Green Fluorescent Protein Derivatives Research Articles

Fluorescence Modulation of ortho-Green Fluorescent Protein Chromophores Following Ultrafast Proton Transfer in Solution

Photophysical and photochemical properties of the green fluorescent protein (GFP) chromophore and derivatives underlie their bioimaging applications. To date, ultrafast spectroscopic tools represent the key for unraveling fluorescence mechanisms toward rational design of this powerful biomimetic framework. To correlate the excited-state intramolecular proton transfer (ESIPT) with chromophore emission properties, we implement experimental and computational tool sets to elucidate real-time electronic and structural dynamics of two archetypal ortho-GFP chromophores (o-HBDI and o-LHBDI) possessing an intramolecular hydrogen bond to undergo efficient ESIPT, only differing in a bridge-bond constraint. Using excited-state femtosecond stimulated Raman spectroscopy (FSRS), a low-frequency phenolic (P)-ring-deformation mode (∼562 cm-1) was uncovered to accompany ESIPT. The tautomerized chromophore undergoes either rapid P-ring isomerization to reach the ground state with essentially no fluorescence for o-HBDI or enhanced (up to an impressive 180-fold in acetonitrile) and solvent-polarity-dependent fluorescence by P-ring locking in o-LHBDI. The significant dependence of the fluorescence enhancement ratio on solvent viscosity confirms P-ring isomerization as the dominant nonradiative decay pathway for o-HBDI. This work provides crucial insights into the dynamic solute-solvent electrostatic and steric interactions, enabling the application-specific improvement of ESIPT-capable molecules as versatile fluorescence-based sensors and imaging agents from large Stokes shift emission to brighter probes in physiological environments.

Nanoparticle-Mediated Assembly of Peptoid Nanosheets Functionalized with Solid-Binding Proteins: Designing Heterostructures for Hierarchy.

The fabrication of ordered architectures that intimately integrate polymer, protein, and inorganic components remains difficult. Two promising building blocks to tackle this challenge are peptoids, peptide mimics capable of self-assembly into well-defined structures, and solid-binding peptides, which offer a biological path to controlled inorganic assembly. Here, we report on the synthesis of 3.3-nm-thick thiol-reactive peptoid nanosheets from equimolar mixtures of unmodified and maleimide-derivatized versions of the Nbpe6Nce6 oligomer, optimize the location of engineered cysteine residues in silica-binding derivatives of superfolder green fluorescent protein for maleimide conjugation, and react the two components to form protein-peptoid hybrids exhibiting partial or uniform protein coverage on both of their sides. Using 10 nm silica nanoparticles, we trigger the stacking of these 2D structures into a multilayered material composed of alternating peptoid, protein, and organic layers. This simple and modular approach to hierarchical hybrid synthesis should prove useful in bioimaging and photocatalysis applications.

Ultrafast excited-state proton transfer dynamics in dihalogenated non-fluorescent and fluorescent GFP chromophores.

Green fluorescent protein (GFP) has enabled a myriad of bioimaging advances due to its photophysical and photochemical properties. To deepen the mechanistic understanding of such light-induced processes, novel derivatives of GFP chromophore p-HBDI were engineered by fluorination or bromination of the phenolic moiety into superphotoacids, which efficiently undergo excited-state proton transfer (ESPT) in aqueous solution within the short lifetime of the excited state, as opposed to p-HBDI where efficient ESPT is not observed. In addition, we tuned the excited-state lifetime from picoseconds to nanoseconds by conformational locking of the p-HBDI backbone, essentially transforming the nonfluorescent chromophores into highly fluorescent ones. The unlocked superphotoacids undergo a barrierless ESPT without much solvent activity, whereas the locked counterparts exhibit two distinct solvent-involved ESPT pathways. Comparative analysis of femtosecond transient absorption spectra of these unlocked and locked superphotoacids reveals that the ESPT rates adopt an "inverted" kinetic behavior as the thermodynamic driving force increases upon locking the backbone. Further experimental and theoretical investigations are expected to shed more light on the interplay between the modified electronic structure (mainly by dihalogenation) and nuclear motions (by conformational locking) of the functionalized GFP derivatives (e.g., fluorescence on and off).

Red-Shifted Substrates for FAST Fluorogen-Activating Protein Based on the GFP-Like Chromophores.

