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.
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•Subunit deletions reveal association dependencies in the budding yeast CCAN network•The Cnn1 histone-fold extension is required for assembly of a complex with Ctf3c•Cnn1 localizes to the core yeast centromere in a Ctf3-dependent manner•The Cnn1-Wip1-Ctf3c complex binds two Ndc80 molecules in a parallel configuration
Pekgöz Altunkaya, Malvezzi et al. investigate the assembly of the kinetochore, a large complex connecting chromosomes to the mitotic spindle. They identify protein-protein interactions involving histone-fold domains, which allow the binding of multiple molecules of the key microtubule-binding complex Ndc80 in order to generate robust attachments.
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.
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•Helical arrangement of VipA/B tubules resembles that of contracted phage tails•Homologous VipB and viral tail sheath core regions stabilize the contracted tubule•VipA and a unique VipB N-terminal domain adapt the T6SS for bacterial secretion•Exposed recognition motif mediates contraction-state-specific disassembly by ClpV
Type VI secretion systems (T6SSs) are involved in bacterial competition and virulence. Here, Kube et al. report the structure of the Vibrio cholerae VipA/B T6SS tubule in its contracted state. The overall architecture of the tubule resembles bacteriophage tails. However, further similarities are restricted to the inner-architecture-defining domains of VipB. The structure indicates a mechanism for contraction-state-specific recycling by a unique N-terminal domain, which is only accessible in the contracted state.
Kinetochores are chromatin‐bound multi‐protein complexes that allow high‐fidelity chromosome segregation during mitosis and meiosis. Kinetochore assembly is exclusively initiated at chromatin ...containing Cse4/CENP‐A nucleosomes. The molecular mechanisms ensuring that subcomplexes assemble efficiently into kinetochores only at centromeres, but not anywhere else, are incompletely understood. Here, we combine biochemical and genetic experiments to demonstrate that auto‐inhibition of the conserved kinetochore subunit Mif2/CENP‐C contributes to preventing unscheduled kinetochore assembly in budding yeast cells. We show that wild‐type Mif2 is attenuated in its ability to bind a key downstream component in the assembly pathway, the Mtw1 complex, and that addition of Cse4 nucleosomes overcomes this inhibition. By exchanging the N‐terminus of Mif2 with its functional counterpart from Ame1/CENP‐U, we have created a Mif2 mutant which bypasses the Cse4 requirement for Mtw1 binding in vitro, thereby shortcutting kinetochore assembly. Expression of this Mif2 mutant in cells leads to mis‐localization of the Mtw1 complex and causes pronounced chromosome segregation defects. We propose that auto‐inhibition of Mif2/CENP‐C constitutes a key concept underlying the molecular logic of kinetochore assembly.
Synopsis
What mechanisms ensure that kinetochore protein complexes assemble only at centromeres? Here, an auto‐inhibited conformation of budding yeast Mif2/CENP‐C is found to allow binding to the outer kinetochore Mtw1/MIS12 complex exclusively in the presence of Cse4/CENP‐A‐containing centromeric nucleosomes.
N‐terminal sequences of Mif2/CENP‐C and Ame1/CENP‐U compete for binding to the Mtw1 complex.
Genetic swap experiments indicate different functionalities of chimeric Mtw1 complex receptors.
Mif2 binding to the Mtw1 complex is attenuated by autoinhibition.
Mif2 autoinhibition is released upon binding to Cse4 nucleosomes.
Expression of Mif2 mutants lacking autoinhibition compromises kinetochore assembly in vivo.
Only the presence of Cse4/CENP‐A‐containing centromeric nucleosomes allows Mif2/CENP‐C binding and recruitment of the outer kinetochore Mtw1/MIS12 complex.
One-loop jet functions by geometric subtraction Basdew-Sharma, Avanish; Herzog, Franz; van Velzen, Solange Schrijnder ...
The journal of high energy physics,
10/2020, Letnik:
2020, Številka:
10
Journal Article
Recenzirano
Odprti dostop
A
bstract
In factorization formulae for cross sections of scattering processes, final-state jets are described by jet functions, which are a crucial ingredient in the resummation of large logarithms. ...We present an approach to calculate generic one-loop jet functions, by using the geometric subtraction scheme. This method leads to local counterterms generated from a slicing procedure; and whose analytic integration is particularly simple. The poles are obtained analytically, up to an integration over the azimuthal angle for the observable- dependent soft counterterm. The poles depend only on the soft limit of the observable, characterized by a power law, and the finite term is written as a numerical integral. We illustrate our method by reproducing the known expressions for the jet function for angularities, the jet shape, and jets defined through a cone or
k
T
algorithm. As a new result, we obtain the one-loop jet function for an angularity measurement in
e
+
e
−
collisions, that accounts for the formally power-suppressed but potentially large effect of recoil. An implementation of our approach is made available as the GOJet Mathematica package accompanying this paper.
A
bstract
We present the inclusive Higgs boson cross-section at the LHC with collision energy of 8 TeV. Our predictions are obtained using our publicly available program iHixs which incorporates NNLO ...QCD corrections and electroweak corrections. We review the convergence of the QCD perturbative expansion at this new energy and examine the impact of finite Higgs width effects. We also study the impact of different parton distribution functions on the cross-section. We present tables with the cross-section values and estimates for their uncertainty due to uncalculated higher orders in the perturbative expansion and parton densities.
