Publications by authors named "Josh Lawrimore"

21 Publications

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The rDNA is biomolecular condensate formed by polymer-polymer phase separation and is sequestered in the nucleolus by transcription and R-loops.

Nucleic Acids Res 2021 05;49(8):4586-4598

Biology Department, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

The nucleolus is the site of ribosome biosynthesis encompassing the ribosomal DNA (rDNA) locus in a phase separated state within the nucleus. In budding yeast, we find the rDNA locus and Cdc14, a protein phosphatase that co-localizes with the rDNA, behave like a condensate formed by polymer-polymer phase separation, while ribonucleoproteins behave like a condensate formed by liquid-liquid phase separation. The compaction of the rDNA and Cdc14's nucleolar distribution are dependent on the concentration of DNA cross-linkers. In contrast, ribonucleoprotein nucleolar distribution is independent of the concentration of DNA cross-linkers and resembles droplets in vivo upon replacement of the endogenous rDNA locus with high-copy plasmids. When ribosomal RNA is transcribed from the plasmids by Pol II, the rDNA-binding proteins and ribonucleoprotein signals are weakly correlated, but upon repression of transcription, ribonucleoproteins form a single, stable droplet that excludes rDNA-binding proteins from its center. Degradation of RNA-DNA hybrid structures, known as R-loops, by overexpression of RNase H1 results in the physical exclusion of the rDNA locus from the nucleolar center. Thus, the rDNA locus is a polymer-polymer phase separated condensate that relies on transcription and physical contact with RNA transcripts to remain encapsulated within the nucleolus.
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http://dx.doi.org/10.1093/nar/gkab229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8096216PMC
May 2021

Performance of deep learning restoration methods for the extraction of particle dynamics in noisy microscopy image sequences.

Mol Biol Cell 2021 04 27;32(9):903-914. Epub 2021 Jan 27.

Department of Physics, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202.

Particle tracking in living systems requires low light exposure and short exposure times to avoid phototoxicity and photobleaching and to fully capture particle motion with high-speed imaging. Low-excitation light comes at the expense of tracking accuracy. Image restoration methods based on deep learning dramatically improve the signal-to-noise ratio in low-exposure data sets, qualitatively improving the images. However, it is not clear whether images generated by these methods yield accurate quantitative measurements such as diffusion parameters in (single) particle tracking experiments. Here, we evaluate the performance of two popular deep learning denoising software packages for particle tracking, using synthetic data sets and movies of diffusing chromatin as biological examples. With synthetic data, both supervised and unsupervised deep learning restored particle motions with high accuracy in two-dimensional data sets, whereas artifacts were introduced by the denoisers in three-dimensional data sets. Experimentally, we found that, while both supervised and unsupervised approaches improved tracking results compared with the original noisy images, supervised learning generally outperformed the unsupervised approach. We find that nicer-looking image sequences are not synonymous with more precise tracking results and highlight that deep learning algorithms can produce deceiving artifacts with extremely noisy images. Finally, we address the challenge of selecting parameters to train convolutional neural networks by implementing a frugal Bayesian optimizer that rapidly explores multidimensional parameter spaces, identifying networks yielding optimal particle tracking accuracy. Our study provides quantitative outcome measures of image restoration using deep learning. We anticipate broad application of this approach to critically evaluate artificial intelligence solutions for quantitative microscopy.
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http://dx.doi.org/10.1091/mbc.E20-11-0689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8108534PMC
April 2021

Statistical mechanics of chromosomes: in vivo and in silico approaches reveal high-level organization and structure arise exclusively through mechanical feedback between loop extruders and chromatin substrate properties.

Nucleic Acids Res 2020 11;48(20):11284-11303

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

The revolution in understanding higher order chromosome dynamics and organization derives from treating the chromosome as a chain polymer and adapting appropriate polymer-based physical principles. Using basic principles, such as entropic fluctuations and timescales of relaxation of Rouse polymer chains, one can recapitulate the dominant features of chromatin motion observed in vivo. An emerging challenge is to relate the mechanical properties of chromatin to more nuanced organizational principles such as ubiquitous DNA loops. Toward this goal, we introduce a real-time numerical simulation model of a long chain polymer in the presence of histones and condensin, encoding physical principles of chromosome dynamics with coupled histone and condensin sources of transient loop generation. An exact experimental correlate of the model was obtained through analysis of a model-matching fluorescently labeled circular chromosome in live yeast cells. We show that experimentally observed chromosome compaction and variance in compaction are reproduced only with tandem interactions between histone and condensin, not from either individually. The hierarchical loop structures that emerge upon incorporation of histone and condensin activities significantly impact the dynamic and structural properties of chromatin. Moreover, simulations reveal that tandem condensin-histone activity is responsible for higher order chromosomal structures, including recently observed Z-loops.
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http://dx.doi.org/10.1093/nar/gkaa871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672462PMC
November 2020

Polymer perspective of genome mobilization.

