demonstrieren, dass für die MT – Bindungsaktivität von Bim1 sowohl die Dimerisierung des Moleküls als auch das Vorhandensein der N-terminalen Calponin Homo-logie (CH) - Domäne Grundvoraussetzungen sind. Darüber hinaus haben die flexible Linkerregion im Molekül und die CH-Domäne eine synergistische Wirkung auf die MT- Bindungaktivität von Bim1, die durch Phosphorylierung reguliert wird. Zusätzlich habe ich die Interaktion zwischen Bim1 und dynamischen Mikrotubuli mit total internal reflection fluorescence (TIRF) - Mikroskopie in vitro rekonstruiert. Bim1-Dimere lokali-sieren autonom an das Plus-Ende der Mikrotubuli. Weiters haben die TIRF - Experimente ge-zeigt, dass die Assoziation von Bim1 mit dynamischen Mikrotubuli von Ipl1 reguliert wird. Um die Rolle Bim1-abhängiger Rekrutierung von Ipl1 an das Plus-Ende der Mikrotubuli im Detail zu studieren, habe ich Ipl1-Mutanten hergestellt die ihre Fähigkeit an Bim1 zu binden ver-loren haben. Eine Serie an Experimenten zeigt, dass die physische Interaktion zwischen Ipl1 und Bim1 notwendig ist für die effiziente Lokalisation von Ipl1 an Mikrotubuli in vitro und die Kinase-Aktivität von Ipl1 in vivo beeinflusst. Zusammenfassend zeigen meine Daten, dass die Linkerdomäne von Bim1 eine wichtige Rolle spielt bezüglich den MT-Bindungseigenschaften des Proteins, und der Fähigkeit das Plus-Ende eines Mikrotubulus zu erkennen. Hieraus ergibt sich ein generelles und ein mögliches Konzept für die Funktion und Regulierung von CH-Domänen in einem EB1-Protein.Dynamic instability of microtubules (MTs) is regulated by several MT-associated proteins. A special subgroup of MT-associated factors is constituted by the plus-end tracking proteins (+TIPs), which are characterized by their selective association with the growing ends of tubulin polymers thus linking cellular components to MT plus ends. +TIPs are highly conserved and among them, EB1 (end binding) proteins have emerged as key regulators of microtubules in all eukaryotes. Despite their widespread importance, molecular mechanisms regulating the activity of these proteins still remain unclear. Here, I performed a comprehensive structure-function analysis of the budding yeast EB1 protein Bim1p. My study shows that Bim1p is regulated by Aurora B/Ipl1 phosphorylation. Bim1p directly associates with the Ipl1p kinase and upon activation by Sli15p, becomes phosphorylated on a cluster of six serine residues in the flexible linker domain. Bim1p phosphorylation occurs during anaphase in vivo, and it is required for normal spindle elongation kinetics and an efficient disassembly of the spindle midzone. Recombinant Bim1p phosphorylated with Ipl1p-Sli15p complex in vitro or a mutant mimicking constitutive phosphorylation display reduced affinity for taxol-stabilized microtubules. By engineering different Bim1 mutants I could furthermore demonstrate that the MT binding activity of the molecule depends on the dimerization properties of EB1 and that the N-terminal calponin homology (CH) domain of Bim1p is necessary, but not sufficient for stable microtubule association. In contrast, the flexible linker region of the Bim1 molecule synergistically with the CH domain enhances Bim1p MT binding and is critical for the regulation of this EB1 family member. By using total internal reflection fluorescence microscopy (TIRF) I could visualize dynamic microtubules in vitro and show that Bim1p, like other EB1-proteins, tracks microtubule plus ends autonomously. My data show that the interaction between Bim1p and dynamic microtubules de-pends on the dimeric form of molecule and that these associations are negatively regulated by Ipl1p phosphorylation of the linker domain. Finally, I dissected the interaction between the Ipl1 kinase and Bim1p, which is critical for proper kinase function in vivo. I identified two functionally redundant SxIP motifs in the N-terminus of Ipl1p, which mediate the association with the Bim1 EBH domain. The interaction between Bim1p and Ipl1p is negatively regulated by Cdk1 phosphorylation on serine residues immediately adjacent to the SxIP motifs. This suggests a role for Bim1p-mediated translocation of the kinase to the spindle during anaphase

xVis: a web server for the schematic visualization and interpretation of crosslink-derived spatial restraints

