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1.
bioRxiv ; 2024 Apr 03.
Article in English | MEDLINE | ID: mdl-38617353

ABSTRACT

Centrosomes are the principal microtubule-organizing centers of the cell and play an essential role in mitotic spindle function. Centrosome biogenesis is achieved by strict control of protein acquisition and phosphorylation prior to mitosis. Defects in this process promote fragmentation of pericentriolar material culminating in multipolar spindles and chromosome missegregation. Centriolar satellites, membrane-less aggrupations of proteins involved in the trafficking of proteins toward and away from the centrosome, are thought to contribute to centrosome biogenesis. Here we show that the microtubule plus-end directed kinesin motor Kif9 localizes to centriolar satellites and regulates their pericentrosomal localization during interphase. Lack of Kif9 leads to aggregation of satellites closer to the centrosome and increased centrosomal protein degradation that disrupts centrosome maturation and results in chromosome congression and segregation defects during mitosis. Our data reveal roles for Kif9 and centriolar satellites in the regulation of cellular proteostasis and mitosis.

2.
EMBO J ; 42(15): e113565, 2023 08 01.
Article in English | MEDLINE | ID: mdl-37305927

ABSTRACT

BRCA1/BARD1 is a tumor suppressor E3 ubiquitin (Ub) ligase with roles in DNA damage repair and in transcriptional regulation. BRCA1/BARD1 RING domains interact with nucleosomes to facilitate mono-ubiquitylation of distinct residues on the C-terminal tail of histone H2A. These enzymatic domains constitute a small fraction of the heterodimer, raising the possibility of functional chromatin interactions involving other regions such as the BARD1 C-terminal domains that bind nucleosomes containing the DNA damage signal H2A K15-Ub and H4 K20me0, or portions of the expansive intrinsically disordered regions found in both subunits. Herein, we reveal novel interactions that support robust H2A ubiquitylation activity mediated through a high-affinity, intrinsically disordered DNA-binding region of BARD1. These interactions support BRCA1/BARD1 recruitment to chromatin and sites of DNA damage in cells and contribute to their survival. We also reveal distinct BRCA1/BARD1 complexes that depend on the presence of H2A K15-Ub, including a complex where a single BARD1 subunit spans adjacent nucleosome units. Our findings identify an extensive network of multivalent BARD1-nucleosome interactions that serve as a platform for BRCA1/BARD1-associated functions on chromatin.


Subject(s)
Nucleosomes , Tumor Suppressor Proteins , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , Ubiquitination , Histones/genetics , Histones/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Chromatin
3.
J Proteome Res ; 22(2): 647-655, 2023 02 03.
Article in English | MEDLINE | ID: mdl-36629399

ABSTRACT

Fragmentation ion spectral analysis of chemically cross-linked proteins is an established technology in the proteomics research repertoire for determining protein interactions, spatial orientation, and structure. Here we present Kojak version 2.0, a major update to the original Kojak algorithm, which was developed to identify cross-linked peptides from fragment ion spectra using a database search approach. A substantially improved algorithm with updated scoring metrics, support for cleavable cross-linkers, and identification of cross-links between 15N-labeled homomultimers are among the newest features of Kojak 2.0 presented here. Kojak 2.0 is now integrated into the Trans-Proteomic Pipeline, enabling access to dozens of additional tools within that suite. In particular, the PeptideProphet and iProphet tools for validation of cross-links improve the sensitivity and accuracy of correct cross-link identifications at user-defined thresholds. These new features improve the versatility of the algorithm, enabling its use in a wider range of experimental designs and analysis pipelines. Kojak 2.0 remains open-source and multiplatform.


Subject(s)
Proteomics , Tandem Mass Spectrometry , Proteomics/methods , Tandem Mass Spectrometry/methods , Peptides/analysis , Proteins/chemistry , Software , Cross-Linking Reagents/chemistry
4.
EMBO J ; 42(8): e112600, 2023 04 17.
Article in English | MEDLINE | ID: mdl-36651597

