共查询到20条相似文献,搜索用时 31 毫秒
1.
Combining protein complementation assays with resonance energy transfer to detect multipartner protein complexes in living cells 总被引:1,自引:0,他引:1
Rebois RV Robitaille M Pétrin D Zylbergold P Trieu P Hébert TE 《Methods (San Diego, Calif.)》2008,45(3):214-218
A variety of fluorescent proteins with different spectral properties have been created by mutating green fluorescent protein. When these proteins are split in two, neither fragment is fluorescent per se, nor can a fluorescent protein be reconstituted by co-expressing the complementary N- and C-terminal fragments. However, when these fragments are genetically fused to proteins that associate with each other in cellulo, the N- and C-terminal fragments of the fluorescent protein are brought together and can reconstitute a fluorescent protein. A similar protein complementation assay (PCA) can be performed with two complementary fragments of various luciferase isoforms. This makes these assays useful tools for detecting the association of two proteins in living cells. Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) occurs when energy from, respectively, a luminescent or fluorescent donor protein is non-radiatively transferred to a fluorescent acceptor protein. This transfer of energy can only occur if the proteins are within 100 Å of each other. Thus, BRET and FRET are also useful tools for detecting the association of two proteins in living cells. By combining different protein fragment complementation assays (PCA) with BRET or FRET it is possible to demonstrate that three or more proteins are simultaneous parts of the same protein complex in living cells. As an example of the utility of this approach, we show that as many as four different proteins are simultaneously associated as part of a G protein-coupled receptor signalling complex. 相似文献
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Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins
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Gautier I Tramier M Durieux C Coppey J Pansu RB Nicolas JC Kemnitz K Coppey-Moisan M 《Biophysical journal》2001,80(6):3000-3008
Fluorescence anisotropy decay microscopy was used to determine, in individual living cells, the spatial monomer-dimer distribution of proteins, as exemplified by herpes simplex virus thymidine kinase (TK) fused to green fluorescent protein (GFP). Accordingly, the fluorescence anisotropy dynamics of two fusion proteins (TK27GFP and TK366GFP) was recorded in the confocal mode by ultra-sensitive time-correlated single-photon counting. This provided a measurement of the rotational time of these proteins, which, by comparing with GFP, allowed the determination of their oligomeric state in both the cytoplasm and the nucleus. It also revealed energy homo-transfer within aggregates that TK366GFP progressively formed. Using a symmetric dimer model, structural parameters were estimated; the mutual orientation of the transition dipoles of the two GFP chromophores, calculated from the residual anisotropy, was 44.6 +/- 1.6 degrees, and the upper intermolecular limit between the two fluorescent tags, calculated from the energy transfer rate, was 70 A. Acquisition of the fluorescence steady-state intensity, lifetime, and anisotropy decay in the same cells, at different times after transfection, indicated that TK366GFP was initially in a monomeric state and then formed dimers that grew into aggregates. Picosecond time-resolved fluorescence anisotropy microscopy opens a promising avenue for obtaining structural information on proteins in individual living cells, even when expression levels are very low. 相似文献
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Subcellular localization of biomacromolecules (nucleotides and proteins) is the base for their proper function in bacterial cells. One model to explain the localization of biomacromolecules, particularly proteins, is “diffusion and capture”. In this model, proteins are localized by diffusion through the cytoplasm or the membrane until binding to another protein or proteins that were already previously sequestered in cells. The use of fusions with fluorescent proteins to follow the fate of biomacromolecules has given new insight into the molecular localization mechanisms in living cells. Here, several proteins following a diffusion and capture mechanism to reach their proper location in the cells are presented. Some RNAs also seem to localize by this mechanism. It is an intrinsic feature that the information for molecular localization should exist in the sequences of protein itself. However, very little information has been available in this field to date. 相似文献
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Experiments with fluorescence recovery after photobleaching (FRAP) started 30 years ago to visualize the lateral mobility and dynamics of fluorescent proteins in living cells. Its popularity increased when non-invasive fluorescent tagging became possible with the green fluorescent protein (GFP). Many researchers use GFP to study the localization of fusion proteins in fixed or living cells, but the same fluorescent proteins can also be used to study protein mobility in living cells. Here we review the potential of FRAP to study protein dynamics and activity within a single living cell. These measurements can be made with most standard confocal laser-scanning microscopes equipped with photobleaching protocols. 相似文献
7.
Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins 总被引:3,自引:0,他引:3
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Quantitative characterization of protein interactions under physiological conditions is vital for systems biology. Fluorescence photobleaching/activation experiments of GFP-tagged proteins are frequently used for this purpose, but robust analysis methods to extract physicochemical parameters from such data are lacking. Here, we implemented a reaction-diffusion model to determine the contributions of protein interaction and diffusion on fluorescence redistribution. The model was validated and applied to five chromatin-interacting proteins probed by photoactivation in living cells. We found that very transient interactions are common for chromatin proteins. Their observed mobility was limited by the amount of free protein available for diffusion but not by the short residence time of the bound proteins. Individual proteins thus locally scan chromatin for binding sites, rather than diffusing globally before rebinding at random nuclear positions. By taking the real cellular geometry and the inhomogeneous distribution of binding sites into account, our model provides a general framework to analyze the mobility of fluorescently tagged factors. Furthermore, it defines the experimental limitations of fluorescence perturbation experiments and highlights the need for complementary methods to measure transient biochemical interactions in living cells. 相似文献
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By using fluorescent labelling techniques, the distribution and dynamics of proteins can be measured within living cells, allowing to study in vivo the response of cells to a triggering event, such as DNA damage. In order to evaluate the reaction rate constants and to identify the proteins and reactions that are essential for the investigated process, mechanistic models are used, which often contain many proteins and associated parameters and are therefore underdetermined by the data. In order to establish criteria for assessing the significance of a model, we present here a systematic investigation of the information that can be reliably deduced from protein recruitment data, assuming that the complete set of reactions that affect the data of the considered protein species is not known. To this purpose, we study in detail models where one or two proteins that influence each other are recruited to a substrate. We show that in many cases the kind of interaction between the proteins can be deduced by analyzing the shape of the recruitment curves of one protein. Furthermore, we discuss in general in which cases it is possible to discriminate between different models and in which cases it is impossible based on the data. Finally, we argue that if different models fit experimental data equally well, conducting experiments with different protein concentrations would allow discrimination between the alternative models in many cases. 相似文献
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Nina Senutovitch Lawrence Vernetti Robert Boltz Richard DeBiasio Albert Gough D Lansing Taylor 《Experimental biology and medicine (Maywood, N.J.)》2015,240(6):795-808
This mini-review discusses the evolution of fluorescence as a tool to study living cells and tissues in vitro and the present role of fluorescent protein biosensors (FPBs) in microphysiological systems (MPSs). FPBs allow the measurement of temporal and spatial dynamics of targeted cellular events involved in normal and perturbed cellular assay systems and MPSs in real time. FPBs evolved from fluorescent analog cytochemistry (FAC) that permitted the measurement of the dynamics of purified proteins covalently labeled with environmentally insensitive fluorescent dyes and then incorporated into living cells, as well as a large list of diffusible fluorescent probes engineered to measure environmental changes in living cells. In parallel, a wide range of fluorescence microscopy methods were developed to measure the chemical and molecular activities of the labeled cells, including ratio imaging, fluorescence lifetime, total internal reflection, 3D imaging, including super-resolution, as well as high-content screening. FPBs evolved from FAC by combining environmentally sensitive fluorescent dyes with proteins in order to monitor specific physiological events such as post-translational modifications, production of metabolites, changes in various ion concentrations, and the dynamic interaction of proteins with defined macromolecules in time and space within cells. Original FPBs involved the engineering of fluorescent dyes to sense specific activities when covalently attached to particular domains of the targeted protein. The subsequent development of fluorescent proteins (FPs), such as the green fluorescent protein, dramatically accelerated the adoption of studying living cells, since the genetic “labeling” of proteins became a relatively simple method that permitted the analysis of temporal–spatial dynamics of a wide range of proteins. Investigators subsequently engineered the fluorescence properties of the FPs for environmental sensitivity that, when combined with targeted proteins/peptides, created a new generation of FPBs. Examples of FPBs that are useful in MPS are presented, including the design, testing, and application in a liver MPS. 相似文献
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The bimolecular fluorescence complementation (BiFC) assay is a powerful tool for visualizing and identifying protein interactions in living cells. This assay is based on the principle of protein-fragment complementation, using two nonfluorescent fragments derived from fluorescent proteins. When two fragments are brought together in living cells by tethering each to one of a pair of interacting proteins, fluorescence is restored. Here, we provide a protocol for a Venus-based BiFC assay to visualize protein interactions in the living nematode, Caenorhabditis elegans. We discuss how to design appropriate C. elegans BiFC cloning vectors to enable visualization of protein interactions using either inducible heat shock promoters or native promoters; transform the constructs into worms by microinjection; and analyze and interpret the resulting data. When expression of BiFC fusion proteins is induced by heat shock, the fluorescent signals can be visualized as early as 30 min after induction and last for 24 h in transgenic animals. The entire procedure takes 2-3 weeks to complete. 相似文献