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 sub-complexes 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/

xVis: a web server for the schematic visualization and interpretation of crosslink-derived spatial restraints

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 sub-complexes 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/

Structure of the VipA/B Type VI Secretion Complex Suggests a Contraction-State-Specific Recycling Mechanism

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 A 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

Phosphoregulation of the budding yeast EB1 homologue Bim1p by Aurora/Ipl1p

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

A cooperative mechanism drives budding yeast kinetochore assembly downstream of CENP-A

Kinetochores are megadalton-sized protein complexes that mediate chromosome–microtubule interactions in eukaryotes. How kinetochore assembly is triggered specifically on centromeric chromatin is poorly understood. Here we use biochemical reconstitution experiments alongside genetic and structural analysis to delineate the contributions of centromere-associated proteins to kinetochore assembly in yeast. We show that the conserved kinetochore subunits Ame1$^{CENP-U}$ and Okp1$^{CENP-Q}$ form a DNA-binding complex that associates with the microtubule-binding KMN network via a short Mtw1 recruitment motif in the N terminus of Ame1. Point mutations in the Ame1 motif disrupt kinetochore function by preventing KMN assembly on chromatin. Ame1–Okp1 directly associates with the centromere protein C (CENP-C) homologue Mif2 to form a cooperative binding platform for outer kinetochore assembly. Our results indicate that the key assembly steps, CENP-A recognition and outer kinetochore recruitment, are executed through different yeast constitutive centromere-associated network subunits. This two-step mechanism may protect against inappropriate kinetochore assembly similar to rate-limiting nucleation steps used by cytoskeletal polymers

C-Terminal Motifs of the MTW1 Complex Cooperatively Stabilize Outer Kinetochore Assembly in Budding Yeast

Kinetochores are macromolecular protein assemblies at centromeres that mediate accurate chromosome segregation during cell division. The outer kinetochore KNL1SPC105, MIS12MTW1, and NDC80NDC80 complexes assemble the KMN network, which harbors the sites of microtubule binding and spindle assembly checkpoint signaling. The buildup of the KMN network that transmits microtubule pulling forces to budding yeast point centromeres is poorly understood. Here, we identify 225 inter-protein crosslinks by mass spectrometry on KMN complexes isolated from Saccharomyces cerevisiae that delineate the KMN subunit connectivity for outer kinetochore assembly. C-Terminal motifs of Nsl1 and Mtw1 recruit the SPC105 complex through Kre28, and both motifs aid tethering of the NDC80 complex by the previously reported Dsn1 C terminus. We show that a hub of three C-terminal MTW1 subunit motifs mediates the cooperative stabilization of the KMN network, which is augmented by a direct NDC80-SPC105 association

A cooperative mechanism drives budding yeast kinetochore assembly downstream of CENP-A

Kinetochores are megadalton-sized protein complexes that mediate chromosome–microtubule interactions in eukaryotes. How kinetochore assembly is triggered specifically on centromeric chromatin is poorly understood. Here we use biochemical reconstitution experiments alongside genetic and structural analysis to delineate the contributions of centromere-associated proteins to kinetochore assembly in yeast. We show that the conserved kinetochore subunits Ame1$^{CENP-U}$ and Okp1$^{CENP-Q}$ form a DNA-binding complex that associates with the microtubule-binding KMN network via a short Mtw1 recruitment motif in the N terminus of Ame1. Point mutations in the Ame1 motif disrupt kinetochore function by preventing KMN assembly on chromatin. Ame1–Okp1 directly associates with the centromere protein C (CENP-C) homologue Mif2 to form a cooperative binding platform for outer kinetochore assembly. Our results indicate that the key assembly steps, CENP-A recognition and outer kinetochore recruitment, are executed through different yeast constitutive centromere-associated network subunits. This two-step mechanism may protect against inappropriate kinetochore assembly similar to rate-limiting nucleation steps used by cytoskeletal polymers