ABSTRACT

Forcing budding yeast to chromatinize their DNA with human histones manifests an abrupt fitness cost. We previously proposed chromosomal aneuploidy and missense mutations as two potential modes of adaptation to histone humanization. Here, we show that aneuploidy in histone-humanized yeasts is specific to a subset of chromosomes that are defined by their centromeric evolutionary origins but that these aneuploidies are not adaptive. Instead, we find that a set of missense mutations in outer kinetochore proteins drives adaptation to human histones. Furthermore, we characterize the molecular mechanism underlying adaptation in two mutants of the outer kinetochore DASH/Dam1 complex, which reduce aneuploidy by suppression of chromosome instability. Molecular modeling and biochemical experiments show that these two mutants likely disrupt a conserved oligomerization interface thereby weakening microtubule attachments. We propose a model through which weakened microtubule attachments promote increased kinetochore-microtubule turnover and thus suppress chromosome instability. In sum, our data show how a set of point mutations evolved in histone-humanized yeasts to counterbalance human histone-induced chromosomal instability through weakening microtubule interactions, eventually promoting a return to euploidy.


Subject(s)
Kinetochores , Saccharomyces cerevisiae Proteins , Humans , Kinetochores/metabolism , Histones/genetics , Histones/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Cell Cycle Proteins/metabolism , Microtubules/metabolism , Chromosome Segregation/genetics , Ploidies , Aneuploidy
5.
Structure ; 30(9): 1269-1284.e6, 2022 09 01.
Article in English | MEDLINE | ID: mdl-35716664

ABSTRACT

RING-between-RING (RBR) E3 ligases mediate ubiquitin transfer through an obligate E3-ubiquitin thioester intermediate prior to substrate ubiquitination. Although RBRs share a conserved catalytic module, substrate recruitment mechanisms remain enigmatic, and the relevant domains have yet to be identified for any member of the class. Here we characterize the interaction between the auto-inhibited RBR, HHARI (AriH1), and its target protein, 4EHP, using a combination of XL-MS, HDX-MS, NMR, and biochemical studies. The results show that (1) a di-aromatic surface on the catalytic HHARI Rcat domain forms a binding platform for substrates and (2) a phosphomimetic mutation on the auto-inhibitory Ariadne domain of HHARI promotes release and reorientation of Rcat for transthiolation and substrate modification. The findings identify a direct binding interaction between a RING-between-RING ligase and its substrate and suggest a general model for RBR substrate recognition.


Subject(s)
Cullin Proteins , Ubiquitin , Catalytic Domain , Cullin Proteins/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitination
6.
J Cell Biol ; 221(5)2022 05 02.
Article in English | MEDLINE | ID: mdl-35353161

ABSTRACT

Accurate mitosis requires kinetochores to make persistent, load-bearing attachments to dynamic microtubule tips, thereby coupling chromosome movements to tip growth and shortening. This tip-coupling behavior depends on the conserved Ndc80 complex and, in budding yeast, on the Dam1 complex, which bind each other directly via three distinct interacting regions. The functional relevance of these multiple interactions was mysterious. Here we show that interactions between two of these regions support the high rupture strengths that occur when applied force is rapidly increased and also support the stability of tip-coupling when force is held constant over longer durations. The contribution of either of these two regions to tip-coupling is reduced by phosphorylation by Aurora B kinase. The third interaction region makes no apparent contribution to rupture strength, but its phosphorylation by Aurora B kinase specifically decreases the long-term stability of tip-coupling. The specific reduction of long-term stability relative to short-term strength might have important implications for mitotic error correction.


Subject(s)
Kinetochores , Microtubule-Associated Proteins , Microtubules , Mitosis , Saccharomyces cerevisiae Proteins , Aurora Kinase B/genetics , Aurora Kinase B/metabolism , Cell Cycle Proteins , Chromosome Segregation , Kinetochores/metabolism , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Nuclear Proteins , Phosphorylation , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
7.
Anal Chem ; 94(8): 3501-3509, 2022 03 01.
Article in English | MEDLINE | ID: mdl-35184559

ABSTRACT

Drugs are often metabolized to reactive intermediates that form protein adducts. Adducts can inhibit protein activity, elicit immune responses, and cause life-threatening adverse drug reactions. The masses of reactive metabolites are frequently unknown, rendering traditional mass spectrometry-based proteomics approaches incapable of adduct identification. Here, we present Magnum, an open-mass search algorithm optimized for adduct identification, and Limelight, a web-based data processing package for analysis and visualization of data from all existing algorithms. Limelight incorporates tools for sample comparisons and xenobiotic-adduct discovery. We validate our tools with three drug/protein combinations and apply our label-free workflow to identify novel xenobiotic-protein adducts in CYP3A4. Our new methods and software enable accurate identification of xenobiotic-protein adducts with no prior knowledge of adduct masses or protein targets. Magnum outperforms existing label-free tools in xenobiotic-protein adduct discovery, while Limelight fulfills a major need in the rapidly developing field of open-mass searching, which until now lacked comprehensive data visualization tools.