A genetically encoded fluorescent tag for live cell microscopy is presented. This tag is composed of previously published fluorogen-activating protein FAST and a novel fluorogenic derivative of green fluorescent protein (GFP)-like chromophore with red fluorescence. The reversible binding of the novel fluorogen and FAST is accompanied by three orders of magnitude increase in red fluorescence (580-650 nm). The proposed dye instantly stains target cellular proteins fused with FAST, washes out in a minute timescale, and exhibits higher photostability of the fluorescence signal in confocal and widefield microscopy, in contrast with previously published fluorogen:FAST complexes.

Annals of Neurology: Volume 82, Number 6, December 2017

A stack of five 5 micron‐thick confocal photomicrographs showing the striatum in a rat injected into the frontal cortex with a viral agent causing neurons to express channelrhodopsin 2 fused to a derivative of green fluorescent protein. Note the bundles of descending cortical axons, and the ramifications and small axonal boutons scattered within the striatum. A few large dendrites covered with spines are seen in the upper part of the field, due to leakage of virus to transfect some medium spiny neurons in the striatum as well. In rats with brain iron deficiency, these synaptic terminals become hypersensitive in their ability to release glutamate upon direct light stimulation. See Yepes et al., p. 951 for details. Ann Neurol 2017;82:1–1

Biological nitrogen fixation and plant growth promotion of lodgepole pine by an endophytic diazotroph Paenibacillus polymyxa and its GFP-tagged derivative

Paenibacillus polymyxa P2b-2R is an endophytic diazotroph originally isolated from lodgepole pine. It is reported to fix significant amounts of nitrogen (N) and promote plant growth. To evaluate the endophytic colonization sites, a green fluorescent protein (GFP) derivative of P2b-2R was generated (P2b–2Rgfp), but the effects of GFP modification on the functioning of P2b-2R have not been fully elucidated yet. In this study, we wanted to confirm and contrast the N-fixing and plant-growth-promoting abilities of P2b-2Rgfp with those of the wild-type P2b-2R in lodgepole pine. Pine seedlings were grown in an N-limited environment and harvested 2, 4, 8, and 12 months after inoculation to evaluate the endophytic and rhizospheric colonization by both strains, the amount of N-fixed, and seedling growth enhancement. Both P2b-2R and P2b-2Rgfp strains formed persistent rhizospheric and endophytic populations; fixed N; and enhanced seedling growth continuously after 4 months. P2b-2Rgfp-treated seedlings outperformed the P2b-2R-treated seedlings in terms of biomass only during the initial stages of plant development, but the differences decreased during the trial and were not significant towards the end. To the best of our knowledge, this is the first study that reports the effects of GFP-tagging of an endophyte when inoculated into a gymnosperm tree species.

Expression and Secretion of Cyan Fluorescent Protein (CFP) in B. subtilis using the Chitinase Promoter from Bacillus pumilus SG2

Background:Improved cyan fluorescent protein (ICFP) is a monochromic, green fluorescent protein (GFP) derivative produced by Aequorea macrodactyla in a process similar to GFP. This protein has strong absorption spectra at wavelengths 426-446 nm. ICFP can be used in cell, organelle or intracellular protein labeling, investigating the protein-protein interactions as well as assessing the promoter activities.Methods:In our previous study, the promoters of two chitinases (ChiS and ChiL) from Bacillus pumilus SG2 were assessed in B. subtilis and their regulatory elements were characterized. In the present study, icfp was cloned downstream of several truncated promoters obtained in the former study, and ICFP expression was evaluated in B. subtilis.Results:Extracellular expression and secretion of ICFP were analyzed under the control of different truncated versions of ChiSL promoters grown on different media. Results from SDS-PAGE and fluorimetric analyses showed that there were different expression rates of CFP; however, the UPChi-ICFP3 construct exhibited a higher level of expression and secretion in the culture medium.Conclusion:Our presented results revealed that inserting this truncated form of Chi promoter upstream of the ICFP, as a reporter gene, in B. subtilis led to an approximately ten fold increase in ICFP expression.

Making Fluorescent Streptococci and Enterococci for Live Imaging.

