RESUMEN
The formation of multicellular tissues during development is governed by mechanical forces that drive cell shape and tissue architecture. Protein complexes at sites of adhesion to the extracellular matrix (ECM) and cell neighbors, not only transmit these mechanical forces, but also allow cells to respond to changes in force by inducing biochemical feedback pathways. Such force-induced signaling processes are termed mechanotransduction. Vinculin is a central protein in mechanotransduction that in both integrin-mediated cell-ECM and cadherin-mediated cell-cell adhesions mediates force-induced cytoskeletal remodeling and adhesion strengthening. Vinculin was found to be important for the integrity and remodeling of epithelial tissues in cell culture models and could therefore be expected to be of broad importance in epithelial morphogenesis in vivo. Besides a function in mouse heart development, however, the importance of vinculin in morphogenesis of other vertebrate tissues has remained unclear. To investigate this further, we knocked out vinculin functioning in zebrafish, which contain two fully functional isoforms designated as vinculin A and vinculin B that both show high sequence conservation with higher vertebrates. Using TALEN and CRISPR-Cas gene editing technology we generated vinculin-deficient zebrafish. While single vinculin A mutants are viable and able to reproduce, additional loss of zygotic vinculin B was lethal after embryonic stages. Remarkably, vinculin-deficient embryos do not show major developmental defects, apart from mild pericardial edemas. These results lead to the conclusion that vinculin is not of broad importance for the development and morphogenesis of zebrafish tissues.
Asunto(s)
Vinculina/genética , Vinculina/metabolismo , Pez Cebra/crecimiento & desarrollo , Animales , Adhesión Celular , Perros , Desarrollo Embrionario , Matriz Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Células de Riñón Canino Madin Darby , Mecanotransducción Celular , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
We recently reported on CFP-Epac-YFP, an Epac-based single polypeptide FRET reporter to resolve cAMP levels in living cells. In this study, we compared and optimized the fluorescent protein donor/acceptor pairs for use in biosensors such as CFP-Epac-YFP. Our strategy was to prepare a wide range of constructs consisting of different donor and acceptor fluorescent proteins separated by a short linker. Constructs were expressed in HEK293 cells and tested for FRET and other relevant properties. The most promising pairs were subsequently used in an attempt to improve the FRET span of the Epac-based cAMP sensor. The results show significant albeit not perfect correlation between performance in the spacer construct and in the Epac sensor. Finally, this strategy enabled us to identify improved sensors both for detection by sensitized emission and by fluorescent lifetime imaging. The present overview should be helpful in guiding development of future FRET sensors.
Asunto(s)
AMP Cíclico/metabolismo , Transferencia Resonante de Energía de Fluorescencia/instrumentación , Transferencia Resonante de Energía de Fluorescencia/métodos , Factores de Intercambio de Guanina Nucleótido/fisiología , Proteínas Bacterianas/química , Técnicas Biosensibles , Línea Celular , Dimerización , Genes Reporteros , Factores de Intercambio de Guanina Nucleótido/química , Humanos , Luz , Proteínas Luminiscentes/química , Modelos Biológicos , Programas InformáticosRESUMEN
Fluorescent proteins, such as green fluorescent protein and red fluorescent protein (DsRED), have become frequently used reporters in plant biology. However, their potential to monitor dynamic gene regulation is limited by their high stability. The recently made DsRED-E5 variant overcame this problem. DsRED-E5 changes its emission spectrum over time from green to red in a concentration independent manner. Therefore, the green to red fluorescence ratio indicates the age of the protein and can be used as a fluorescent timer to monitor dynamics of gene expression. Here, we analyzed the potential of DsRED-E5 as reporter in plant cells. We showed that in cowpea (Vigna unguiculata) mesophyll protoplasts, DsRED-E5 changes its fluorescence in a way similar to animal cells. Moreover, the timing of this shift is suitable to study developmental processes in plants. To test whether DsRed-E5 can be used to monitor gene regulation in plant organs, we placed DsRED-E5 under the control of promoters that are either up- or down-regulated (MtACT4 and LeEXT1 promoters) or constitutively expressed (MtACT2 promoter) during root hair development in Medicago truncatula. Analysis of the fluorescence ratios clearly provided more accurate insight into the timing of promoter activity.
Asunto(s)
Fabaceae/genética , Proteínas Luminiscentes/análisis , Cartilla de ADN , Genes Reporteros , Proteínas Luminiscentes/genética , Microscopía Fluorescente/métodos , Biología Molecular/métodos , Datos de Secuencia Molecular , Protoplastos , Mapeo Restrictivo , Transfección/métodos , Proteína Fluorescente RojaRESUMEN
The movement protein (MP) of cowpea mosaic virus (CPMV) forms tubules on infected protoplasts and through plasmodesmata in infected plants. In protoplasts the MP fused to GFP (MP-GFP) was shown to localize in peripheral punctate structures and in long tubular structures extending from the protoplast surface. Using cytoskeletal assembly inhibitors (latrunculin B and oryzalin) and an inhibitor of the secretory pathway (brefeldin A), targeting of the MP to the peripheral punctate structures was demonstrated not to be dependent on an intact cytoskeleton or functional secretion pathway. Furthermore it was shown that a disrupted cytoskeleton had no effect on tubule formation but that the addition of brefeldin A severely inhibited tubule formation. The results presented in this paper suggest a role for a plasma membrane host factor in tubule formation of plant viral MPs.
Asunto(s)
Comovirus/metabolismo , Protoplastos/metabolismo , Sulfanilamidas , Proteínas Virales/metabolismo , Brefeldino A/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Dinitrobencenos/farmacología , Fabaceae/virología , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes , Herbicidas/farmacología , Proteínas Luminiscentes , Proteínas de Movimiento Viral en Plantas , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/metabolismo , Tiazoles/farmacología , Tiazolidinas , Transfección , Proteínas Virales/genéticaRESUMEN
Circular dichroism (CD) spectra have been obtained from several variants of green fluorescent protein: blue fluorescent protein (BFP), enhanced cyan fluorescent protein (CFP), enhanced green fluorescent protein (GFP), enhanced yellow fluorescent protein (YFP), all from Aequorea victoria, and the red fluorescent protein from the coral species Discosoma (DsRed). We demonstrate that CD spectra in the spectral fingerprint region of the chromophore yield spectra that after normalization are not coincident with the normalized absorbance spectra of GFP, YFP and DsRed. On the other hand, the CD spectra of BFP and CFP coincide with the absorbance spectra. The resolution of absorption and CD spectra into Gaussian bands confirmed the location of the different electronic band positions of GFP and YFP as reported in the literature using other techniques. In the case of BFP and CFP the location of Gaussian bands provided information of the vibrational progression of the electronic absorption bands. The CD spectrum of DsRed is anomalous in the sense that the major CD band has a clear excitonic character. Far-UV CD spectra of GFP confirmed the presence of the high beta-sheet content of the polypeptide chain in the three-dimensional structure.