RESUMEN
Cell-cell contact formation of polarized epithelial cells is a multi-step process that involves the co-ordinated activities of Rho family small GTPases. Consistent with the central role of Rho GTPases, a number of Rho guanine nucleotide exchange factors (GEFs) and Rho GTPase-activating proteins (GAPs) have been identified at cell-cell junctions at various stages of junction maturation. As opposed to RhoGEFs and RhoGAPs, the role of Rho GDP dissociation inhibitors (GDIs) during cell-cell contact formation is poorly understood. Here, we have analyzed the role of RhoGDI1/ARHGDIA, a member of the RhoGDI family, during cell-cell contact formation of polarized epithelial cells. Depletion of RhoGDI1 delays the development of linear cell-cell junctions and the formation of barrier-forming tight junctions. In addition, RhoGDI1 depletion impairs the ability of cells to stop migration in response to cell collision and increases the migration velocity of collectively migrating cells. We also find that the cell adhesion receptor JAM-A promotes the recruitment of RhoGDI1 to cell-cell contacts. Our findings implicate RhoGDI1 in various processes involving the dynamic reorganization of cell-cell junctions.
RESUMEN
We have shown that in murine cardiomyopathy caused by overexpression of the ß1-adrenoceptor, Gαi2-deficiency is detrimental. Given the growing evidence for isoform-specific Gαi-functions, we now examined the consequences of Gαi3 deficiency in the same heart-failure model. Mice overexpressing cardiac ß1-adrenoceptors with (ß1-tg) or without Gαi3-expression (ß1-tg/Gαi3-/-) were compared to C57BL/6 wildtypes and global Gαi3-knockouts (Gαi3-/-). The life span of ß1-tg mice was significantly shortened but improved when Gαi3 was lacking (95% CI: 592-655 vs. 644-747 days). At 300 days of age, left-ventricular function and survival rate were similar in all groups. At 550 days of age, ß1-tg but not ß1-tg/Gαi3-/- mice displayed impaired ejection fraction (35 ± 18% vs. 52 ± 16%) compared to wildtype (59 ± 4%) and Gαi3-/- mice (60 ± 5%). Diastolic dysfunction of ß1-tg mice was prevented by Gαi3 deficiency, too. The increase of ANP mRNA levels and ventricular fibrosis observed in ß1-tg hearts was significantly attenuated in ß1-tg/Gαi3-/- mice. Transcript levels of phospholamban, ryanodine receptor 2, and cardiac troponin I were similar in all groups. However, Western blots and phospho-proteomic analyses showed that in ß1-tg, but not ß1-tg/Gαi3-/- ventricles, phospholamban protein was reduced while its phosphorylation increased. Here, we show that in mice overexpressing the cardiac ß1-adrenoceptor, Gαi3 deficiency slows or even prevents cardiomyopathy and increases shortened life span. Previously, we found Gαi2 deficiency to aggravate cardiac dysfunction and mortality in the same heart-failure model. Our findings indicate isoform-specific interventions into Gi-dependent signaling to be promising cardio-protective strategies.
Asunto(s)
Cardiomiopatías , Insuficiencia Cardíaca , Animales , Ratones , Cardiomiopatías/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Miocardio/metabolismo , Isoformas de Proteínas/metabolismo , ProteómicaRESUMEN
Epithelia maintain a functional barrier during tissue turnover while facing varying mechanical stress. This maintenance requires both dynamic cell rearrangements driven by actomyosin-linked intercellular adherens junctions and ability to adapt to and resist extrinsic mechanical forces enabled by keratin filament-linked desmosomes. How these two systems crosstalk to coordinate cellular movement and mechanical resilience is not known. Here we show that in stratifying epithelia the polarity protein aPKCλ controls the reorganization from stress fibers to cortical actomyosin during differentiation and upward movement of cells. Without aPKC, stress fibers are retained resulting in increased contractile prestress. This aberrant stress is counterbalanced by reorganization and bundling of keratins, thereby increasing mechanical resilience. Inhibiting contractility in aPKCλ-/- cells restores normal cortical keratin networks but also normalizes resilience. Consistently, increasing contractile stress is sufficient to induce keratin bundling and enhance resilience, mimicking aPKC loss. In conclusion, our data indicate that keratins sense the contractile stress state of stratified epithelia and balance increased contractility by mounting a protective response to maintain tissue integrity.