Since the discovery of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria, outstanding fluorescent labeling tools with numerous applications in vastly different areas of life sciences have been developed. To optimize GFP for diverse life science applications, a large variety of GFP derivatives with different environmental characteristics have been generated by mutagenesis. The enhanced green fluorescent protein (EGFP) is a well-known GFP derivative with highly increased fluorescence intensity compared to the GFP wild-type molecule. Further optimization strategies include numerous GFP derivatives with blue- and yellow-shifted fluorescence and increased pH-stability. The methods reported herein describe in detail the construction of customized fluorescent GFP reporter plasmids where the fluorescence gene is expressed under the control of a certain bacterial promoter of interest. Special attention is given to the GFP derivatives EGFP and Sirius. We explain how to generate EGFP/Sirius expressing streptococci and how to employ recombinantly labeled streptococci in different downstream fluorescent applications.

Filling a GAP—An Optimized Probe for ER Ca2+ Imaging In Vivo

In this issue of Cell Chemical Biology, Navas-Navarro etal. (2016) demonstrate that fusion of engineered derivatives of the long-known jellyfish proteins green fluorescent protein (GFP) and aequorin yield optimized genetically encoded fluorescent probes for detecting Ca(2+) signals within the endoplasmic reticulum (ER) of living animals.

Seedling growth promotion and nitrogen fixation by a bacterial endophyte Paenibacillus polymyxa P2b-2R and its GFP derivative in corn in a long-term trial

A plant growth promoting endophyte, Paenibacillus polymyxa P2b-2R, originally isolated form a lodgepole pine seedling and its green fluorescent protein (GFP) derivative, P2b-2Rgfp, were evaluated for their ability to survive, fix atmospheric nitrogen (N) and promote plant growth when inoculated into corn (Zea Mays L.) in a long-term trial. We were also interested to see the effects of GFP-tagging of P2b-2R on its ability to promote growth of corn seedlings in a long-term study. Corn seedlings were inoculated with either strain P2b-2R or P2b-2Rgfp and non-inoculated seedlings were treated as controls. Seedlings were harvested after 3 months and evaluated for plant growth promotion (length and biomass) and N fixation (15N foliar dilution assay). Colonization and survival of P2b-2R and P2b-2Rgfp outside (rhizosphere) and inside (internal tissues) the inoculated seedlings were also determined. Both strains survived inside and outside corn seedlings forming rhizospheric and endophytic colonies in stem and root tissues. Inoculation by P2b-2R strain promoted corn plant growth via enhancing seedling length and biomass by 52 % and 53 %, respectively. Similarly, P2b-2Rgfp inoculation enhanced seedling length by 68 % and biomass by 67 %. Corn seedlings inoculated with strain P2b-2R derived 30 % of foliar N from the atmosphere and seedlings inoculated with P2b-2Rgfp derived 32 % of foliar N from the atmosphere. But there was no statistically significant difference between P2b-2R and P2b-2Rgfp treated seedlings in terms of overall seedling length, biomass and amount of N fixed in this long-term trial. These results combined with the results from an earlier study suggest that P. polymyxa P2b-2R and its GFP-tagged derivative is capable of enhancing overall plant growth throughout the life cycle of corn plant.

Brighter Fluorescent Derivatives of UTI89 Utilizing a Monomeric vGFP.

Fluorescent proteins, especially green fluorescent protein (GFP), have been instrumental in understanding urinary tract infection pathogenesis by uropathogenic Escherichia coli (UPEC). We have used a recently developed GFP variant, vsfGFP-9, to create new plasmid- and chromosome-based GFP derivatives of the UPEC strain UTI89. The vsfGFP-9 strains are nearly 10× brighter with no in vitro growth or in vivo virulence defects compared to previously reported GFP-expressing UTI89 strains. The chromosomal vsfGFP-9 strain is equivalent to the wild type UTI89 during in vivo UTI, while both plasmid GFP constructs have an equivalent virulence defect compared to non-plasmid carrying UTI89. These new vsfGFP-9 expressing strains should be useful for further studies of the pathogenesis of UTI89, and similar strategies can be used to create improved fluorescent derivatives of other UPEC strains.

