RESUMO
Fluorescent proteins are standard tools for addressing biological questions in a cell biology laboratory. The genetic tagging of protein of interest with fluorescent proteins opens the opportunity to follow them in vivo and to understand their interactions and dynamics. In addition, the latest advances in optical microscopy image acquisition and processing allow us to study many cellular processes in vivo. Techniques such as fluorescence lifetime microscopy and hyperspectral imaging provide valuable tools for understanding fluorescent protein interactions and their photophysics. Finally, fluorescence fluctuation analysis opens the possibility to address questions of molecular diffusion, protein-protein interactions, and oligomerization, among others, yielding quantitative information on the subject of study. This chapter will cover some of the more important advances in cutting-edge technologies and methods that, combined with fluorescent proteins, open new frontiers for biological studies.
Assuntos
Corantes , Proteínas , Fenômenos Fisiológicos Celulares , Microscopia de Fluorescência/métodosRESUMO
For long time, studies on ectomycorrhiza (ECM) have been limited by inefficient expression of fluorescent proteins (FPs) in the fungal partner. To convert this situation, we have evaluated the basic requirements of FP expression in the model ECM homobasidiomycete Laccaria bicolor and established eGFP and mCherry as functional FP markers. Comparison of intron-containing and intronless FP-expression cassettes confirmed that intron-processing is indispensable for efficient FP expression in Laccaria. Nuclear FP localization was obtained via in-frame fusion of FPs between the intron-containing genomic gene sequences of Laccaria histone H2B, while cytosolic FP expression was produced by incorporating the intron-containing 5' fragment of the glyceraldehyde-3-phosphate dehydrogenase encoding gene. In addition, we have characterized the consensus Kozak sequence of strongly expressed genes in Laccaria and demonstrated its boosting effect on transgene mRNA accumulation. Based on these results, an Agrobacterium-mediated transformation compatible plasmid set was designed for easy use of FPs in Laccaria. The four cloning plasmids presented here allow fast and highly flexible construction of C-terminal in-frame fusions between the sequences of interest and the two FPs, expressed either from the endogenous gene promoter, allowing thus evaluation of the native regulation modes of the gene under study, or alternatively, from the constitutive Agaricus bisporus gpdII promoter for enhanced cellular protein localization assays. The molecular tools described here for cell-biological studies in Laccaria can also be exploited in studies of other biotrophic or saprotrophic basidiomycete species susceptible to genetic transformation.
Assuntos
Proteínas de Fluorescência Verde/genética , Laccaria/genética , Proteínas Luminescentes/genética , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Agrobacterium/genética , Basidiomycota/genética , Núcleo Celular/genética , Citosol/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Histonas/genética , Laccaria/metabolismo , Proteínas Luminescentes/metabolismo , Microrganismos Geneticamente Modificados , Microscopia de Fluorescência , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Transformação Genética , Proteína Vermelha FluorescenteRESUMO
The enhanced green fluorescent protein (eGFP) is one of the most employed variants of fluorescent proteins. Nonetheless little is known about the oxidative modifications that this protein can undergo in the cellular milieu. The present work explored the consequences of the exposure of eGFP to free radicals derived from γ-radiolysis of water, and AAPH thermolysis. Results demonstrated that protein crosslinking was the major pathway of modification of eGFP towards these oxidants. As evidenced by HPLC-FLD and UPLC-MS, eGFP crosslinking would occur as consequence of a mixture of pathways including the recombination of two protein radicals, as well as secondary reactions between nucleophilic residues (e.g. lysine, Lys) with protein carbonyls. The first mechanism was supported by detection of dityrosine and cysteine-tyrosine bonds, whilst evidence of formation of protein carbonyls, along with Lys consumption, would suggest the formation and participation of Schiff bases in the crosslinking process. Despite of the degree of oxidative modifications elicited by peroxyl radicals (ROOâ¢) generated from the thermolysis of AAPH, and free radicals generated from γ-radiolysis of water, that were evidenced at amino acidic level, only the highest dose of γ-irradiation (10 kGy) triggered significant changes in the secondary structure of eGFP. These results were accompanied by the complete loss of fluorescence arising from the chromophore unit of eGFP in γ-irradiation-treated samples, whereas it was conserved in ROOâ¢-treated samples. These data have potential biological significance, as this fluorescent protein is widely employed to study interactions between cytosolic proteins; consequently, the formation of fluorescent eGFP dimers could act as artifacts in such experiments.