Mutat Res 2020 May - Dec;821:111706. Epub 2020 May 26.

Department of Biology, 623 Fordham Hall CB#3280, University of North Carolina, Chapel Hill, NC 27599-3280, United States. Electronic address:

Chromosome motion is an intrinsic feature of all DNA-based metabolic processes and is a particularly well-documented response to both DNA damage and repair. By using both biological and polymer physics approaches, many of the contributing factors of chromatin motility have been elucidated. These include the intrinsic properties of chromatin, such as stiffness, as well as the loop modulators condensin and cohesin. Various biological factors such as external tethering to nuclear domains, ATP-dependent processes, and nucleofilaments further impact chromatin motion. DNA damaging agents that induce double-stranded breaks also cause increased chromatin motion that is modulated by recruitment of repair and checkpoint proteins. Approaches that integrate biological experimentation in conjunction with models from polymer physics provide mechanistic insights into the role of chromatin dynamics in biological function. In this review we discuss the polymer models and the effects of both DNA damage and repair on chromatin motion as well as mechanisms that may underlie these effects.
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http://dx.doi.org/10.1016/j.mrfmmm.2020.111706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721199PMC
December 2020

AI-Assisted Forward Modeling of Biological Structures.

Front Cell Dev Biol 2019 14;7:279. Epub 2019 Nov 14.

Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, United States.

The rise of machine learning and deep learning technologies have allowed researchers to automate image classification. We describe a method that incorporates automated image classification and principal component analysis to evaluate computational models of biological structures. We use a computational model of the kinetochore to demonstrate our artificial-intelligence (AI)-assisted modeling method. The kinetochore is a large protein complex that connects chromosomes to the mitotic spindle to facilitate proper cell division. The kinetochore can be divided into two regions: the inner kinetochore, including proteins that interact with DNA; and the outer kinetochore, comprised of microtubule-binding proteins. These two kinetochore regions have been shown to have different distributions during metaphase in live budding yeast and therefore act as a test case for our forward modeling technique. We find that a simple convolutional neural net (CNN) can correctly classify fluorescent images of inner and outer kinetochore proteins and show a CNN trained on simulated, fluorescent images can detect difference in experimental images. A polymer model of the ribosomal DNA locus serves as a second test for the method. The nucleolus surrounds the ribosomal DNA locus and appears amorphous in live-cell, fluorescent microscopy experiments in budding yeast, making detection of morphological changes challenging. We show a simple CNN can detect subtle differences in simulated images of the ribosomal DNA locus, demonstrating our CNN-based classification technique can be used on a variety of biological structures.
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http://dx.doi.org/10.3389/fcell.2019.00279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868055PMC
November 2019

The regulation of chromosome segregation via centromere loops.

Crit Rev Biochem Mol Biol 2019 08 1;54(4):352-370. Epub 2019 Oct 1.

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Biophysical studies of the yeast centromere have shown that the organization of the centromeric chromatin plays a crucial role in maintaining proper tension between sister kinetochores during mitosis. While centromeric chromatin has traditionally been considered a simple spring, recent work reveals the centromere as a multifaceted, tunable shock absorber. Centromeres can differ from other regions of the genome in their heterochromatin state, supercoiling state, and enrichment of structural maintenance of chromosomes (SMC) protein complexes. Each of these differences can be utilized to alter the effective stiffness of centromeric chromatin. In budding yeast, the SMC protein complexes condensin and cohesin stiffen chromatin by forming and cross-linking chromatin loops, respectively, into a fibrous structure resembling a bottlebrush. The high density of the loops compacts chromatin while spatially isolating the tension from spindle pulling forces to a subset of the chromatin. Paradoxically, the molecular crowding of chromatin via cohesin and condensin also causes an outward/poleward force. The structure allows the centromere to act as a shock absorber that buffers the variable forces generated by dynamic spindle microtubules. Based on the distribution of SMCs from bacteria to human and the conserved distance between sister kinetochores in a wide variety of organisms (0.4 to 1 micron), we propose that the bottlebrush mechanism is the foundational principle for centromere function in eukaryotes.
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http://dx.doi.org/10.1080/10409238.2019.1670130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856439PMC
August 2019

Transient crosslinking kinetics optimize gene cluster interactions.