Subject(s)
Proteins , Proteomics , Algorithms , DNA Adducts , Mass Spectrometry/methods , Proteins/analysis , Proteomics/methods , Software
8.
Proc Natl Acad Sci U S A ; 119(8)2022 02 22.
Article in English | MEDLINE | ID: mdl-35173049

ABSTRACT

Kinesin-14 molecular motors represent an essential class of proteins that bind microtubules and walk toward their minus-ends. Previous studies have described important roles for Kinesin-14 motors at microtubule minus-ends, but their role in regulating plus-end dynamics remains controversial. Kinesin-14 motors have been shown to bind the EB family of microtubule plus-end binding proteins, suggesting that these minus-end-directed motors could interact with growing microtubule plus-ends. In this work, we explored the role of minus-end-directed Kinesin-14 motor forces in controlling plus-end microtubule dynamics. In cells, a Kinesin-14 mutant with reduced affinity to EB proteins led to increased microtubule lengths. Cell-free biophysical microscopy assays were performed using Kinesin-14 motors and an EB family marker of growing microtubule plus-ends, Mal3, which revealed that when Kinesin-14 motors bound to Mal3 at growing microtubule plus-ends, the motors subsequently walked toward the minus-end, and Mal3 was pulled away from the growing microtubule tip. Strikingly, these interactions resulted in an approximately twofold decrease in the expected postinteraction microtubule lifetime. Furthermore, generic minus-end-directed tension forces, generated by tethering growing plus-ends to the coverslip using λ-DNA, led to an approximately sevenfold decrease in the expected postinteraction microtubule growth length. In contrast, the inhibition of Kinesin-14 minus-end-directed motility led to extended tip interactions and to an increase in the expected postinteraction microtubule lifetime, indicating that plus-ends were stabilized by nonmotile Kinesin-14 motors. Together, we find that Kinesin-14 motors participate in a force balance at microtubule plus-ends to regulate microtubule lengths in cells.


Subject(s)
Kinesins/metabolism , Microtubules/physiology , Chromosome Segregation , Kinesins/physiology , Microtubule Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Protein Binding , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Spindle Apparatus/metabolism
9.
Oncoscience ; 8: 134-153, 2021.
Article in English | MEDLINE | ID: mdl-34926718

ABSTRACT

Hec1 (Highly expressed in cancer 1) resides in the outer kinetochore where it works to facilitate proper kinetochore-microtubule interactions during mitosis. Hec1 is overexpressed in various cancers and its expression shows correlation with high tumour grade and poor patient prognosis. Chemical perturbation of Hec1 is anticipated to impair kinetochore-microtubule binding, activate the spindle assembly checkpoint (spindle checkpoint) and thereby suppress cell proliferation. In this study, we performed high-throughput screen to identify novel small molecules that target the Hec1 calponin homology domain (CHD), which is needed for normal microtubule attachments. 4 million compounds were first virtually fitted against the CHD, and the best hit molecules were evaluated in vitro. These approaches led to the identification of VTT-006, a 1,2-disubstituted-tetrahydro-beta-carboline derivative, which showed binding to recombinant Ndc80 complex and modulated Hec1 association with microtubules in vitro. VTT-006 treatment resulted in chromosome congression defects, reduced chromosome oscillations and induced loss of inter-kinetochore tension. Cells remained arrested in mitosis with an active spindle checkpoint for several hours before undergoing cell death. VTT-006 suppressed the growth of several cancer cell lines and enhanced the sensitivity of HeLa cells to Taxol. Our findings propose that VTT-006 is a potential anti-mitotic compound that disrupts M phase, impairs kinetochore-microtubule interactions, and activates the spindle checkpoint.

10.
Science ; 374(6564): 157, 2021 Oct 08.
Article in English | MEDLINE | ID: mdl-34618572

ABSTRACT

A biochemical virtuoso.