Turnip crinkle virus with nonviral gene cancels the effect of silencing suppressors of P19 and 2b in Arabidopsis thaliana

In plants, green fluorescent protein (GFP) has become a preferred molecular marker for gene expression and cellular localization, and plant viral vectors are valuable tools for heterologous gene expression. Some plant viruses have been used for expression of GFP, and the activities of these viruses are barely affected by the extra GFP gene. In contrast, the packaging and the length of Turnip crinkle virus (TCV) genome is strictly limited when foreign genes are inserted into the coding sequences of TCV genome. In this report, we removed the silencing suppressor p38 from TCV, and constructed GFP derivatives of TCV. Then the resulting TCV mutants were used to infect Arabidopsis plants containing mutations in key silencing pathway genes, including triple dcl2/dcl3/dcl4, dcl2, dcl4 and ago mutant plants. Our results demonstrate that the activity of TCV is affected by nonviral GFP insert in Arabidopsis plants, and RNA silencing appears not play an important role. AGOs appear to be more efficient at slicing RNAs of viral origin, especially AGO2 and AGO7. Although the viral suppressors of RNA silencing (VSRs) P19 and 2b can enhance the accumulation of viral RNAs, neither P19 nor 2b can significantly increase the expression of TCV mutants with nonviral genes. TCV is an example of an RNA virus that is recalcitrant to add nonviral gene sequences.

Channelrhodopsin Photochromic Reactions Provide Multicolor Optogenetic Control

In this issue of the Biophysical Journal, Venkatachalam and Cohen (1) present an innovative combination of opsin-based optogenetic voltage control with simultaneous imaging of green fluorescent protein (GFP)-based sensors. The challenge is that the overlapping absorption spectra of GFP derivatives and the commonly used blue-light activated ChR2-based channelrhodopsins prevent photoactivation of neuron firing without simultaneously perturbing the fluorescent reporter. The authors elegantly circumvent this overlap by taking advantage of the color-sensitive photoreactions of spectral intermediates of ChR2. An interesting twist is that in the “stoplight” technique that they developed, optical engineering creatively mimics nature. Evolution produced a mechanism using the same principle to enable color-discriminating motility responses mediated by the phototaxis receptor sensory rhodopsin I in haloarchaea.

An evaluation of genetically encoded FRET-based biosensors for quantitative metabolite analyses in vivo

A broad range of genetically-encoded fluorescence biosensors has been developed, allowing the detection of signaling intermediates and metabolites in real time. Many of these biosensors are based on Foerster Resonance Energy Transfer (FRET). The two biosensors of the well-known “Venus-flytrap” type exemplarily studied in this work are composed of a central sugar binding protein flanked by two green fluorescent protein derivatives, namely ECFP as well as Citrine and EYFP, respectively. In order to evaluate FRET-based biosensors as an in vivo tool for quantitative metabolite analyses, we have thoroughly studied the effects of pH, buffer salts, ionic strength, temperature and several intracellular metabolites on the signal intensity of both biosensors and both fluorescence proteins. Almost all micro-environmental variations led to considerably different FRET signals, because either the fluorescent proteins or the metabolite binding domains were affected by the tested parameters. This resulted not only in altered FRET ratios between the apo state and the saturated state but also in significant shifts of the apparent binding constant. This underlines the necessity of careful controls in order to allow reliable quantitative measurements in vivo.

Modification of enhanced green fluorescent protein for secretion out of cells

Since its discovery approximately 20 years ago, green fluorescent protein (GFP) has become one of the most widely used reporter proteins. GFP has been used in a variety of living organisms, ranging from E. coli to higher eukaryotes, such as plants and animals. The biggest advantage of using this reporter protein is that it can be used to monitor in vitro and in vivo gene expression. One important limitation, however, is its inability to be secreted out of cells. For this reason, it has been difficult to directly measure the expression level of the regulatory sequence of a gene of interest quantitatively. To overcome this drawback, we have modified the enhanced green fluorescent protein gene (EGFP), a derivative of GFP, by adding a signal peptide sequence that encodes a rat follicle-stimulating hormone (FSH) β-subunit upstream of EGFP. Following the expression of this modified gene in several cell types, we have found efficient secretion of EGFP. Consequently, with the secreted protein, we could easily quantify the gene expression level with high reliability. Therefore, the use of our modified EGFP expression cassette would greatly facilitate the evaluation of regulatory sequences, such as promoters and enhancers. Further, it will also be very helpful in the study of transgenic livestock intended to use as bioreactors for mass production of pharmaceuticals.