PLoS Comput Biol 2019 08 21;15(8):e1007124. Epub 2019 Aug 21.

Department of Mathematics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

Our understanding of how chromosomes structurally organize and dynamically interact has been revolutionized through the lens of long-chain polymer physics. Major protein contributors to chromosome structure and dynamics are condensin and cohesin that stochastically generate loops within and between chains, and entrap proximal strands of sister chromatids. In this paper, we explore the ability of transient, protein-mediated, gene-gene crosslinks to induce clusters of genes, thereby dynamic architecture, within the highly repeated ribosomal DNA that comprises the nucleolus of budding yeast. We implement three approaches: live cell microscopy; computational modeling of the full genome during G1 in budding yeast, exploring four decades of timescales for transient crosslinks between 5kbp domains (genes) in the nucleolus on Chromosome XII; and, temporal network models with automated community (cluster) detection algorithms applied to the full range of 4D modeling datasets. The data analysis tools detect and track gene clusters, their size, number, persistence time, and their plasticity (deformation). Of biological significance, our analysis reveals an optimal mean crosslink lifetime that promotes pairwise and cluster gene interactions through "flexible" clustering. In this state, large gene clusters self-assemble yet frequently interact (merge and separate), marked by gene exchanges between clusters, which in turn maximizes global gene interactions in the nucleolus. This regime stands between two limiting cases each with far less global gene interactions: with shorter crosslink lifetimes, "rigid" clustering emerges with clusters that interact infrequently; with longer crosslink lifetimes, there is a dissolution of clusters. These observations are compared with imaging experiments on a normal yeast strain and two condensin-modified mutant cell strains. We apply the same image analysis pipeline to the experimental and simulated datasets, providing support for the modeling predictions.
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http://dx.doi.org/10.1371/journal.pcbi.1007124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6730938PMC
August 2019

Three-Dimensional Thermodynamic Simulation of Condensin as a DNA-Based Translocase.

Methods Mol Biol 2019 ;2004:291-318

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Chromatin dynamics and organization can be altered by condensin complexes. In turn, the molecular behavior of a condensin complex changes based on the tension of the substrate to which condensin is bound. This interplay between chromatin organization and condensin behavior demonstrates the need for tools that allows condensin complexes to be observed on a variety of chromatin organizations. We provide a method for simulating condensin complexes on a dynamic polymer substrate using the polymer dynamics simulator ChromoShake and the condensin simulator RotoStep. These simulations can be converted into simulated fluorescent images that are able to be directly compared to experimental images of condensin and fluorescently labeled chromatin. Our pipeline enables users to explore how changes in condensin behavior alters chromatin dynamics and vice versa while providing simulated image datasets that can be directly compared to experimental observations.
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http://dx.doi.org/10.1007/978-1-4939-9520-2_21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904244PMC
March 2020

tRNA Genes Affect Chromosome Structure and Function via Local Effects.

Mol Cell Biol 2019 04 2;39(8). Epub 2019 Apr 2.

Department of MCD Biology, University of California, Santa Cruz, California, USA

The genome is packaged and organized in an ordered, nonrandom manner, and specific chromatin segments contact nuclear substructures to mediate this organization. tRNA genes (tDNAs) are binding sites for transcription factors and architectural proteins and are thought to play an important role in the organization of the genome. In this study, we investigate the roles of tDNAs in genomic organization and chromosome function by editing a chromosome so that it lacked any tDNAs. Surprisingly our analyses of this tDNA-less chromosome show that loss of tDNAs does not grossly affect chromatin architecture or chromosome tethering and mobility. However, loss of tDNAs affects local nucleosome positioning and the binding of SMC proteins at these loci. The absence of tDNAs also leads to changes in centromere clustering and a reduction in the frequency of long-range heterochromatin clustering with concomitant effects on gene silencing. We propose that the tDNAs primarily affect local chromatin structure, which results in effects on long-range chromosome architecture.
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http://dx.doi.org/10.1128/MCB.00432-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6447413PMC
April 2019

Fork pausing allows centromere DNA loop formation and kinetochore assembly.

Proc Natl Acad Sci U S A 2018 11 29;115(46):11784-11789. Epub 2018 Oct 29.