Assuntos
Cisteína , Água , Amidinas , Cromatografia Líquida , Dipeptídeos , Radicais Livres , Proteínas de Fluorescência Verde , Oxirredução , Estresse Oxidativo , Espectrometria de Massas em Tandem , TirosinaRESUMO
ABSTRACT Purpose: To identify the fetal stem cell (FSC) response to maternal renal injury with emphasis on renal integrity improvement and Y chromosome detection in damaged maternal kidney. Materials and Methods: Eight non-green fluorescent protein (GFP) transgenic Sprague-Dawley rats were mated with GFP-positive transgenic male rats. Renal damage was induced on the right kidney at gestational day 11. The same procedure was performed in eight non-pregnant rats as control group. Three months after delivery, right ne- phrectomy was performed in order to evaluate the injured kidney. The fresh perfused kidneys were stained with anti-GFP antibody. Polymerase chain reaction (PCR) assay was also performed for the Y chromosome detection. Cell culture was performed to detect the GFP-positive cells. Technetium-99m-DMSA renal scan and single-photon emission computed tomography (SPECT) were performed after renal damage induction and 3 months later to evaluate the improvement of renal integrity. Results: The presence of FSCs was confirmed by immune histochemical staining as well as immunofluorescent imaging of the damaged part. Gradient PCR of female rat purified DNA demonstrated the presence of Y-chromosome in the damaged maternal kidney. Moreover, the culture of kidney cells showed GPF- positive cells by immuno- fluorescence microscopy. The acute renal scar was repaired and the integrity of dam- aged kidney reached to near normal levels in experimental group as shown in DMSA scan. However, no significant improvement was observed in control group. Conclusion: FSC seems to be the main mechanism in repairing of the maternal renal injury during pregnancy as indicated by Y chromosome and GFP-positive cells in the sub-cultured medium.
Assuntos
Animais , Masculino , Feminino , Gravidez , Cicatrização/fisiologia , Quimerismo , Células-Tronco Fetais/fisiologia , Nefropatias/fisiopatologia , Troca Materno-Fetal/fisiologia , Fatores de Tempo , Cromossomo Y , Imuno-Histoquímica , Tomografia Computadorizada de Emissão de Fóton Único , Células Cultivadas , Reação em Cadeia da Polimerase , Imunofluorescência , Ratos Sprague-Dawley , Compostos Radiofarmacêuticos , Ácido Dimercaptossuccínico Tecnécio Tc 99m , Modelos Animais de Doenças , Nefropatias/patologia , Nefropatias/diagnóstico por imagemRESUMO
PURPOSE: To identify the fetal stem cell (FSC) response to maternal renal injury with emphasis on renal integrity improvement and Y chromosome detection in damaged maternal kidney. MATERIALS AND METHODS: Eight non-green fluorescent protein (GFP) transgenic Sprague- Dawley rats were mated with GFP-positive transgenic male rats. Renal damage was induced on the right kidney at gestational day 11. The same procedure was performed in eight non-pregnant rats as control group. Three months after delivery, right nephrectomy was performed in order to evaluate the injured kidney. The fresh perfused kidneys were stained with anti-GFP antibody. Polymerase chain reaction (PCR) assay was also performed for the Y chromosome detection. Cell culture was performed to detect the GFP-positive cells. Technetium-99m-DMSA renal scan and single-photon emission computed tomography (SPECT) were performed after renal damage induction and 3 months later to evaluate the improvement of renal integrity. RESULTS: The presence of FSCs was confirmed by immune histochemical staining as well as immunofluorescent imaging of the damaged part. Gradient PCR of female rat purified DNA demonstrated the presence of Y-chromosome in the damaged maternal kidney. Moreover, the culture of kidney cells showed GPF- positive cells by immunofluorescence microscopy. The acute renal scar was repaired and the integrity of damaged kidney reached to near normal levels in experimental group as shown in DMSA scan. However, no significant improvement was observed in control group. CONCLUSION: FSC seems to be the main mechanism in repairing of the maternal renal injury during pregnancy as indicated by Y chromosome and GFP-positive cells in the sub-cultured medium.
