Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 10 de 10
Filter
Add more filters










Publication year range
1.
Cell Rep ; 35(3): 109011, 2021 04 20.
Article in English | MEDLINE | ID: mdl-33882306

ABSTRACT

Pulmonary neuroendocrine cells (PNECs) have crucial roles in airway physiology and immunity by producing bioactive amines and neuropeptides (NPs). A variety of human diseases exhibit PNEC hyperplasia. Given accumulated evidence that PNECs represent a heterogenous population of cells, we investigate how PNECs differ, whether the heterogeneity is similarly present in mouse and human cells, and whether specific disease involves discrete PNECs. Herein, we identify three distinct types of PNECs in human and mouse airways based on single and double positivity for TUBB3 and the established NP markers. We show that the three PNEC types exhibit significant differences in NP expression, homeostatic turnover, and response to injury and disease. We provide evidence that these differences parallel their distinct cell of origin from basal stem cells (BSCs) or other airway epithelial progenitors.


Subject(s)
Cell Lineage/genetics , Epithelial Cells/pathology , Neuroendocrine Cells/pathology , Stem Cells/pathology , Tubulin/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation , Epithelial Cells/classification , Epithelial Cells/metabolism , Female , Gene Expression Regulation , Humans , Hyperplasia/genetics , Hyperplasia/metabolism , Hyperplasia/pathology , Infant , Influenza A Virus, H1N1 Subtype/growth & development , Influenza A Virus, H1N1 Subtype/pathogenicity , Lung , Male , Mice , Mice, Transgenic , Neuroendocrine Cells/classification , Neuroendocrine Cells/metabolism , Neuropeptides/genetics , Neuropeptides/metabolism , Orthomyxoviridae Infections/genetics , Orthomyxoviridae Infections/metabolism , Orthomyxoviridae Infections/pathology , Orthomyxoviridae Infections/virology , Signal Transduction , Stem Cells/classification , Stem Cells/metabolism , Sudden Infant Death/genetics , Sudden Infant Death/pathology , Transcription Factors/genetics , Transcription Factors/metabolism , Tubulin/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism
2.
Science ; 371(6524): 52-57, 2021 01 01.
Article in English | MEDLINE | ID: mdl-33384370

ABSTRACT

Neuroendocrine (NE) cells are epithelial cells that possess many of the characteristics of neurons, including the presence of secretory vesicles and the ability to sense environmental stimuli. The normal physiologic functions of solitary airway NE cells remain a mystery. We show that mouse and human airway basal stem cells sense hypoxia. Hypoxia triggers the direct differentiation of these stem cells into solitary NE cells. Ablation of these solitary NE cells during hypoxia results in increased epithelial injury, whereas the administration of the NE cell peptide CGRP rescues this excess damage. Thus, we identify stem cells that directly sense hypoxia and respond by differentiating into solitary NE cells that secrete a protective peptide that mitigates hypoxic injury.


Subject(s)
Cell Differentiation , Hypoxia/pathology , Neuroendocrine Cells/physiology , Oxygen/physiology , Stem Cells/physiology , Trachea/cytology , Anaerobiosis , Animals , Calcitonin Gene-Related Peptide/metabolism , Calcitonin Gene-Related Peptide/pharmacology , Calcitonin Receptor-Like Protein/metabolism , Cell Count , Gene Deletion , Humans , Hypoxia/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice , Mice, Mutant Strains , Neuroendocrine Cells/cytology , Prolyl Hydroxylases/metabolism , Stem Cells/cytology , Stem Cells/drug effects , Trans-Activators/genetics
3.
Nature ; 560(7718): 319-324, 2018 08.
Article in English | MEDLINE | ID: mdl-30069044

ABSTRACT

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.


Subject(s)
Cell Differentiation/genetics , Cell Lineage/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/genetics , Epithelial Cells/metabolism , Animals , Asthma/genetics , Epithelial Cells/cytology , Female , Forkhead Transcription Factors/deficiency , Forkhead Transcription Factors/genetics , Gene Expression Profiling , Gene Expression Regulation , Goblet Cells/cytology , Goblet Cells/metabolism , Humans , Lung/cytology , Male , Mice , Sequence Analysis, RNA , Single-Cell Analysis , Trachea/cytology
4.
Nature ; 555(7697): 475-482, 2018 03 22.
Article in English | MEDLINE | ID: mdl-29539637

ABSTRACT

Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility. These cables also tie together all other elements of the nuclear pore complex, including membrane-interacting regions, outer rings and RNA-processing platforms. Inwardly directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized into distinct functional units. This integrative structure enables us to rationalize the architecture, transport mechanism and evolutionary origins of the nuclear pore complex.


Subject(s)
Nuclear Pore Complex Proteins/chemistry , Nuclear Pore Complex Proteins/metabolism , Nuclear Pore/chemistry , Nuclear Pore/metabolism , Saccharomyces cerevisiae/chemistry , Cross-Linking Reagents/chemistry , Mass Spectrometry , Models, Molecular , Protein Stability , Protein Transport , RNA Transport
5.
J Cell Biol ; 216(11): 3551-3570, 2017 11 06.
Article in English | MEDLINE | ID: mdl-28939613

ABSTRACT

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.