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Actomiosina , Transducción de Señal , Actomiosina/metabolismo , Epitelio/metabolismo , Citoesqueleto/metabolismo , Queratinas/metabolismo , Células Epiteliales/metabolismoRESUMEN
Autophagy provides nutrients during starvation and eliminates detrimental cellular components. However, accumulating evidence indicates that autophagy is not merely a housekeeping process. Here, by combining mouse models of neuron-specific ATG5 deficiency in either excitatory or inhibitory neurons with quantitative proteomics, high-content microscopy, and live-imaging approaches, we show that autophagy protein ATG5 functions in neurons to regulate cAMP-dependent protein kinase A (PKA)-mediated phosphorylation of a synapse-confined proteome. This function of ATG5 is independent of bulk turnover of synaptic proteins and requires the targeting of PKA inhibitory R1 subunits to autophagosomes. Neuronal loss of ATG5 causes synaptic accumulation of PKA-R1, which sequesters the PKA catalytic subunit and diminishes cAMP/PKA-dependent phosphorylation of postsynaptic cytoskeletal proteins that mediate AMPAR trafficking. Furthermore, ATG5 deletion in glutamatergic neurons augments AMPAR-dependent excitatory neurotransmission and causes the appearance of spontaneous recurrent seizures in mice. Our findings identify a novel role of autophagy in regulating PKA signaling at glutamatergic synapses and suggest the PKA as a target for restoration of synaptic function in neurodegenerative conditions with autophagy dysfunction.
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Neuronas , Sinapsis , Ratones , Animales , Sinapsis/metabolismo , Neuronas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Transducción de Señal , AutofagiaRESUMEN
Communication between individuals via molecules, termed chemosignaling, is widespread among animal and plant species. However, we lack knowledge on the specific functions of the substances involved for most systems. The femoral gland is an organ that secretes a waxy substance involved in chemical communication in lizards. Although the lipids and volatile substances secreted by the femoral glands have been investigated in several biochemical studies, the protein composition and functions of secretions remain completely unknown. Applying a proteomic approach, we provide the first attempt to comprehensively characterize the protein composition of femoral gland secretions from the Galápagos marine iguana. Using samples from several organs, the marine iguana proteome was assembled by next-generation sequencing and MS, resulting in 7513 proteins. Of these, 4305 proteins were present in the femoral gland, including keratins, small serum proteins, and fatty acid-binding proteins. Surprisingly, no proteins with discernible roles in partner recognition or inter-species communication could be identified. However, we did find several proteins with direct associations to the innate immune system, including lysozyme C, antileukoproteinase (ALP), pulmonary surfactant protein (SFTPD), and galectin (LGALS1) suggesting that the femoral glands function as an important barrier to infection. Furthermore, we report several novel anti-microbial peptides from the femoral glands that show similar action against Escherichia coli and Bacillus subtilis such as oncocin, a peptide known for its effectiveness against Gram-negative pathogens. This proteomics data set is a valuable resource for future functional protein analysis and demonstrates that femoral gland secretions also perform functions of the innate immune system.
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Antiinfecciosos/metabolismo , Antiinfecciosos/farmacología , Iguanas/metabolismo , Sistema Inmunológico/metabolismo , Inmunidad Innata , Proteoma/metabolismo , Transcriptoma , Animales , Apoproteínas/genética , Apoproteínas/metabolismo , Bacillus subtilis/efectos de los fármacos , Encéfalo/metabolismo , Factores Quimiotácticos/genética , Factores Quimiotácticos/metabolismo , Ecuador , Endopeptidasas/genética , Endopeptidasas/metabolismo , Escherichia coli/efectos de los fármacos , Galectinas/genética , Galectinas/metabolismo , Corazón/fisiología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Iguanas/genética , Iguanas/inmunología , Inmunidad Innata/genética , Pulmón/metabolismo , Muramidasa/genética , Muramidasa/metabolismo , Músculos/metabolismo , Miocardio/metabolismo , Especificidad de Órganos , Proteoma/genética , Proteoma/inmunología , Proteómica , Proteínas Asociadas a Surfactante Pulmonar/genética , Proteínas Asociadas a Surfactante Pulmonar/metabolismo , Piel/metabolismo , Espectrometría de Masas en Tándem , Transcriptoma/genéticaRESUMEN
Epidermal barrier dysfunction is associated with a wide range of highly prevalent inflammatory skin diseases. However, the molecular processes that drive epidermal barrier maintenance are still largely unknown. Here, using quantitative proteomics, lipidomics, and mouse genetics, we characterize epidermal barrier maintenance versus a newly established barrier and functionally identify differential ceramide synthase 4 protein expression as one key difference. We show that epidermal loss of ceramide synthase 4 first disturbs epidermal lipid metabolism and adult epidermal barrier function, ultimately resulting in chronic skin barrier disease characterized by acanthosis, hyperkeratosis, and immune cell accumulation. Importantly, prolonged barrier dysfunction induced by loss of ceramide synthase 4 induced a barrier repair response that largely recapitulates molecular programs of barrier establishment. Collectively, this study provides an unbiased temporal proteomic characterization of barrier maintenance and disturbed homeostasis and shows that lipid homeostasis is essential to maintain adult skin barrier function to prevent disease.