Assuntos
Quimerismo , Células-Tronco Fetais/fisiologia , Nefropatias/fisiopatologia , Troca Materno-Fetal/fisiologia , Cicatrização/fisiologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Imunofluorescência , Imuno-Histoquímica , Nefropatias/diagnóstico por imagem , Nefropatias/patologia , Masculino , Reação em Cadeia da Polimerase , Gravidez , Compostos Radiofarmacêuticos , Ratos Sprague-Dawley , Ácido Dimercaptossuccínico Tecnécio Tc 99m , Fatores de Tempo , Tomografia Computadorizada de Emissão de Fóton Único , Cromossomo YRESUMO
Cellular functions emerge from the collective action of a large number of different proteins. Understanding how these protein networks operate requires monitoring their components in intact cells. Due to intercellular and intracellular molecular variability, it is important to monitor simultaneously multiple components at high spatiotemporal resolution. However, inherent trade-offs narrow the boundaries of achievable multiplexed imaging. Pushing these boundaries is essential for a better understanding of cellular processes. Here the motivations, challenges and approaches for multiplexed imaging of intracellular protein networks are discussed. © 2016 International Society for Advancement of Cytometry.
Assuntos
Citoplasma/química , Proteínas de Fluorescência Verde/química , Imagem Molecular/métodos , Mapas de Interação de Proteínas , Citoplasma/genética , Microscopia de FluorescênciaRESUMO
Blister spot (Colletotrichum gloeosporioides) is now widespread in most coffee producing states of Brazil, becoming a limiting factor for production. The lack of data relating to the reproduction of typical symptoms (light green, oily patches) leaves a gap within the pathosystem, forcing the search for new methodologies for monitoring the disease. Monitoring of genetically modified organisms has proven to be an effective tool in understanding the host x pathogen interactions. Thus, the present study was carried out to evaluate the effectiveness of two systems of genetic transformation in obtaining mutants using the gfp reporter gene. Using the two transformation systems (PEG and electroporation) revealed the efficiency of both, confirmed by fluorescence microscopy and resistance to the antibiotic hygromycin-B, when incorporated into the culture medium. The fungus maintained its cultural and morphological characteristics when compared to wild strains. When inoculated on coffee seedlings, it was found that the pathogenicity of the processed isolates had not changed.
Assuntos
Colletotrichum/genética , Colletotrichum/isolamento & purificação , Resistência Microbiana a Medicamentos , Proteínas Fúngicas/análise , Metodologia como Assunto , Microscopia de Fluorescência , Fungos Mitospóricos , VirulênciaRESUMO
Blister spot (Colletotrichum gloeosporioides) is now widespread in most coffee producing states of Brazil, becoming a limiting factor for production. The lack of data relating to the reproduction of typical symptoms (light green, oily patches) leaves a gap within the pathosystem, forcing the search for new methodologies for monitoring the disease. Monitoring of genetically modified organisms has proven to be an effective tool in understanding the host × pathogen interactions. Thus, the present study was carried out to evaluate the effectiveness of two systems of genetic transformation in obtaining mutants using the gfp reporter gene. Using the two transformation systems (PEG and electroporation) revealed the efficiency of both, confirmed by fluorescence microscopy and resistance to the antibiotic hygromycin-B, when incorporated into the culture medium. The fungus maintained its cultural and morphological characteristics when compared to wild strains. When inoculated on coffee seedlings, it was found that the pathogenicity of the processed isolates had not changed.
RESUMO
Blister spot (Colletotrichum gloeosporioides) is now widespread in most coffee producing states of Brazil, becoming a limiting factor for production. The lack of data relating to the reproduction of typical symptoms (light green, oily patches) leaves a gap within the pathosystem, forcing the search for new methodologies for monitoring the disease. Monitoring of genetically modified organisms has proven to be an effective tool in understanding the host x pathogen interactions. Thus, the present study was carried out to evaluate the effectiveness of two systems of genetic transformation in obtaining mutants using the gfp reporter gene. Using the two transformation systems (PEG and electroporation) revealed the efficiency of both, confirmed by fluorescence microscopy and resistance to the antibiotic hygromycin-B, when incorporated into the culture medium. The fungus maintained its cultural and morphological characteristics when compared to wild strains. When inoculated on coffee seedlings, it was found that the pathogenicity of the processed isolates had not changed.