Strengths and Weaknesses of Recently Engineered Red Fluorescent Proteins Evaluated in Live Cells Using Fluorescence Correlation Spectroscopy

The scientific community is still looking for a bright, stable red fluorescent protein (FP) as functional as the current best derivatives of green fluorescent protein (GFP). The red FPs exploit the reduced background of cells imaged in the red region of the visible spectrum, but photophysical short comings have limited their use for some spectroscopic approaches. Introduced nearly a decade ago, mCherry remains the most often used red FP for fluorescence correlation spectroscopy (FCS) and other single molecule techniques, despite the advent of many newer red FPs. All red FPs suffer from complex photophysics involving reversible conversions to a dark state (flickering), a property that results in fairly low red FP quantum yields and potential interference with spectroscopic analyses including FCS. The current report describes assays developed to determine the best working conditions for, and to uncover the shortcoming of, four recently engineered red FPs for use in FCS and other diffusion and spectroscopic studies. All five red FPs assayed had potential shortcomings leading to the conclusion that the current best red FP for FCS is still mCherry. The assays developed here aim to enable the rapid evaluation of new red FPs and their smooth adaptation to live cell spectroscopic microscopy and nanoscopy.

Construction of two ureolytic model organisms for the study of microbially induced calcium carbonate precipitation

Two bacterial strains, Pseudomonas aeruginosa MJK1 and Escherichia coli MJK2, were constructed that both express green fluorescent protein (GFP) and carry out ureolysis. These two novel model organisms are useful for studying bacterial carbonate mineral precipitation processes and specifically ureolysis-driven microbially induced calcium carbonate precipitation (MICP). The strains were constructed by adding plasmid-borne urease genes (ureABC, ureD and ureFG) to the strains P. aeruginosa AH298 and E. coli AF504gfp, both of which already carried unstable GFP derivatives. The ureolytic activities of the two new strains were compared to the common, non-GFP expressing, model organism Sporosarcina pasteurii in planktonic culture under standard laboratory growth conditions. It was found that the engineered strains exhibited a lower ureolysis rate per cell but were able to grow faster and to a higher population density under the conditions of this study. Both engineered strains were successfully grown as biofilms in capillary flow cell reactors and ureolysis-induced calcium carbonate mineral precipitation was observed microscopically. The undisturbed spatiotemporal distribution of biomass and calcium carbonate minerals were successfully resolved in 3D using confocal laser scanning microscopy. Observations of this nature were not possible previously because no obligate urease producer that expresses GFP had been available. Future observations using these organisms will allow researchers to further improve engineered application of MICP as well as study natural mineralization processes in model systems.

Right-hemispheric Dominance Of Dentate Granule Cell Activity After Spatial Exploration In Split-brain Mice But Not In Acallosal Mutant Mice

