Biology subjects

Davis, T. N.

Publications and source records attributed to Davis, T. N..

11 recordsLinked to original sources

bioRxiv2024

Force transmission through the inner kinetochore is enhanced by centromeric DNA sequences

Previously, we reconstituted a minimal functional kinetochore from recombinant Saccharomyces cerevisiae proteins that was capable of transmitting force from dynamic microtubules to nucleosomes containing the centromere-specific histone variant Cse4 (Hamilton et al. 2020). This work revealed two paths of force transmission through the inner kinetochore: through Mif2 and through the Okp1/Ame1 complex (OA). Here, using a chimeric DNA sequence that contains crucial centromere-determining elements of the budding yeast point centromere, we demonstrate that the presence of centromeric DNA sequences in Cse4-containing nucleosomes significantly strengthens OA-mediated linkages. Our findings indicate that centromeric sequences are important for the transmission of microtubule-based forces to the chromosome.

biochemistry↗

bioRxiv2024

Limelight - An open, web-based tool for visualizing, sharing, and analyzing mass spectrometry data from DDA pipelines

Liquid chromatography-tandem mass spectrometry employing data-dependent acquisition (DDA) is a mature, widely used proteomics technique routinely applied to proteome profiling, protein-protein interaction studies, biomarker discovery, and protein modification analysis. Numerous tools exist for searching DDA data and myriad file formats are output as results. While some search and post processing tools include data visualization features to aid biological interpretation, they are often limited or tied to specific software pipelines. This restricts the accessibility, sharing and interpretation of data, and hinders comparison of results between different software pipelines. We developed Limelight, an easy-to-use, open-source, freely available tool that provides data sharing, analysis and visualization and is not tied to any specific software pipeline. Limelight is a data visualization tool specifically designed to provide access to the whole "data stack", from raw and annotated scan data to peptide-spectrum matches, quality control, peptides, proteins, and modifications. Limelight is designed from the ground up for sharing and collaboration and to support data from any DDA workflow. We provide tools to import data from many widely used open-mass and closed-mass search software workflows. Limelight helps maximize the utility of data by providing an easy-to-use interface for finding and interpreting data, all using the native scores from respective workflows.

bioinformatics↗

bioRxiv2022

DASH/Dam1 complex mutants stabilize ploidy by weakening kinetochore-microtubule attachments

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, defined by their centromeric evolutionary origins, however, they are not adaptive. Instead we show that a set of missense mutations in outer kineto-chore proteins drive adaptation to human histones. Further, we characterize the molecular mechanism of 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. Lastly, we show that one mutant, DAD1E50D, while suppressing chromosome instability in mitosis, leads to gross defects in meiosis. In sum, our data show how a set of point mutations evolved in the histone-humanized yeasts to counterbalance human histone induced chromosomal instability through weakening microtubule interactions, eventually promoting a return to euploidy.

genetics↗

bioRxiv2022

Multivalent BARD1-nucleosome interactions facilitate H2A ubiquitylation by BRCA1/BARD1

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.

biochemistry↗

bioRxiv2021

Discovery and visualization of uncharacterized drug-protein adducts using mass spectrometry.

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 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 two drug/protein combinations and apply our 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 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.

pharmacology and toxicology↗

bioRxiv2021

Microtubule associated proteins and motors required for ectopic microtubule array formation in S. cerevisiae

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 {gamma}-tubulin complex, nucleation and elongation of microtubules, and the organization of microtubules 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. Further, 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.

cell biology↗

bioRxiv2020

CM1-driven assembly and activation of Yeast γ-Tubulin Small Complex underlies microtubule nucleation

Microtubule (MT) nucleation is regulated by the {gamma}-tubulin ring complex ({gamma}TuRC), conserved from yeast to humans. In Saccharomyces cerevisiae, {gamma}TuRC is composed of seven identical {gamma}-tubulin small complex ({gamma}TuSC) sub-assemblies which associate helically to template microtubule growth. {gamma}TuRC assembly provides a key point of regulation for the MT cytoskeleton. Here we combine cross-linking mass spectrometry (XL-MS), X-ray crystallography and cryo-EM structures of both monomeric and dimeric {gamma}TuSCs, and open and closed helical {gamma}TuRC assemblies in complex with Spc110p to elucidate the mechanisms of {gamma}TuRC assembly. {gamma}TuRC assembly is substantially aided by the evolutionarily conserved CM1 motif in Spc110p spanning a pair of adjacent {gamma}TuSCs. By providing the highest resolution and most complete views of any {gamma}TuSC 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 {gamma}TuRC structure at the interface between GCP2 and GCP6 allows for the interpretation of significant structural changes arising from CM1 helix binding to metazoan {gamma}TuRC.

biochemistry↗

bioRxiv2020

XMAP215 and γ-tubulin additively promote microtubule nucleation in purified solutions

Microtubule nucleation is spatiotemporally regulated in cells by several molecules, including the template {gamma}-tubulin and the polymerase XMAP215. Recently, XMAP215 and the {gamma}-tubulin ring complex were reported to function synergistically, and this synergy was hypothesized to be due to direct binding between XMAP215 and {gamma}-tubulin. Here, we address this hypothesis by 1) probing domain requirements for XMAP215 to promote microtubule nucleation and 2) testing whether XMAP215 functions synergistically with {gamma}-tubulin in the absence of the other ring complex proteins. We confirm that {gamma}-tubulin and XMAP215 are classically defined nucleators that reduce the nucleation lag seen in bulk tubulin assembly. Then, using deletion constructs, we show that XMAP215s ability to nucleate microtubules in purified solutions correlates with its ability to elongate existing microtubules and does not depend on the number of TOG domains. Finally, we show that XMAP215 and {gamma}-tubulin promote {beta}-tubulin assembly in an additive, not synergistic, manner. Thus, their modes of action during microtubule nucleation are distinct, and the synergy reported between XMAP215 and the {gamma}-tubulin ring complex is not due to {gamma}-tubulin alone.