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Misfolded proteins in the endoplasmic reticulum (ER) are dislocated to the cytosol to be degraded by the proteasomes. Various plant and bacterial toxins and certain viruses hijack this dislocation pathway to exert their toxicity or to infect cells. In this study, we report a dislocation-dependent reconstituted GFP (drGFP) assay that allows, for the first time, imaging proteins dislocated from the ER lumen to the cytosol in living cells. Our results indicate that both luminal and membrane-spanning ER proteins can be fully dislocated from the ER to the cytosol. By combining the drGFP assay with RNAi or chemical inhibitors of proteins in the Hrd1 ubiquitin ligase complex, we demonstrate that the Sel1L, Hrd1, p97/VCP, and importin β proteins are required for the dislocation of misfolded luminal α-1 antitrypsin. The strategy described in this work is broadly applicable to the study of other types of transmembrane transport of proteins and likely also of viruses and toxins in living cells. 相似文献
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Our previous studies showed that, unlike tissue extracts, the cells of living organs secrete substances capable of inducing the same organ rudiments in the early gastrula ectoderm (EGE). In this work, the molecular nature of these substances was studied. The porcine lens epithelium was chosen for the initial analysis. When cultivated, this epithelium secreted a mixture of proteins, which were separated by gel-filtration. Both the total protein mixture and its individual fractions were tested for their inducing capacity using the early gastrula ectoderm of Rana temporaria. Unexpected results were obtained, which indicated that (a) the mixture of native proteins secreted by lens epithelium has a selective inducing capacity differing from those of individual fractions isolated from this mixture and (b) each fraction has a specific effect, but all of them cause the induction of neural tissue or sensory organs. These results (obtained for the first time) suggest that the inducing capacity of individual protein fractions is wider than that of the total protein mixture secreted by lens epithelium. This fact raises a question concerning the relationships between the mechanisms underlying the corresponding inducing effects. 相似文献
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Building and breeding molecules to spy on cells and tumors 总被引:8,自引:0,他引:8
Tsien RY 《FEBS letters》2005,579(4):927-932
Imaging of biochemical processes in living cells and organisms is essential for understanding how genes and gene products work together in space and time and in health and disease. Such imaging depends crucially on indicator molecules designed to maximize sensitivity and specificity. These molecules can be entirely synthetic, entirely genetically encoded macromolecules, or hybrid combinations, each approach having its own pros and cons. Recent examples from the author's laboratory include peptides whose uptake into cells is triggered by proteases typical of tumors, monomeric red fluorescent proteins and biarsenical-tetracysteine systems for determining the age and electron-microscopic location of proteins. 相似文献
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Fluorescence recovery after photobleaching (FRAP) has become a popular technique to investigate the behavior of proteins in living cells. Although the technique is relatively old, its application to studying endogenous intracellular proteins in living cells is relatively recent and is a consequence of the newly developed fluorescent protein-based living cell protein tags. This is particularly true for nuclear proteins, in which endogenous protein mobility has only recently been studied. Here we examine the experimental design and analysis of FRAP experiments. Mathematical modeling of FRAP data enables the experimentalist to extract information such as the association and dissociation constants, distribution of a protein between mobile and immobilized pools, and the effective diffusion coefficient of the molecule under study. As experimentalists begin to dissect the relative influence of protein domains within individual proteins, this approach will allow a quantitative assessment of the relative influences of different molecular interactions on the steady-state distribution and protein function in vivo. 相似文献
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《Biochimica et Biophysica Acta (BBA)/General Subjects》2020,1864(2):129364
BackgroundAccumulating evidence from the experimental and computational studies indicated that the functional properties of proteins are different between in vitro and living cells, raising the necessity to examine the protein structure under the native intracellular milieu. To gain structural information of the proteins inside the living cells at an atomic resolution, in-cell NMR method has been developed for the past two decades.Scope of reviewIn this review, we will overview the recent progress in the methodological developments and the biological applications of in-cell NMR, and discuss the advances and challenges in this filed.Major conclusionsA number of methods were developed to enrich the isotope-labeled proteins inside the cells, enabling the in-cell NMR observation of bacterial cells as well as eukaryotic cells. In-cell NMR has been applied to various biological systems, including de novo structure determinations, protein/protein or protein/drug interactions, and monitoring of chemical reactions exerted by the endogenous enzymes. The bioreactor system, in which the cells in the NMR tube are perfused by fresh culture medium, enabled the long-term in-cell NMR measurements, and the real-time observations of intracellular responses upon external stimuli.General significanceIn-cell NMR has become a unique technology for its ability to obtain the function-related structural information of the target proteins under the physiological or pathological cellular environments, which cannot be reconstituted in vitro. 相似文献
17.