11.
Elife ; 102021 05 05.
Article in English | MEDLINE | ID: mdl-33949948

ABSTRACT

Microtubule (MT) nucleation is regulated by the γ-tubulin ring complex (γTuRC), conserved from yeast to humans. In Saccharomyces cerevisiae, γTuRC is composed of seven identical γ-tubulin small complex (γTuSC) sub-assemblies, which associate helically to template MT growth. γTuRC assembly provides a key point of regulation for the MT cytoskeleton. Here, we combine crosslinking mass spectrometry, X-ray crystallography, and cryo-EM structures of both monomeric and dimeric γTuSCs, and open and closed helical γTuRC assemblies in complex with Spc110p to elucidate the mechanisms of γTuRC assembly. γTuRC assembly is substantially aided by the evolutionarily conserved CM1 motif in Spc110p spanning a pair of adjacent γTuSCs. By providing the highest resolution and most complete views of any γTuSC assembly, our structures allow phosphorylation sites to be mapped, surprisingly suggesting that they are mostly inhibitory. A comparison of our structures with the CM1 binding site in the human γTuRC structure at the interface between GCP2 and GCP6 allows for the interpretation of significant structural changes arising from CM1 helix binding to metazoan γTuRC.


Subject(s)
Antigens, Nuclear/genetics , Microtubules/physiology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Tubulin/chemistry , Tubulin/genetics , Binding Sites , Calmodulin-Binding Proteins/genetics , Calmodulin-Binding Proteins/metabolism , Cryoelectron Microscopy/methods , Crystallography, X-Ray/methods , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Humans , Mass Spectrometry/methods , Microtubule-Organizing Center , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Tubulin/classification , Tubulin/metabolism
12.
Genetics ; 218(2)2021 06 24.
Article in English | MEDLINE | ID: mdl-33752231

ABSTRACT

The mitotic spindle is resilient to perturbation due to the concerted, and sometimes redundant, action of motors and microtubule-associated proteins. Here, we utilize an inducible ectopic microtubule nucleation site in the nucleus of Saccharomyces cerevisiae to study three necessary steps in the formation of a bipolar array: the recruitment of the γ-tubulin complex, nucleation and elongation of microtubules (MTs), and the organization of MTs relative to each other. This novel tool, an Spc110 chimera, reveals previously unreported roles of the microtubule-associated proteins Stu2, Bim1, and Bik1, and the motors Vik1 and Kip3. We report that Stu2 and Bim1 are required for nucleation and that Bik1 and Kip3 promote nucleation at the ectopic site. Stu2, Bim1, and Kip3 join their homologs XMAP215, EB1 and kinesin-8 as promoters of microtubule nucleation, while Bik1 promotes MT nucleation indirectly via its role in SPB positioning. Furthermore, we find that the nucleation activity of Stu2 in vivo correlates with its polymerase activity in vitro. Finally, we provide the first evidence that Vik1, a subunit of Kar3/Vik1 kinesin-14, promotes microtubule minus end focusing at the ectopic site.


Subject(s)
Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Molecular Motor Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/physiology , Cell Nucleus/metabolism , Microtubule-Associated Proteins/genetics , Mitosis , Molecular Motor Proteins/genetics , Mutation , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae Proteins/genetics , Spindle Apparatus/metabolism
13.
J Cell Biol ; 220(3)2021 03 01.
Article in English | MEDLINE | ID: mdl-33464308

ABSTRACT

To assemble a bipolar spindle, microtubules emanating from two poles must bundle into an antiparallel midzone, where plus end-directed motors generate outward pushing forces to drive pole separation. Midzone cross-linkers and motors display only modest preferences for antiparallel filaments, and duplicated poles are initially tethered together, an arrangement that instead favors parallel interactions. Pivoting of microtubules around spindle poles might help overcome this geometric bias, but the intrinsic pivoting flexibility of the microtubule-pole interface has not been directly measured, nor has its importance during early spindle assembly been tested. By measuring the pivoting of microtubules around isolated yeast spindle poles, we show that pivoting flexibility can be modified by mutating a microtubule-anchoring pole component, Spc110. By engineering mutants with different flexibilities, we establish the importance of pivoting in vivo for timely pole separation. Our results suggest that passive thermal pivoting can bring microtubules from side-by-side poles into initial contact, but active minus end-directed force generation will be needed to achieve antiparallel alignment.