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280

De novo kinetochore assembly, but not template-directed assembly, is dependent on COMA, the kinetochore complex engaged in cohesin recruitment. The slowing of replication fork progression by treatment with phleomycin (PHL), hydroxyurea, or deletion of the replication fork protection protein Csm3 can activate de novo kinetochore assembly in COMA mutants. Centromere DNA looping at the site of de novo kinetochore assembly can be detected shortly after exposure to PHL. Using simulations to explore the thermodynamics of DNA loops, we propose that loop formation is disfavored during bidirectional replication fork migration. One function of replication fork stalling upon encounters with DNA damage or other blockades may be to allow time for thermal fluctuations of the DNA chain to explore numerous configurations. Biasing thermodynamics provides a mechanism to facilitate macromolecular assembly, DNA repair, and other nucleic acid transactions at the replication fork. These loop configurations are essential for sister centromere separation and kinetochore assembly in the absence of the COMA complex.
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http://dx.doi.org/10.1073/pnas.1806791115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6243264PMC
November 2018

Geometric partitioning of cohesin and condensin is a consequence of chromatin loops.

Mol Biol Cell 2018 11 12;29(22):2737-2750. Epub 2018 Sep 12.

Biology Department, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.

SMC (structural maintenance of chromosomes) complexes condensin and cohesin are crucial for proper chromosome organization. Condensin has been reported to be a mechanochemical motor capable of forming chromatin loops, while cohesin passively diffuses along chromatin to tether sister chromatids. In budding yeast, the pericentric region is enriched in both condensin and cohesin. As in higher-eukaryotic chromosomes, condensin is localized to the axial chromatin of the pericentric region, while cohesin is enriched in the radial chromatin. Thus, the pericentric region serves as an ideal model for deducing the role of SMC complexes in chromosome organization. We find condensin-mediated chromatin loops establish a robust chromatin organization, while cohesin limits the area that chromatin loops can explore. Upon biorientation, extensional force from the mitotic spindle aggregates condensin-bound chromatin from its equilibrium position to the axial core of pericentric chromatin, resulting in amplified axial tension. The axial localization of condensin depends on condensin's ability to bind to chromatin to form loops, while the radial localization of cohesin depends on cohesin's ability to diffuse along chromatin. The different chromatin-tethering modalities of condensin and cohesin result in their geometric partitioning in the presence of an extensional force on chromatin.
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http://dx.doi.org/10.1091/mbc.E18-02-0131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249845PMC
November 2018

RotoStep: A Chromosome Dynamics Simulator Reveals Mechanisms of Loop Extrusion.

Cold Spring Harb Symp Quant Biol 2017 22;82:101-109. Epub 2017 Nov 22.

Department of Biology, University of North Carolina at Chapel Hill, North Carolina 27599-3280.

ChromoShake is a three-dimensional simulator designed to explore the range of configurational states a chromosome can adopt based on thermodynamic fluctuations of the polymer chain. Here, we refine ChromoShake to generate dynamic simulations of a DNA-based motor protein such as condensin walking along the chromatin substrate. We model walking as a rotation of DNA-binding heat-repeat proteins around one another. The simulation is applied to several configurations of DNA to reveal the consequences of mechanical stepping on taut chromatin under tension versus loop extrusion on single-tethered, floppy chromatin substrates. These simulations provide testable hypotheses for condensin and other DNA-based motors functioning along interphase chromosomes. Our model reveals a novel mechanism for condensin enrichment in the pericentromeric region of mitotic chromosomes. Increased condensin dwell time at centromeres results in a high density of pericentric loops that in turn provide substrate for additional condensin.
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http://dx.doi.org/10.1101/sqb.2017.82.033696DOI Listing
November 2017

Enrichment of dynamic chromosomal crosslinks drive phase separation of the nucleolus.

Nucleic Acids Res 2017 Nov;45(19):11159-11173

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Regions of highly repetitive DNA, such as those found in the nucleolus, show a self-organization that is marked by spatial segregation and frequent self-interaction. The mechanisms that underlie the sequestration of these sub-domains are largely unknown. Using a stochastic, bead-spring representation of chromatin in budding yeast, we find enrichment of protein-mediated, dynamic chromosomal cross-links recapitulates the segregation, morphology and self-interaction of the nucleolus. Rates and enrichment of dynamic crosslinking have profound consequences on domain morphology. Our model demonstrates the nucleolus is phase separated from other chromatin in the nucleus and predicts that multiple rDNA loci will form a single nucleolus independent of their location within the genome. Fluorescent labeling of budding yeast nucleoli with CDC14-GFP revealed that a split rDNA locus indeed forms a single nucleolus. We propose that nuclear sub-domains, such as the nucleolus, result from phase separations within the nucleus, which are driven by the enrichment of protein-mediated, dynamic chromosomal crosslinks.
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http://dx.doi.org/10.1093/nar/gkx741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737219PMC
November 2017

Microtubule dynamics drive enhanced chromatin motion and mobilize telomeres in response to DNA damage.

