Regulators of Centromeric Nucleosomes in <i>Saccharomyces cerevisiae</i>

The centromere is a specialized chromosomal structure that regulates faithful chromosome segregation during cell division, as it dictates the site of assembly of the kineto-chore. In all organisms, the centromeric nucleosomes are specified by a H3 variant, known as Cse4 in budding yeast. How these centromeric nucleosomes are assembled and perpetuated is only beginning to be understood. Scm3 is an evolutionarily conserved essential inner kinetochore protein that has been shown to be important for centromere specification. Plasmid supercoiling assays performed in vitro with recombinant proteins demonstrate that Scm3 can act as a Cse4-specific nucleosome assembly factor. Assembly activity depends on an evolutionarily conserved domain of Scm3 and the centromere targeting domain (CATD) of Cse4, but is sequence independent. Interestingly, micrococcal nuclease digestion of Scm3 assembled Cse4 nucleosomes reveals that less DNA is protected compared to Nap 1 assembled Cse4 nucleosomes, suggesting structural differences. Fluorescence correlation spectroscopy in combination with brightness measurements and confocal imaging experiments in live cells revealed that centromeres at G1 phase have one copy of Cse4 per centromeric nucleosome whereas two copies are detected at anaphase. The apparent structural change occurs at the “metaphase to anaphase” transition. We propose a model in which the structure and composition of centromeric nucleosomes is cell cycle regulated. Our model reconciles experimental evidence for the existence of both the hemisome and the octasome

Structural plasticity of the living kinetochore

The kinetochore is a large, evolutionarily conserved protein structure that connects chromosomes with microtubules. During chromosome segregation, outer kinetochore components track depolymerizing ends of microtubules to facilitate the separation of chromosomes into two cells. In budding yeast, each chromosome has a point centromere upon which a single kinetochore is built, which attaches to a single microtubule. This defined architecture facilitates quantitative examination of kinetochores during the cell cycle. Using three independent measures-calibrated imaging, FRAP, and photoconversion-we find that the Dam1 submodule is unchanged during anaphase, whereas MIND and Ndc80 submodules add copies to form an "anaphase configuration" kinetochore. Microtubule depolymerization and kinesin-related motors contribute to copy addition. Mathematical simulations indicate that the addition of microtubule attachments could facilitate tracking during rapid microtubule depolymerization. We speculate that the minimal kinetochore configuration, which exists from G1 through metaphase, allows for correction of misattachments. Our study provides insight into dynamics and plasticity of the kinetochore structure during chromosome segregation in living cells

A revised airway epithelial hierarchy includes CFTR-expressing ionocytes

The airways of the lung are the primary sites of disease in asthma and cystic fibrosis. Here we study the cellular composition and hierarchy of the mouse tracheal epithelium by single-cell RNA-sequencing (scRNA-seq) and in vivo lineage tracing. We identify a rare cell type, the Foxi1+ pulmonary ionocyte; functional variations in club cells based on their location; a distinct cell type in high turnover squamous epithelial structures that we term ‘hillocks’; and disease-relevant subsets of tuft and goblet cells. We developed ‘pulse-seq’, combining scRNA-seq and lineage tracing, to show that tuft, neuroendocrine and ionocyte cells are continually and directly replenished by basal progenitor cells. Ionocytes are the major source of transcripts of the cystic fibrosis transmembrane conductance regulator in both mouse (Cftr) and human (CFTR). Knockout of Foxi1 in mouse ionocytes causes loss of Cftr expression and disrupts airway fluid and mucus physiology, phenotypes that are characteristic of cystic fibrosis. By associating cell-type-specific expression programs with key disease genes, we establish a new cellular narrative for airways disease

Integrative structure and functional anatomy of a nuclear pore complex

International audienc

Scm3 Is a Centromeric Nucleosome Assembly Factor*

The Cse4 nucleosome at each budding yeast centromere must be faithfully assembled each cell cycle to specify the site of kinetochore assembly and microtubule attachment for chromosome segregation. Although Scm3 is required for the localization of the centromeric H3 histone variant Cse4 to centromeres, its role in nucleosome assembly has not been tested. We demonstrate that Scm3 is able to mediate the assembly of Cse4 nucleosomes in vitro, but not H3 nucleosomes, as measured by a supercoiling assay. Localization of Cse4 to centromeres and the assembly activity depend on an evolutionarily conserved core motif in Scm3, but localization of the CBF3 subunit Ndc10 to centromeres does not depend on this motif. The centromere targeting domain of Cse4 is sufficient for Scm3 nucleosome assembly activity. Assembly does not depend on centromeric sequence. We propose that Scm3 plays an active role in centromeric nucleosome assembly

Modeling of the yeast Nuclear Pore Complex

<p>These scripts demonstrate the use of <a href="http://salilab.org/imp">IMP</a> in the modeling of the yeast NPC complex using diverse types of data as described in Seung Joong Kim, et al.'s 2018 NPC article published in Nature.</p> <p><strong>For more information</strong> about how to reproduce this modeling, see the <a href="https://salilab.org/npc2018/">Sali lab website</a> or the README file.</p

Brownian Dynamics simulations of yeast Nuclear Pore Complex FG repeats

<p>The files in this folder can be used to reproduce the Brownian Dynamics simulations of FG repeats using IMP as described in Kim et al., 2018.</p