RESUMEN
A key step in regulation of Hippo pathway signaling in response to mechanical tension is recruitment of the LIM domain proteins TRIP6 and LIMD1 to adherens junctions. Mechanical tension also triggers TRIP6 and LIMD1 to bind and inhibit the Hippo pathway kinase LATS1. How TRIP6 and LIMD1 are recruited to adherens junctions in response to tension is not clear, but previous studies suggested that they could be regulated by the known mechanosensory proteins α-catenin and vinculin at adherens junctions. We found that the three LIM domains of TRIP6 and LIMD1 are necessary and sufficient for tension-dependent localization to adherens junctions. The LIM domains of TRIP6, LIMD1, and certain other LIM domain proteins have been shown to bind to actin networks under strain/tension. Consistent with this, we show that TRIP6 and LIMD1 colocalize with the ends of actin fibers at adherens junctions. Point mutations in a key conserved residue in each LIM domain that are predicted to impair binding to f-actin under strain inhibits TRIP6 and LIMD1 localization to adherens junctions and their ability to bind to and recruit LATS1 to adherens junctions. Together these results show that the ability of TRIP6 and LIMD1 to bind to strained actin underlies their ability to localize to adherens junctions and regulate LATS1 in response to mechanical tension.
RESUMEN
Single nucleotide polymorphisms (SNPs) in the gene encoding kinesin family member 3A, KIF3A, have been associated with atopic dermatitis (AD), a chronic inflammatory skin disorder. We find that KIF3A SNP rs11740584 and rs2299007 risk alleles create cytosine-phosphate-guanine sites, which are highly methylated and result in lower KIF3A expression, and this methylation is associated with increased transepidermal water loss (TEWL) in risk allele carriers. Kif3aK14∆/∆ mice have increased TEWL, disrupted junctional proteins, and increased susceptibility to develop AD. Thus, KIF3A is required for skin barrier homeostasis whereby decreased KIF3A skin expression causes disrupted skin barrier function and promotes development of AD.
Asunto(s)
Dermatitis Atópica/metabolismo , Cinesinas/metabolismo , Piel/metabolismo , Adolescente , Adulto , Alelos , Animales , Niño , Dermatitis Atópica/genética , Dermatitis Atópica/patología , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Cinesinas/genética , Masculino , Metilación , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Piel/patología , Adulto JovenRESUMEN
The respiratory system undergoes a diversity of structural, biochemical, and functional changes necessary for adaptation to air breathing at birth. To identify the heterogeneity of pulmonary cell types and dynamic changes in gene expression mediating adaptation to respiration, here we perform single cell RNA analyses of mouse lung on postnatal day 1. Using an iterative cell type identification strategy we unbiasedly identify the heterogeneity of murine pulmonary cell types. We identify distinct populations of epithelial, endothelial, mesenchymal, and immune cells, each containing distinct subpopulations. Furthermore we compare temporal changes in RNA expression patterns before and after birth to identify signaling pathways selectively activated in specific pulmonary cell types, including activation of cell stress and the unfolded protein response during perinatal adaptation of the lung. The present data provide a single cell view of the adaptation to air breathing after birth.
Asunto(s)
Adaptación Fisiológica/genética , Pulmón/citología , ARN/metabolismo , Fenómenos Fisiológicos Respiratorios , Análisis de la Célula Individual/métodos , Animales , Animales Recién Nacidos , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Pulmón/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Embarazo , ARN/aislamiento & purificación , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Análisis de Secuencia de ARN , Respuesta de Proteína Desplegada/fisiologíaRESUMEN
Recent studies have implicated keratin 5 (KRT5)+ cells in repopulation of damaged lung tissue following severe H1N1 influenza virus infection. However, the origins of the cells repopulating the injured alveolar region remain controversial. We sought to determine the cellular dynamics of lung repair following influenza infection and define whether nascent KRT5+ cells repopulating alveolar epithelium were derived from pre-existing alveolar or airway progenitor cells. We found that the wound-healing response begins with proliferation of SOX2+ SCGB1A1- KRT5- progenitor cells in airways. These cells generate nascent KRT5+ cells as an early response to airway injury and yield progeny that colonize damaged alveolar parenchyma. Moreover, we show that local alveolar progenitors do not contribute to nascent KRT5+ cells after injury. Repopulation of injured airway and alveolar regions leads to proximalization of distal airways by pseudostratified epithelium and of alveoli by airway-derived epithelial cells that lack the normal characteristics of mature airway or alveolar epithelium.