Mol Biol Cell 2017 Jun 27;28(12):1701-1711. Epub 2017 Apr 27.

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599

Chromatin exhibits increased mobility on DNA damage, but the biophysical basis for this behavior remains unknown. To explore the mechanisms that drive DNA damage-induced chromosome mobility, we use single-particle tracking of tagged chromosomal loci during interphase in live yeast cells together with polymer models of chromatin chains. Telomeres become mobilized from sites on the nuclear envelope and the pericentromere expands after exposure to DNA-damaging agents. The magnitude of chromatin mobility induced by a single double-strand break requires active microtubule function. These findings reveal how relaxation of external tethers to the nuclear envelope and internal chromatin-chromatin tethers, together with microtubule dynamics, can mobilize the genome in response to DNA damage.
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http://dx.doi.org/10.1091/mbc.E16-12-0846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5469612PMC
June 2017

Entropy gives rise to topologically associating domains.

Nucleic Acids Res 2016 07 2;44(12):5540-9. Epub 2016 Jun 2.

Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA

We investigate chromosome organization within the nucleus using polymer models whose formulation is closely guided by experiments in live yeast cells. We employ bead-spring chromosome models together with loop formation within the chains and the presence of nuclear bodies to quantify the extent to which these mechanisms shape the topological landscape in the interphase nucleus. By investigating the genome as a dynamical system, we show that domains of high chromosomal interactions can arise solely from the polymeric nature of the chromosome arms due to entropic interactions and nuclear confinement. In this view, the role of bio-chemical related processes is to modulate and extend the duration of the interacting domains.
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http://dx.doi.org/10.1093/nar/gkw510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4937343PMC
July 2016

SUMO-Targeted Ubiquitin Ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin.

Mol Biol Cell 2016 Mar 9. Epub 2016 Mar 9.

Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institute of Health, Bethesda, MD 20892.

Centromeric histone H3, CENP-A, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-A restricts its localization to centromeres. Mislocalization of CENP-A is associated with aneuploidy in yeast, flies and tumorigenesis in human cells; thus, defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast has shown that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here, we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by Small Ubiquitin-related Modifier (SUMO)-Targeted Ubiquitin Ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5∆ strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1 since slx5∆ psh1∆ strains exhibit higher levels of Cse4 stability and mislocalization compared to either slx5∆ or psh1∆ strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability.
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http://dx.doi.org/10.1091/mbc.E15-12-0827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850037PMC
March 2016

ChromoShake: a chromosome dynamics simulator reveals that chromatin loops stiffen centromeric chromatin.

Mol Biol Cell 2016 Jan 4;27(1):153-66. Epub 2015 Nov 4.

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280

ChromoShake is a three-dimensional simulator designed to find the thermodynamically favored states for given chromosome geometries. The simulator has been applied to a geometric model based on experimentally determined positions and fluctuations of DNA and the distribution of cohesin and condensin in the budding yeast centromere. Simulations of chromatin in differing initial configurations reveal novel principles for understanding the structure and function of a eukaryotic centromere. The entropic position of DNA loops mirrors their experimental position, consistent with their radial displacement from the spindle axis. The barrel-like distribution of cohesin complexes surrounding the central spindle in metaphase is a consequence of the size of the DNA loops within the pericentromere to which cohesin is bound. Linkage between DNA loops of different centromeres is requisite to recapitulate experimentally determined correlations in DNA motion. The consequences of radial loops and cohesin and condensin binding are to stiffen the DNA along the spindle axis, imparting an active function to the centromere in mitosis.
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http://dx.doi.org/10.1091/mbc.E15-08-0575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4694754PMC
January 2016

DNA loops generate intracentromere tension in mitosis.