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Homeostasis/fisiología , Piel/metabolismo , Esfingosina N-Aciltransferasa/fisiología , Animales , Epidermis/metabolismo , Metabolismo de los Lípidos , Ratones , Ratones Endogámicos C57BL , Morfogénesis , ProteómicaRESUMEN
The development of modern high-throughput instrumentation and improved core facility infrastructures leads to an accumulation of large amounts of scientific data. However, for a majority of scientists the comprehensive analysis and visualization of their data goes beyond their expertise. To reduce this hurdle, we developed a software suite called Instant Clue that helps scientists to visually analyze data and to gain insights into biological processes from their high-dimensional dataset. Instant Clue combines the power of visual and statistical analytics using a straight forward drag & drop approach making the software highly intuitive. Additionally, it offers a comprehensive portfolio of statistical tools for systematic analysis such as dimensional reduction, (un)-supervised learning, clustering, multi-block (omics) integration and curve fitting. Charts can be combined with high flexibility into a main figure template for direct usage in scientific publications. Even though Instant Clue was developed with the omics-sciences in mind, users can analyze any kind of data from low to high dimensional data sets. The open-source software is available for Windows and Mac OS ( http://www.instantclue.uni-koeln.de ) and is accompanied by a detailed video tutorial series.
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Programas Informáticos , Algoritmos , Visualización de DatosRESUMEN
Tissue mechanics drive morphogenesis, but how forces are sensed and transmitted to control stem cell fate and self-organization remains unclear. We show that a mechanosensory complex of emerin (Emd), non-muscle myosin IIA (NMIIA) and actin controls gene silencing and chromatin compaction, thereby regulating lineage commitment. Force-driven enrichment of Emd at the outer nuclear membrane of epidermal stem cells leads to defective heterochromatin anchoring to the nuclear lamina and a switch from H3K9me2,3 to H3K27me3 occupancy at constitutive heterochromatin. Emd enrichment is accompanied by the recruitment of NMIIA to promote local actin polymerization that reduces nuclear actin levels, resulting in attenuation of transcription and subsequent accumulation of H3K27me3 at facultative heterochromatin. Perturbing this mechanosensory pathway by deleting NMIIA in mouse epidermis leads to attenuated H3K27me3-mediated silencing and precocious lineage commitment, abrogating morphogenesis. Our results reveal how mechanics integrate nuclear architecture and chromatin organization to control lineage commitment and tissue morphogenesis.
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Diferenciación Celular/genética , Linaje de la Célula/fisiología , Heterocromatina/metabolismo , Proteínas del Grupo Polycomb/genética , Animales , Linaje de la Célula/genética , Cromatina/metabolismo , Silenciador del Gen , Heterocromatina/genética , Histonas/metabolismo , Ratones Transgénicos , Morfogénesis , Miosina Tipo IIA no Muscular/deficiencia , Unión Proteica/genéticaRESUMEN
Polarity signaling through the atypical PKC (aPKC)-Par polarity complex is essential for the development and maintenance of the podocyte architecture and the function of the glomerular filtration barrier of the kidney. To study the contribution of Par3A in this complex, we generated a novel Pard3 podocyte-specific knockout mouse model by targeting exon 6 of the Pard3 gene. Genetic deletion of Pard3a did not impair renal function, neither at birth nor later in life. Even challenging the animals did not result in glomerular disease. Despite its well-established role in aPKC-mediated signaling, Par3A appears to be dispensable for the function of the glomerular filtration barrier. Moreover, its homolog Pard3b, and not Pard3a, is the dominant Par3 gene expressed in podocytes and found at the basis of the slit diaphragm, where it partially colocalizes with podocin. In conclusion, Par3A function is either dispensable for slit diaphragm integrity, or compensatory mechanisms and a high redundancy of the different polarity proteins, including Par3B, Lgl, or PALS1, maintain the function of the glomerular filtration barrier, even in the absence of Par3A.
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Moléculas de Adhesión Celular/metabolismo , Barrera de Filtración Glomerular/fisiología , Riñón/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Moléculas de Adhesión Celular/genética , Proteínas de Ciclo Celular , Células Cultivadas , Femenino , Riñón/patología , Lipopolisacáridos/toxicidad , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Nucleósido-Fosfato Quinasa/genética , Nucleósido-Fosfato Quinasa/metabolismo , Podocitos/efectos de los fármacos , Podocitos/metabolismo , Podocitos/patología , Cultivo Primario de Células , Albúmina Sérica Bovina/toxicidadAsunto(s)
Linaje de la Célula , Dermatología , Genes Reporteros , Técnicas Genéticas , Integrasas , Animales , Educación Médica Continua , Humanos , Ratones DesnudosRESUMEN
The epidermis of the skin is a highly polarized, metabolic tissue with important innate immune functions. The polarity of the epidermis is, for example, reflected in controlled changes in cell shape that accompany differentiation, oriented cell division, and the planar orientation of hair follicles and cilia. The establishment and maintenance of polarity is organized by a diverse set of polarity proteins that include transmembrane adhesion proteins, cytoskeletal scaffold proteins, and kinases. Although polarity proteins have been extensively studied in cell culture and in vivo in simple epithelia of lower organisms, their role in mammalian tissue biology is only slowly evolving. This article will address the importance of polarizing processes and their molecular regulators in epidermal morphogenesis and homeostasis and discuss how alterations in polarity may contribute to skin disease.