Asunto(s)
Células Epiteliales Alveolares/metabolismo , Diferenciación Celular , Queratina-5/metabolismo , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Factores de Transcripción SOXB1/metabolismo , Células Madre/citología , Células Madre/metabolismo , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/virología , Animales , Biomarcadores , Linaje de la Célula , Autorrenovación de las Células/genética , Subtipo H1N1 del Virus de la Influenza A , Ratones , Ratones Transgénicos , Modelos Biológicos , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Mucosa Respiratoria/virología , Factores de Transcripción SOXB1/genéticaRESUMEN
The epidermis provides an essential seal from the external environment and retains fluids within the body. To form an effective barrier, cells in the epidermis must form tight junctions and terminally differentiate into cornified envelopes. Here, we demonstrate that the branched actin nucleator, the actin-related protein (Arp)2/3 complex, is unexpectedly required for both these activities. Loss of the ArpC3 subunit of the Arp2/3 complex resulted in minimal changes in the morphogenesis and architecture of this stratified squamous epithelium, but resulted in profound defects in its physiology. Mutant embryos did not develop an effective barrier to the external environment and died within hours of birth. We discovered two underlying causes for these effects. First, ArpC3 was essential for robust assembly and function of tight junctions, specialized cell-cell adhesions that restrict water loss in the epidermis. Second, there were defects in differentiation of the epidermis and the production of cornified envelopes, structures essential for barrier activity. Underlying this defect, we found that YAP was inappropriately active not only in the ArpC3 mutant tissue, but also in cultured cells. Inhibition of YAP activity rescued the differentiation and barrier defects caused by loss of ArpC3. These results demonstrate previously unappreciated roles for the Arp2/3 complex and highlight the functions of branched actin networks in a complex tissue.
Asunto(s)
Proteína 2 Relacionada con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/metabolismo , Actinas/metabolismo , Epidermis/fisiología , Complejos Multiproteicos/metabolismo , Uniones Estrechas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Benzotiazoles , Proteínas de Ciclo Celular , Diaminas , Epidermis/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Indoles/farmacología , Queratinocitos , Listeria monocytogenes/fisiología , Ratones , Análisis por Micromatrices , Complejos Multiproteicos/antagonistas & inhibidores , Compuestos Orgánicos , Fosfoproteínas/metabolismo , Quinolinas , Reacción en Cadena en Tiempo Real de la Polimerasa , Tiofenos/farmacología , Proteínas Señalizadoras YAPRESUMEN
Many tissues in our body experience mechanical stresses caused by both internal and external forces. The skin, for example, must tolerate diverse mechanical insults. In this paper, we report a role for ß-catenin in providing stability to epithelia under stress. Loss of ß-catenin during epidermal development caused perinatal lethality. Mutant embryos up-regulated stress responses at sites of active morphogenesis, which became more widespread after the stresses associated with birth. In addition, selective loss of tight junctions occurred in focal regions. This was recapitulated in cultured ß-catenin-null cells exposed to externally applied forces. In addition, mutant cells were defective in tension-induced engagement of adherens junctions. We found that ß-catenin was required to recruit vinculin to the cell cortex and to strengthen the junction's association with the underlying cytoskeleton in response to tension. These data demonstrate that a complete understanding of the functions of cell adhesion proteins must take into account their roles in response to mechanical stresses.
Asunto(s)
Epidermis/metabolismo , Regulación del Desarrollo de la Expresión Génica , Estrés Mecánico , beta Catenina/metabolismo , Uniones Adherentes/genética , Uniones Adherentes/metabolismo , Animales , Animales Recién Nacidos , Fenómenos Biomecánicos , Adhesión Celular , Diferenciación Celular , Células Cultivadas , Citoesqueleto , Pérdida del Embrión/genética , Pérdida del Embrión/metabolismo , Pérdida del Embrión/patología , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Queratinocitos/metabolismo , Ratones , Ratones Noqueados , Vinculina/metabolismo , beta Catenina/genéticaRESUMEN
The septum initiation network (SIN) regulates multiple functions during late mitosis to ensure successful completion of cytokinesis in Schizosaccharomyces pombe. One mechanism by which the SIN promotes cytokinesis is by inhibiting a competing polarity pathway called the MOR, which is required for initiation of polarized growth following completion of cytokinesis. Mutual antagonism between the two NDR kinase pathways, SIN and MOR, is required to coordinate cytoskeletal rearrangements during the mitosis-interphase transition. To determine how the SIN regulates the MOR pathway, we developed a proteomics approach that allowed us to identify multiple substrates of the SIN effector kinase Sid2, including the MOR pathway components Nak1 kinase and an associated protein, Sog2. We show that Sid2 phosphorylation of Nak1 causes removal of Nak1 from the spindle pole bodies, which may both relieve Nak1 inhibition of the SIN and block MOR signaling by preventing interaction of Nak1 with the scaffold protein Mor2. Because the SIN and MOR are conserved in mammalian cells (Hippo and Ndr1/2 pathways, respectively), this work may provide important insight into how the activities of these essential pathways are coordinated.