biochemistry↗

bioRxiv2018

Spc110 N-Terminal Domains Act Independently to Mediate Stable γ-Tubulin Small Complex Binding and γ-Tubulin Ring Complex Assembly

Microtubule (MT) nucleation in vivo is regulated by the {gamma}-tubulin ring complex ({gamma}TuRC), an approximately 2-megadalton complex conserved from yeast to humans. In Saccharomyces cerevisiae, {gamma}TuRC assembly is a key point of regulation over the MT cytoskeleton. Budding yeast {gamma}TuRC is composed of seven {gamma}-tubulin small complex ({gamma}TuSC) subassemblies which associate helically to form a template from which microtubules grow. This assembly process requires higher-order oligomers of the coiled-coil protein Spc110 to bind multiple {gamma}TuSCs, thereby stabilizing the otherwise low-affinity interface between {gamma}TuSCs. While Spc110 oligomerization is critical, its N-terminal domain (NTD) also plays a role that is poorly understood both functionally and structurally. In this work, we sought a mechanistic understanding of Spc110 NTD using a combination of structural and biochemical analyses. Through crosslinking-mass spectrometry (XL-MS), we determined that a segment of Spc110 coiled-coil is a major point of contact with {gamma}TuSC. We determined the structure of this coiled-coil segment by X-ray crystallography and used it in combination with our XL-MS dataset to generate an integrative structural model of the {gamma}TuSC-Spc110 complex. This structural model, in combination with biochemical analyses of Spc110 heterodimers lacking one NTD, suggests that the two NTDs within an Spc110 dimer act independently, one stabilizing association between Spc110 and {gamma}TuSC and the other stabilizing the interface between adjacent {gamma}TuSCs.

biochemistry↗

bioRxiv2018

Novel phosphorylation states of the yeast spindle pole body

Phosphorylation regulates yeast spindle pole body (SPB) duplication and separation and likely regulates microtubule nucleation. We report a phosphoproteomic analysis using tandem mass spectrometry of purified Saccharomyces cerevisiae SPBs for two cell cycle arrests, G1/S and the mitotic checkpoint, expanding on previously reported phosphoproteomic data sets. We present a novel phosphoproteomic state of SPBs arrested in G1/S by a cdc4-1 temperature sensitive mutation, with particular interest in phosphorylation events on the {gamma}-tubulin small complex ({gamma}-TuSC). The cdc4-1 arrest is the earliest arrest at which microtubule nucleation has occurred at the newly duplicated SPB. Several novel phosphorylation sites were identified in G1/S and during mitosis on the microtubule nucleating {gamma}-TuSC. These sites were analyzed in vivo by fluorescence microscopy and were shown to be required for proper regulation of spindle length. Additionally, in vivo analysis of two mitotic sites in Spc97 found that phosphorylation of at least one of these sites is required for progression through the cell cycle. This phosphoproteomic data set not only broadens the scope of the phosphoproteome of SPBs, it also identifies several {gamma}-TuSC phosphorylation sites influencing microtubule regulation.

biochemistry↗

bioRxiv2018

The human Ska complex and Ndc80 complex interact to form a load-bearing assembly that strengthens kinetochore-microtubule attachments

Accurate segregation of chromosomes relies on the force-bearing capabilities of the kinetochore to robustly attach chromosomes to dynamic microtubule tips. The human Ska complex and Ndc80 complex are outer-kinetochore components that bind microtubules and are required to fully stabilize kinetochore-microtubule attachments in vivo. While purified Ska complex tracks with disassembling microtubule tips, it remains unclear whether the Ska complex-microtubule interaction is sufficiently strong to make a significant contribution to kinetochore-microtubule coupling. Alternatively, Ska complex might affect kinetochore coupling indirectly, through recruitment of phospho-regulatory factors. Using optical tweezers, we show that the Ska complex itself bears load on microtubule tips, strengthens Ndc80 complex-based tip attachments, and increases the switching dynamics of the attached microtubule tips. Crosslinking mass spectrometry suggests the Ska complex directly binds Ndc80 complex through interactions between the Ska3 unstructured C-terminal region and the coiled-coil regions of each Ndc80 complex subunit. Deletion of the Ska complex microtubule-binding domain or the Ska3 C-terminus prevents Ska complex from strengthening Ndc80 complex-based attachments. Together our results indicate that the Ska complex can directly strengthen the kinetochore microtubule interface and regulate microtubule tip dynamics by forming an additional connection between the Ndc80 complex and the microtubule.\n\nSIGNIFICANCE STATEMENTMicrotubules are dynamic, tube-like structures that drive the segregation of duplicated chromosomes during cell division. The Ska complex is part of a molecular machine that forms force-bearing connections between chromosomes and microtubule ends. Depletion of the Ska complex destabilizes these connections and disrupts cell division. The Ska complex binds microtubules but it is unknown if it directly holds force at microtubules or indirectly stabilizes the connections. Here, we show that the Ska complex makes a direct force-bearing linkage with microtubule ends and assembles with another microtubule binding component, the Ndc80 complex, to strengthen its ability to withstand force. Our results suggest that the Ska and Ndc80 complexes work together to maintain the connections between chromosomes and microtubule ends.

biochemistry↗