J Cell Biol 2015 Aug;210(4):553-64

Department of Biology, University of North Carolina, Chapel Hill, NC 27599

The centromere is the DNA locus that dictates kinetochore formation and is visibly apparent as heterochromatin that bridges sister kinetochores in metaphase. Sister centromeres are compacted and held together by cohesin, condensin, and topoisomerase-mediated entanglements until all sister chromosomes bi-orient along the spindle apparatus. The establishment of tension between sister chromatids is essential for quenching a checkpoint kinase signal generated from kinetochores lacking microtubule attachment or tension. How the centromere chromatin spring is organized and functions as a tensiometer is largely unexplored. We have discovered that centromere chromatin loops generate an extensional/poleward force sufficient to release nucleosomes proximal to the spindle axis. This study describes how the physical consequences of DNA looping directly underlie the biological mechanism for sister centromere separation and the spring-like properties of the centromere in mitosis.
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http://dx.doi.org/10.1083/jcb.201502046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539978PMC
August 2015

Determining absolute protein numbers by quantitative fluorescence microscopy.

Methods Cell Biol 2014 ;123:347-65

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Biological questions are increasingly being addressed using a wide range of quantitative analytical tools to examine protein complex composition. Knowledge of the absolute number of proteins present provides insights into organization, function, and maintenance and is used in mathematical modeling of complex cellular dynamics. In this chapter, we outline and describe three microscopy-based methods for determining absolute protein numbers--fluorescence correlation spectroscopy, stepwise photobleaching, and ratiometric comparison of fluorescence intensity to known standards. In addition, we discuss the various fluorescently labeled proteins that have been used as standards for both stepwise photobleaching and ratiometric comparison analysis. A detailed procedure for determining absolute protein number by ratiometric comparison is outlined in the second half of this chapter. Counting proteins by quantitative microscopy is a relatively simple yet very powerful analytical tool that will increase our understanding of protein complex composition.
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http://dx.doi.org/10.1016/B978-0-12-420138-5.00019-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4221264PMC
February 2015

Spindle assembly checkpoint proteins are positioned close to core microtubule attachment sites at kinetochores.

J Cell Biol 2013 Sep 26;202(5):735-46. Epub 2013 Aug 26.

Department of Biology, The University of North Carolina-Chapel Hill, Chapel Hill, NC 27599, USA.

Spindle assembly checkpoint proteins have been thought to reside in the peripheral corona region of the kinetochore, distal to microtubule attachment sites at the outer plate. However, recent biochemical evidence indicates that checkpoint proteins are closely linked to the core kinetochore microtubule attachment site comprised of the Knl1-Mis12-Ndc80 (KMN) complexes/KMN network. In this paper, we show that the Knl1-Zwint1 complex is required to recruit the Rod-Zwilch-Zw10 (RZZ) and Mad1-Mad2 complexes to the outer kinetochore. Consistent with this, nanometer-scale mapping indicates that RZZ, Mad1-Mad2, and the C terminus of the dynein recruitment factor Spindly are closely juxtaposed with the KMN network in metaphase cells when their dissociation is blocked and the checkpoint is active. In contrast, the N terminus of Spindly is ∼75 nm outside the calponin homology domain of the Ndc80 complex. These results reveal how checkpoint proteins are integrated within the substructure of the kinetochore and will aid in understanding the coordination of microtubule attachment and checkpoint signaling during chromosome segregation.
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http://dx.doi.org/10.1083/jcb.201304197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3760617PMC
September 2013

Point centromeres contain more than a single centromere-specific Cse4 (CENP-A) nucleosome.

J Cell Biol 2011 Nov;195(4):573-82

Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA.

Cse4 is the budding yeast homologue of CENP-A, a modified histone H3 that specifies the base of kinetochores in all eukaryotes. Budding yeast is unique in having only one kinetochore microtubule attachment site per centromere. The centromere is specified by CEN DNA, a sequence-specific binding complex (CBF3), and a Cse4-containing nucleosome. Here we compare the ratio of kinetochore proximal Cse4-GFP fluorescence at anaphase to several standards including purified EGFP molecules in vitro to generate a calibration curve for the copy number of GFP-fusion proteins. Our results yield a mean of ~5 Cse4s, ~3 inner kinetochore CBF3 complexes, and ~20 outer kinetochore Ndc80 complexes. Our calibrated measurements increase 2.5-3-fold protein copy numbers at eukaryotic kinetochores based on previous ratio measurements assuming two Cse4s per budding yeast kinetochore. All approximately five Cse4s may be associated with the CEN nucleosome, but we show that a mean of three Cse4s could be located within flanking nucleosomes at random sites that differ between chromosomes.
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http://dx.doi.org/10.1083/jcb.201106036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257525PMC
November 2011
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