Event Abstract Back to Event Right-hemispheric Dominance Of Dentate Granule Cell Activity After Spatial Exploration In Split-brain Mice But Not In Acallosal Mutant Mice Hassan Ahmed1*, RYOSUKE KAWAKAMI2, Yugo FUKAZAWA3 and Ryuichi Shigemoto4 1 Faculty of veterinary Medicine, South Valley University, Physiology Department, Egypt 2 Research institute for electronic science, Hokkaido University, Laboratory of molecular and cellular biophysics, Japan 3 Nagoya University, Department of anatomy and molecular cell biology, Japan 4 National Institute for Physiological Sciences, Division of Cerebral Structure, Japan In the last decade, left/right brain asymmetry studies have been focused on the molecular level of the hippocampus. First, left/right asymmetry has been reported in GluN2B-NMDA subunit distribution and synaptic plasticity in hippocampal pyramidal cell synapses depending on the side of input (Kawakami et al, 2003). Furthermore, Shinohara et al (2008) found that GluA1-AMPA receptor subunit density, PSD size and spine head volume are larger in CA1 radiatum synapses contacting with presynaptic fibers from right than left CA3. Second, right isomerism in terms of the GluN2B asymmetry was found in inversus viscerum (iv) mice (Kawakami et al, 2008). Third, right-side dominance in spatial learning was reported in split-brain mouse (Shinohara et al, 2012). In the present study, we investigated asymmetry of hippocampal neural activity in wild-type, iv, Arc-Venus transgenic and acallosal mutant mice. Arc-Venus mice have the ability to produce destabilized form of Venus (derivative of green fluorescent protein) controlled by Arc-promoter which is activated by neural stimulation. These mice received transection of ventral hippocampal commissure and corpus callosum with unilateral eye deprivation to force each mice use predominantly either left or right hippocampus during spatial learning. Both left and right eye deprived wild-type mice showed right-side dominance of c-Fos expression in dentate gyrus with a similar tendency in CA1. Venus expression and Venus-immunopositive cells in left eye deprived Arc-Venus mice also showed right-side dominance confirming the dominance of neural activity in right hippocampus. Interestingly, c-Fos positive cells in left eye deprived iv mice also showed right-side dominance regardless of normal or inverted asymmetry in internal organs. On the other hand, left and right eye deprived acallosal mutant mice showed lack of left/right asymmetry in neural activity. These results suggest right-hemispheric dominance in spatial learning in mice and a critical role of corpus callosum for the development of this asymmetry. Keywords: split-brain, asymmetry, Dentate Gyrus, Right-dominance, Acallosal mice Conference: 4th Conference of the Mediterrarnean Neuroscience Society, Istanbul, Turkey, 30 Sep - 3 Oct, 2012. Presentation Type: Poster Presentation Topic: Abstracts Citation: Ahmed H, KAWAKAMI R, FUKAZAWA Y and Shigemoto R (2013). Right-hemispheric Dominance Of Dentate Granule Cell Activity After Spatial Exploration In Split-brain Mice But Not In Acallosal Mutant Mice. Conference Abstract: 4th Conference of the Mediterrarnean Neuroscience Society. doi: 10.3389/conf.fnhum.2013.210.00003 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 06 Feb 2013; Published Online: 11 Apr 2013. * Correspondence: Dr. Hassan Ahmed, Faculty of veterinary Medicine, South Valley University, Physiology Department, Qena, 83523, Egypt, hassan-Younes@vet.svu.edu.eg Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Hassan Ahmed RYOSUKE KAWAKAMI Yugo FUKAZAWA Ryuichi Shigemoto Google Hassan Ahmed RYOSUKE KAWAKAMI Yugo FUKAZAWA Ryuichi Shigemoto Google Scholar Hassan Ahmed RYOSUKE KAWAKAMI Yugo FUKAZAWA Ryuichi Shigemoto PubMed Hassan Ahmed RYOSUKE KAWAKAMI Yugo FUKAZAWA Ryuichi Shigemoto Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

A novel fluorescent reporter system for monitoring and identifying RNase III activity and its target RNAs

Bacteriophage vectors for achieving single-copy gene expression linked to a colorigenic reporter assay have been used successfully for genetic screening applications. However, the limited number of cloning sites in these vectors, combined with the requirement for lac- strains and the time- and/or media-dependence of the chemical-based colorimetric reaction, have limited the range of applications for these vectors. An alternative approach using a fluorescent reporter gene such as green fluorescent protein (GFP) or GFP derivatives could overcome some of these technical issues and facilitate real-time monitoring of promoter and/or protein activity. Here, we report the development of a novel translational bacteriophage fusion vector encoding enhanced GFP (eGFP) that can be incorporated into the chromosome as a single-copy gene. We identified a Bacillus promoter (BP) that is stably expressed in Escherichia coli and drives ~6-fold more expression of eGFP than the T7 promoter in the absence of inducer. Incorporating this BP and RNase III target signals into a single system enabled clear detection of the absence or downregulation of RNase III activity in vivo, thereby establishing a system for screening and identifying novel RNase III targets in a matter of days. An RNase III target signal identified in this manner was confirmed by post-transcriptional analysis. We anticipate that this novel translational fusion vector will be used extensively to study activity of both interesting RNases and related complex or to identify or validate targets of RNases that are otherwise difficult to study due to their sensitivity to environmental stresses and/or autoregulatory processes.

Association between segments of zonula occludens proteins: live‐cell FRET and mass spectrometric analysis

The tight junction protein ZO-1 (zonula occludens protein 1) has recruiting/scaffolding functions in the junctional complex of epithelial and endothelial cells. Homodimerization was proposed to be crucial for ZO-1 function. Here, we investigated the ability of ZO-1 domains to mediate self-interaction in living cells. We expressed ZO-1 truncation mutants as fusions with derivatives of green fluorescent protein in tight junction-free HEK-293 cells and determined self-association by means of fluorescence resonance energy transfer measurements using live-cell imaging. We show that both an SH3-hinge-GuK fusion protein and the PDZ2 domain self-associate in our test system. The recombinant PDZ2 domain also binds to ZO-1 and ZO-2 in tight junction-forming HT29/B6 cell lysates, as demonstrated by coprecipitation. Both interaction types are of relevance for the function of ZO-1 in the regulation of the junctional complex in polar cells.