Subject(s)
Chromosome Segregation , Chromosomes, Fungal/metabolism , Kinetochores/metabolism , Microtubules/metabolism , Saccharomyces cerevisiae/metabolism , Anaphase , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosomes, Fungal/genetics , Computer Simulation , Evolution, Molecular , G1 Phase , Genotype , Metaphase , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Microtubules/genetics , Models, Biological , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phenotype , Protein Binding , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Time Factors
6.
Cell ; 150(2): 304-16, 2012 Jul 20.
Article in English | MEDLINE | ID: mdl-22817893

ABSTRACT

The centromere is a specialized chromosomal structure that regulates chromosome segregation. Centromeres are marked by a histone H3 variant. In budding yeast, the histone H3 variant Cse4 is present in a single centromeric nucleosome. Experimental evidence supports several different models for the structure of centromeric nucleosomes. To investigate Cse4 copy number in live yeast, we developed a method coupling fluorescence correlation spectroscopy and calibrated imaging. We find that centromeric nucleosomes have one copy of Cse4 during most of the cell cycle, whereas two copies are detected at anaphase. The proposal of an anaphase-coupled structural change is supported by Cse4-Cse4 interactions, incorporation of Cse4, and the absence of Scm3 in anaphase. Nucleosome reconstitution and ChIP suggests both Cse4 structures contain H2A/H2B. The increase in Cse4 intensity and deposition at anaphase are also observed in Candida albicans. Our experimental evidence supports a cell-cycle-coupled oscillation of centromeric nucleosome structure in yeast.


Subject(s)
Candida albicans/cytology , Cell Cycle , Centromere/metabolism , Nucleosomes/metabolism , Saccharomyces cerevisiae/cytology , Anaphase , Candida albicans/chemistry , Candida albicans/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/metabolism , Fungal Proteins/metabolism , Green Fluorescent Proteins/analysis , Nuclear Pore Complex Proteins/metabolism , Nucleosome Assembly Protein 1/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism
8.
J Biol Chem ; 286(14): 12016-23, 2011 Apr 08.
Article in English | MEDLINE | ID: mdl-21317428

ABSTRACT

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.


Subject(s)
Centromere/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Nucleosomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Cell Cycle/genetics , Cell Cycle/physiology , Centromere/genetics , Chromatin Immunoprecipitation , Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Flow Cytometry , Histones/genetics , Histones/metabolism , Immunoprecipitation , Kinetochores/metabolism , Mutagenesis, Site-Directed , Mutation , Polymerase Chain Reaction , Saccharomyces cerevisiae Proteins/genetics
9.
Mol Cell ; 40(3): 444-54, 2010 Nov 12.
Article in English | MEDLINE | ID: mdl-21070970

ABSTRACT

Cse4 is a variant of histone H3 that is incorporated into a single nucleosome at each centromere in budding yeast. We have discovered an E3 ubiquitin ligase, called Psh1, which controls the cellular level of Cse4 via ubiquitylation and proteolysis. The activity of Psh1 is dependent on both its RING and zinc finger domains. We demonstrate the specificity of the ubiquitylation activity of Psh1 toward Cse4 in vitro and map the sites of ubiquitylation. Mutation of key lysines prevents ubiquitylation of Cse4 by Psh1 in vitro and stabilizes Cse4 in vivo. While deletion of Psh1 stabilizes Cse4, elimination of the Cse4-specific chaperone Scm3 destabilizes Cse4, and the addition of Scm3 to the Psh1-Cse4 ubiquitylation reaction prevents Cse4 ubiquitylation, together suggesting Scm3 may protect Cse4 from ubiquitylation. Without Psh1, Cse4 overexpression is toxic and Cse4 is found at ectopic locations. Our results suggest Psh1 functions to prevent the mislocalization of Cse4.


Subject(s)
Centromere/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/metabolism , Peptide Elongation Factors/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/enzymology , Ubiquitin-Protein Ligases/metabolism , Chromosomal Proteins, Non-Histone/chemistry , DNA-Binding Proteins/chemistry , Gene Deletion , Histones , Humans , Protein Binding , Protein Isoforms/metabolism , Protein Processing, Post-Translational , Protein Stability , Protein Structure, Tertiary , Protein Transport , Saccharomyces cerevisiae Proteins/chemistry , Ubiquitination
10.
Mol Cell ; 35(6): 794-805, 2009 Sep 24.
Article in English | MEDLINE | ID: mdl-19782029

ABSTRACT

The budding yeast CenH3 histone variant Cse4 localizes to centromeric nucleosomes and is required for kinetochore assembly and chromosome segregation. The exact composition of centromeric Cse4-containing nucleosomes is a subject of debate. Using unbiased biochemical, cell-biological, and genetic approaches, we have tested the composition of Cse4-containing nucleosomes. Using micrococcal nuclease-treated chromatin, we find that Cse4 is associated with the histones H2A, H2B, and H4, but not H3 or the nonhistone protein Scm3. Overexpression of Cse4 rescues the lethality of a scm3 deletion, indicating that Scm3 is not essential for the formation of functional centromeric chromatin. We also find that octameric Cse4 nucleosomes can be reconstituted in vitro. Furthermore, Cse4-Cse4 dimerization occurs in vivo at the centromeric nucleosome, and this requires the predicted Cse4-Cse4 dimerization interface. Taken together, our experimental evidence supports the model that the Cse4 nucleosome is an octamer, containing two copies each of Cse4, H2A, H2B, and H4.


Subject(s)
Centromere/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/metabolism , Histones/metabolism , Nucleosomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Chromatin Immunoprecipitation , Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation, Fungal , Models, Molecular , Multiprotein Complexes , Mutation , Nucleic Acid Conformation , Nucleosomes/genetics , Protein Conformation , Protein Multimerization , Protein Subunits , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...