Subject(s)
Microtubules/metabolism , Mitosis , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Spindle Apparatus/metabolism , Genetic Engineering , Mutation/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/metabolism , Spindle Pole Bodies/metabolism , Torsion, Mechanical
14.
Curr Biol ; 30(22): 4491-4499.e5, 2020 11 16.
Article in English | MEDLINE | ID: mdl-32946748

ABSTRACT

To ensure the faithful inheritance of DNA, a macromolecular protein complex called the kinetochore sustains the connection between chromosomes and force-generating dynamic microtubules during cell division. Defects in this process lead to aneuploidy, a common feature of cancer cells and the cause of many developmental diseases [1-4]. One of the major microtubule-binding activities in the kinetochore is mediated by the conserved Ndc80 complex (Ndc80c) [5-7]. In budding yeast, the retention of kinetochores on dynamic microtubule tips also depends on the essential heterodecameric Dam1 complex (Dam1c) [8-15], which binds to the Ndc80c and is proposed to be a functional ortholog of the metazoan Ska complex [16, 17]. The load-bearing activity of the Dam1c depends on its ability to oligomerize, and the purified complex spontaneously self-assembles into microtubule-encircling oligomeric rings, which are proposed to function as collars that allow kinetochores to processively track the plus-end tips of microtubules and harness the forces generated by disassembling microtubules [10-15, 18-22]. However, it is unknown whether there are specific regulatory events that promote Dam1c oligomerization to ensure accurate segregation. Here, we used a reconstitution system to discover that Cdk1, the major mitotic kinase that drives the cell cycle, phosphorylates the Ask1 component of the Dam1c to increase its residence time on microtubules and enhance kinetochore-microtubule attachment strength. We propose that Cdk1 activity promotes Dam1c oligomerization to ensure that kinetochore-microtubule attachments are stabilized as kinetochores come under tension in mitosis.


Subject(s)
CDC28 Protein Kinase, S cerevisiae/metabolism , Cell Cycle Proteins/metabolism , Kinetochores/metabolism , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Chromosome Segregation , Enzyme Assays , Microtubule-Associated Proteins/genetics , Mitosis , Mutation , Phosphorylation/physiology , Protein Multimerization/physiology , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/genetics
15.
Mol Biol Cell ; 31(20): 2187-2194, 2020 09 15.
Article in English | MEDLINE | ID: mdl-32726183

ABSTRACT

Microtubule nucleation is spatiotemporally regulated in cells by several known molecules, including the template γ-tubulin and the polymerase XMAP215. The role of XMAP215 in nucleation is under debate, specifically whether it acts independently as a polymerase or acts dependently with γ-tubulin. We first confirm XMAP215 as a classically defined nucleator that reduces the nucleation lag seen in bulk tubulin assembly. Secondly, using deletion constructs, we probe the domain requirements for XMAP215 to promote microtubule nucleation. We show that its ability to nucleate microtubules in purified solutions correlates with its ability to elongate existing microtubules and does not depend on the number of tumor overexpressed gene (TOG) domains. Finally, we show that XMAP215 and γ-tubulin promote αß-tubulin assembly in an additive, not synergistic, manner. Thus, their modes of action during microtubule nucleation are distinct. These findings suggest there are at least two independent processes in nucleation, one promoted by γ-tubulin and one promoted by XMAP215. We propose that XMAP215 accelerates the addition of subunits to existing nucleation intermediates formed either spontaneously or by oligomers of γ-tubulin. [Media: see text].


Subject(s)
Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Tubulin/metabolism , Animals , Humans , Microtubule-Associated Proteins/chemistry , Microtubules/chemistry , Protein Aggregates/physiology , Protein Binding/physiology , Tubulin/chemistry
16.
Elife ; 92020 05 14.
Article in English | MEDLINE | ID: mdl-32406818

ABSTRACT

Partitioning duplicated chromosomes equally between daughter cells is a microtubule-mediated process essential to eukaryotic life. A multi-protein machine, the kinetochore, drives chromosome segregation by coupling the chromosomes to dynamic microtubule tips, even as the tips grow and shrink through the gain and loss of subunits. The kinetochore must harness, transmit, and sense mitotic forces, as a lack of tension signals incorrect chromosome-microtubule attachment and precipitates error correction mechanisms. But though the field has arrived at a 'parts list' of dozens of kinetochore proteins organized into subcomplexes, the path of force transmission through these components has remained unclear. Here we report reconstitution of functional Saccharomyces cerevisiae kinetochore assemblies from recombinantly expressed proteins. The reconstituted kinetochores are capable of self-assembling in vitro, coupling centromeric nucleosomes to dynamic microtubules, and withstanding mitotically relevant forces. They reveal two distinct pathways of force transmission and Ndc80c recruitment.