Kinetochores are macromolecular protein complexes at centromeres that ensure accurate chromosome segregation by attaching chromosomes to spindle microtubules and integrating safeguard mechanisms. The ...inner kinetochore is assembled on CENP-A nucleosomes and has been implicated in establishing a kinetochore-associated pool of Aurora B kinase, a chromosomal passenger complex (CPC) subunit, which is essential for chromosome biorientation. By performing crosslink-guided in vitro reconstitution of budding yeast kinetochore complexes we showed that the Ame1/Okp1
heterodimer, which forms the COMA complex with Ctf19/Mcm21
, selectively bound Cse4
nucleosomes through the Cse4 N-terminus. The Sli15/Ipl1
core-CPC interacted with COMA in vitro through the Ctf19 C-terminus whose deletion affected chromosome segregation fidelity in Sli15 wild-type cells. Tethering Sli15 to Ame1/Okp1 rescued synthetic lethality upon Ctf19 depletion in a Sli15 centromere-targeting deficient mutant. This study shows molecular characteristics of the point-centromere kinetochore architecture and suggests a role for the Ctf19 C-terminus in mediating CPC-binding and accurate chromosome segregation.
Cotranscriptional ubiquitination of histone H2B is key to gene regulation. The yeast E3 ubiquitin ligase Bre1 (human RNF20/40) pairs with the E2 ubiquitin conjugating enzyme Rad6 to monoubiquitinate ...H2B at Lys123. How this single lysine residue on the nucleosome core particle (NCP) is targeted by the Rad6–Bre1 machinery is unknown. Using chemical cross-linking and mass spectrometry, we identified the functional interfaces of Rad6, Bre1, and NCPs in a defined in vitro system. The Bre1 RING domain cross-links exclusively with distinct regions of histone H2B and H2A, indicating a spatial alignment of Bre1 with the NCP acidic patch. By docking onto the NCP surface in this distinct orientation, Bre1 positions the Rad6 active site directly over H2B Lys123. The Spt–Ada–Gcn5 acetyltransferase (SAGA) H2B deubiquitinase module competes with Bre1 for binding to the NCP acidic patch, indicating regulatory control. Our study reveals a mechanism that ensures site-specific NCP ubiquitination and fine-tuning of opposing enzymatic activities.
The cohesin subunits Smc1, Smc3 and Scc1 form large tripartite rings which mediate sister chromatid cohesion and chromatin structure. These are thought to entrap DNA with the help of the associated ...proteins SA1/2 and Pds5A/B. Structural information is available for parts of cohesin, but analyses of entire cohesin complexes are limited by their flexibility. Here we generated a more rigid 'bonsai' cohesin by truncating the coiled coils of Smc1 and Smc3 and used single-particle electron microscopy, chemical crosslinking-mass spectrometry and in silico modelling to generate three-dimensional models of cohesin bound to Pds5B. The HEAT-repeat protein Pds5B forms a curved structure around the nucleotide-binding domains of Smc1 and Smc3 and bridges the Smc3-Scc1 and SA1-Scc1 interfaces. These results indicate that Pds5B forms an integral part of the cohesin ring by contacting all other cohesin subunits, a property that may reflect the complex role of Pds5 proteins in controlling cohesin-DNA interactions.
In endothelial cells (ECs), stimulation of Toll-like receptor 4 (TLR4) by the endotoxin lipopolysaccharide (LPS) induces the release of diverse pro-inflammatory mediators, beneficial in controlling ...bacterial infections. However, their systemic secretion is a main driver of sepsis and chronic inflammatory diseases. Since distinct and rapid induction of TLR4 signaling is difficult to achieve with LPS due to the specific and non-specific affinity to other surface molecules and receptors, we engineered new light-oxygen-voltage-sensing (LOV)-domain-based optogenetic endothelial cell lines (opto-TLR4-LOV LECs and opto-TLR4-LOV HUVECs) that allow fast, precise temporal, and reversible activation of TLR4 signaling pathways. Using quantitative mass-spectrometry, RT-qPCR, and Western blot analysis, we show that pro-inflammatory proteins were not only expressed differently, but also had a different time course when the cells were stimulated with light or LPS. Additional functional assays demonstrated that light induction promoted chemotaxis of THP-1 cells, disruption of the EC monolayer and transmigration. In contrast, ECs incorporating a truncated version of the TLR4 extracellular domain (opto-TLR4 ΔECD2-LOV LECs) revealed high basal activity with fast depletion of the cell signaling system upon illumination. We conclude that the established optogenetic cell lines are well suited to induce rapid and precise photoactivation of TLR4, allowing receptor-specific studies.
Transcription regulation in metazoans often involves promoter-proximal pausing of RNA polymerase (Pol) II, which requires the 4-subunit negative elongation factor (NELF). Here we discern the ...functional architecture of human NELF through X-ray crystallography, protein crosslinking, biochemical assays, and RNA crosslinking in cells. We identify a NELF core subcomplex formed by conserved regions in subunits NELF-A and NELF-C, and resolve its crystal structure. The NELF-AC subcomplex binds single-stranded nucleic acids in vitro, and NELF-C associates with RNA in vivo. A positively charged face of NELF-AC is involved in RNA binding, whereas the opposite face of the NELF-AC subcomplex binds NELF-B. NELF-B is predicted to form a HEAT repeat fold, also binds RNA in vivo, and anchors the subunit NELF-E, which is confirmed to bind RNA in vivo. These results reveal the three-dimensional architecture and three RNA-binding faces of NELF.
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