Minchul Kang Charles A. Day Anne K. Kenworthy Emmanuele DiBenedetto 《Traffic (Copenhagen, Denmark)》2012,13(12):1589-1600
Quantitative measurements of diffusion can provide important information about how proteins and lipids interact with their environment within the cell and the effective size of the diffusing species. Confocal fluorescence recovery after photobleaching (FRAP) is one of the most widely accessible approaches to measure protein and lipid diffusion in living cells. However, straightforward approaches to quantify confocal FRAP measurements in terms of absolute diffusion coefficients are currently lacking. Here, we report a simplified equation that can be used to extract diffusion coefficients from confocal FRAP data using the half time of recovery and effective bleach radius for a circular bleach region, and validate this equation for a series of fluorescently labeled soluble and membrane‐bound proteins and lipids. We show that using this approach, diffusion coefficients ranging over three orders of magnitude can be obtained from confocal FRAP measurements performed under standard imaging conditions, highlighting its broad applicability. 相似文献
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In eukaryotes, protein trafficking to and from the nucleus, or shuttling, has been demonstrated to be an important function for proteins that have vital roles in one or both subcellular compartments. Current techniques of detecting protein nuclear shuttling are extremely labor intensive and only statically visualize evidence of shuttling. Fluorescence recovery after photobleaching (FRAP), or fluorescence microphotolysis, has proven to be an effective method of analyzing protein dynamics in live cells, especially when coupled to GFP technology. Here, we describe a relatively simple in vivo protein nuclear shuttling assay that utilizes red fluorescent and green fluorescent protein fusions as substrates for FRAP using a laser confocal microscope. This technique is less time consuming than established shuttle assays, is internally controlled, and visualizes nucleocytoplasmic shuttling in living cells of the same species and cell type. This technique can be potentially used to detect the ability of any nuclear protein to shuttle from the nucleus to any other subcellular compartment for any eukaryotic species in which GFP or dsRed1 fusion protein can be expressed. 相似文献
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Toru Ezure Kei Nanatani Yoko Sato Satomi Suzuki Keishi Aizawa Satoshi Souma Masaaki Ito Takahiro Hohsaka Gunnar von Heijine Toshihiko Utsumi Keietsu Abe Eiji Ando Nobuyuki Uozumi 《PloS one》2014,9(12)
Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins. 相似文献
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Ventura S 《Current molecular medicine》2011,11(7):582-598
Many cellular processes depend on the establishment of selective stable or transient interactions between proteins. Therefore, the ability to identify and characterize these contacts in physiologically relevant environments is crucial to understanding the networks of contacts that allow the transmission and integration of biological information in living cells. Protein-fragment complementation assays (PCA) have emerged as approaches that report on the proximity of two given proteins in the cell at a given location and time. In particular, bimolecular fluorescence complementation (BIFC) allows noninvasive imaging of protein binding in living cells at high spatial resolution and without the requirement for exogenous substrates. In the present review, we discuss PCA and BIFC fundamentals, the implementation of BIFC assays and selected applications of BIFC in drug discovery, developmental studies or neurological disorders. 相似文献