Asunto(s)
Citocinesis/fisiología , Mitosis/fisiología , Schizosaccharomyces/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , División Celular , Proteínas de Microfilamentos/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteómica , Proteínas de Schizosaccharomyces pombe/metabolismo , Transducción de Señal , Huso Acromático/metabolismoRESUMEN
For proper tissue morphogenesis, cell divisions and cell fate decisions must be tightly and coordinately regulated. One elegant way to accomplish this is to couple them with asymmetric cell divisions. Progenitor cells in the developing epidermis undergo both symmetric and asymmetric cell divisions to balance surface area growth with the generation of differentiated cell layers. Here we review the molecular machinery implicated in controlling asymmetric cell division. In addition, we discuss the ability of epidermal progenitors to choose between symmetric and asymmetric divisions and the key regulatory points that control this decision.
RESUMEN
The mechanisms that regulate cytoskeletal remodeling during the transition between mitosis and interphase are poorly understood. In fission yeast the MOR pathway promotes actin polarization to cell tips in interphase, whereas the SIN signaling pathway drives actomyosin ring assembly and cytokinesis. We show that the SIN inhibits MOR signaling in mitosis by interfering with Nak1 kinase-mediated activation of the most downstream MOR component, the NDR family kinase Orb6. Inactivation of the MOR may be a key function of the SIN because attenuation of MOR signaling rescued the cytokinetic defects of SIN mutants and allowed weak SIN signaling to trigger ectopic cytokinesis. Furthermore, failure to inhibit the MOR is toxic when the cell division apparatus is compromised. Together, our results reveal a mutually antagonistic relationship between the SIN and MOR pathways, which is important for completion of cytokinesis and coordination of cytoskeletal remodeling at the mitosis-to-interphase transition.
Asunto(s)
Mitosis , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces , Actinas/genética , Actinas/metabolismo , División Celular , Citocinesis/genética , Citocinesis/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Interfase/genética , Proteínas Recombinantes de Fusión/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/fisiología , Transducción de Señal/genéticaRESUMEN
The septation initiation network (SIN) and mitotic exit network (MEN) signaling pathways regulate cytokinesis and mitotic exit in the yeasts Schizosaccharomyces pombe, and Saccharomyces cerevisiae, respectively. One function of these pathways is to keep the Cdc14-family phosphatase, called Clp1 in S. pombe, from being sequestered and inhibited in the nucleolus. In S. pombe, the SIN and Clp1 act as part of a cytokinesis checkpoint that allows cells to cope with cytokinesis defects. The SIN promotes checkpoint function by 1) keeping Clp1 out of the nucleolus, 2) maintaining the cytokinetic apparatus, and 3) halting the cell cycle until cytokinesis is completed. In a screen for suppressors of the SIN mutant cytokinesis checkpoint defect, we identified a novel nucleolar protein called Dnt1 and other nucleolar proteins, including Rrn5 and Nuc1, which are known to be required for rDNA transcription. Dnt1 shows sequence homology to Net1/Cfi1, which encodes the nucleolar inhibitor of Cdc14 in budding yeast. Like Net1/Cfi1, Dnt1 is required for rDNA silencing and minichromosome maintenance, and both Dnt1 and Net1/Cfi1 negatively regulate the homologous SIN and MEN pathways. Unlike Net1/Cfi1, which regulates the MEN through the Cdc14 phosphatase, Dnt1 can inhibit SIN signaling independently of Clp1, suggesting a novel connection between the nucleolus and the SIN pathway.