Subject(s)
Chromosome Segregation , Chromosomes, Fungal , Kinetochores/metabolism , Mechanotransduction, Cellular , Saccharomyces cerevisiae/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Microtubules/genetics , Microtubules/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleosomes/genetics , Nucleosomes/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Stress, Mechanical
17.
PLoS Genet ; 15(10): e1008423, 2019 10.
Article in English | MEDLINE | ID: mdl-31584935

ABSTRACT

Accurate segregation of chromosomes to daughter cells is a critical aspect of cell division. It requires the kinetochores on duplicated chromosomes to biorient, attaching to microtubules from opposite poles of the cell. Bioriented attachments come under tension, while incorrect attachments lack tension and must be released to allow proper attachments to form. A well-studied error correction pathway is mediated by the Aurora B kinase, which destabilizes low tension-bearing attachments. We recently discovered that in vitro, kinetochores display an additional intrinsic tension-sensing pathway that utilizes Stu2. The contribution of kinetochore-associated Stu2 to error correction in cells, however, was unknown. Here, we identify a Stu2 mutant that abolishes its kinetochore function and show that it causes biorientation defects in vivo. We also show that this Stu2-mediated pathway functions together with the Aurora B-mediated pathway. Altogether, our work indicates that cells employ multiple pathways to ensure biorientation and the accuracy of chromosome segregation.


Subject(s)
Aurora Kinases/metabolism , Chromosome Segregation , Kinetochores/metabolism , Microtubule-Associated Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Aurora Kinases/genetics , Microtubule-Associated Proteins/genetics , Microtubules , Mutation , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics
18.
Anal Chem ; 91(11): 6953-6961, 2019 06 04.
Article in English | MEDLINE | ID: mdl-31045356

ABSTRACT

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.


Subject(s)
Cross-Linking Reagents/chemistry , Mass Spectrometry/methods , Serum Albumin, Bovine/analysis , Serum Albumin, Bovine/chemistry , Laboratories , Mass Spectrometry/instrumentation , Reproducibility of Results
19.
Elife ; 82019 05 02.
Article in English | MEDLINE | ID: mdl-31045495

ABSTRACT

Regulation of the outer kinetochore complex Ndc80 is essential to ensure correct kinetochore-microtubule attachments during mitosis. Here, we present a novel mechanism of regulation that is intrinsic to its structure; tight bending of the Ndc80 complex inhibits its microtubule binding. Using single molecule Förster resonance energy transfer (FRET), we show that the Saccharomyces cerevisiae Ndc80 complex can fluctuate between straight and bent forms, and that binding of the complex to microtubules selects for straightened forms. The loop region of the complex enables its bent conformation, as deletion of the loop promotes straightening. In addition, the kinetochore complex MIND enhances microtubule binding by opposing the tightly bent, auto-inhibited conformation of the Ndc80 complex. We suggest that prior to its assembly at the kinetochore, the Ndc80 complex interchanges between bent (auto-inhibited) and open conformations. Once assembled, its association with MIND stabilizes the Ndc80 complex in a straightened form for higher affinity microtubule binding.


Subject(s)
Kinetochores/chemistry , Kinetochores/metabolism , Microtubules/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Fluorescence Resonance Energy Transfer , Mitosis , Protein Binding , Protein Conformation , Saccharomyces cerevisiae/growth & development , Single Molecule Imaging
20.
Curr Opin Cell Biol ; 60: 44-52, 2019 10.
Article in English | MEDLINE | ID: mdl-31078123

ABSTRACT

This review highlights three recent trends in the field of kinetochore biology: the proliferation of structural data for kinetochore protein complexes (including CBF3, Dam1c, Mis12cMIND, and CENP-NLChl4/Iml3); the growing consensus that the kinetochore is a dynamic structure whose composition changes as the cell cycle progresses; and the mounting evidence of multiple pathways whereby the microtubule-binding elements of the outer kinetochore may be recruited by inner kinetochore proteins. Our focus is on the two best-studied systems in the field: human and budding yeast kinetochores. This review will demonstrate the remarkable similarity of these two systems, as well as their intriguing differences.


Subject(s)
Kinetochores/metabolism , Animals , Cell Cycle , Humans , Models, Biological , Models, Molecular , Saccharomyces cerevisiae Proteins/metabolism , Saccharomycetales/metabolism
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