Josh Lawrimore

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

Quantitative measurement of the number of Cse4, CBF3, and Ndc80 proteins at kinetochores reveals a 2.5–3-fold increased copy number relative to prior estimates.

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

Depletion analyses and nanometer-scale mapping of spindle assembly checkpoint proteins reveal how these proteins are integrated within the substructure of the kinetochore.

DNA loops generate intracentromere tension in mitosis

The geometry and arrangement of DNA loops in the pericentric region of the budding yeast centromere create a DNA-based molecular shock absorber that serves as the basis for how tension is generated between sister centromeres in mitosis.

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

Mechanisms that drive DNA damage-induced chromosome mobility include relaxation of external tethers to the nuclear envelope and internal chromatin–chromatin tethers. Together with microtubule dynamics, these can mobilize the genome in response to DNA damage.

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

A novel chromosome simulator recapitulates the position and dynamics of centromeric chromatin in a model composed of cross-linked intramolecular loops. Simulations reveal that chromatin loops stiffen the centromere and dictate the distribution of pericentric cohesin.

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

A new posttranslational modification is found of centromeric histone H3 variant Cse4. Cse4 is sumoylated by E3 ligases Siz1 and Siz2 and ubiquitinated by Slx5, a Sumo-targeted ubiquitin ligase. Slx5 regulates ubiquitin-mediated proteolysis of Cse4 and prevents it from being mislocalized under normal physiological conditions.

Determining absolute protein numbers by quantitative fluorescence microscopy.

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.

Entropy gives rise to topologically associating domains

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.

Enrichment of dynamic chromosomal crosslinks drive phase separation of the nucleolus

Abstract 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.

RotoStep: A Chromosome Dynamics Simulator Reveals Mechanisms of Loop Extrusion

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.