Fluorescence Resonance Energy Transfer Pair Research Articles

Closely Spatio-Association of TRPC4 with GαI in the TRPC4 Activation Process

Canonical transient receptor potential (TPRC) channels are Ca2+-permeable nonselective cation channels that are widely expressed in numerous cell types. Seven different members of TRPC channels are isolated and canonical type of TRP channel family transduces signals of GPCR with various external stimuli. TRPC4 channels are known to be regulated by Gαi proteins. However, the molecular mechanism how Gαi proteins activate TRPC4 still remains to be questionable. To investigate the mechanism, we used whole patch clamp and FRET (FluorescenceResonance Energy transfer). We tagged mTRPC4 and G protein with CFP and YFP, respectively, and transiently transfected HEK293 cells with FRET pair. FRET efficiency between TRPC4 and Gα was 8.08 2.24% (n = 11) and was greater than those between TRPC4 and Gβγ (4.00 1.67 (n = 11)). At the HEK293 cell transfected with M2 muscarinic receptor, application of carbachol (CCh) increased FRET efficieny from 9.66 4.64 % (n = 7) to 26.27 10.09 % (n = 7). Intracellular 0.2mM GTPγS also increased FRET efficiency and TRPC4 current. In conclusion, we suggest that Gαi closely locates near TRPC4 and regulates TRPC4 channel activity.∗ This research was supported by the National Research Foundation of Korea (NRF) funded by the Korea government (MEST) (2008-2005948 and 2010-0019472).

Metal‐Induced Specific and Nonspecific Oligonucleotide Folding Studied by FRET and Related Biophysical and Bioanalytical Implications

Metal induced nucleic acid folding has been extensively studied with ribozymes, DNAzymes, tRNA and riboswitches. These RNA/DNA molecules usually have a high content of double-stranded regions to support a rigid scaffold. On the other hand, such rigid structural features are not available for many in vitro selected or rationally designed DNA aptamers; they adopt flexible random coil structures in the absence of target molecules. Upon target binding, these aptamers adaptively fold into a compact structure with a reduced end-to-end distance, making fluorescence resonance energy transfer (FRET) a popular signaling mechanism. However, nonspecific folding induced by mono- or divalent metal ions can also reduce the end-to-end distance and thus lead to false positive results. In this study we used a FRET pair labeled Hg(II) binding DNA and monitored metal-induced folding in the presence of various cations. While nonspecific electrostatically mediated folding can be very significant, at each tested salt condition, Hg(II) induced folding was still observed with a similar sensitivity. We also studied the biophysical meaning of the acceptor/donor fluorescence ratio that allowed us to explain the experimental observations. Potential solutions for this ionic strength problem have been discussed. For example, probes designed to signal the formation of double-stranded DNA showed a lower dependency on ionic strength.

Quantification of protein interaction in living cells by two‐photon spectral imaging with fluorescent protein fluorescence resonance energy transfer pair devoid of acceptor bleed‐through

Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful method to visualize and quantify protein-protein interaction in living cells. Unfortunately, the emission bleed-through of FPs limits the usage of this complex technique. To circumvent undesirable excitation of the acceptor fluorophore, using two-photon excitation, we searched for FRET pairs that show selective excitation of the donor but not of the acceptor fluorescent molecule. We found this property in the fluorescent cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) and YFP/mCherry FRET pairs and performed two-photon excited FRET spectral imaging to quantify protein interactions on the later pair that shows better spectral discrimination. Applying non-negative matrix factorization to unmix two-photon excited spectral imaging data, we were able to eliminate the donor bleed-through as well as the autofluorescence. As a result, we achieved FRET quantification by means of a single spectral acquisition, making the FRET approach not only easy and straightforward but also less prone to calculation artifacts. As an application of our approach, the intermolecular interaction of amyloid precursor protein and the adaptor protein Fe65 associated with Alzheimer's disease was quantified. We believe that the FRET approach using two-photon and fluorescent YFP/mCherry pair is a promising method to monitor protein interaction in living cells.

Ratiometric fluorescence imaging of dual bio-molecular events in single living cells using a new FRET pair mVenus/mKOκ-based biosensor and a single fluorescent protein biosensor

Genetically coded fluorescent protein (FP)-based biosensors are powerful tools for the non-invasive tracking of molecular events in living cells. Although a variety of FP biosensors are available, the simultaneous imaging of multiple biosensors (multi-parameter imaging) in single living cells remains a challenge and is far from routinely used to elucidate the intricate networks of molecular events. In this study, we established a novel combination of FP biosensors for dual-parameter ratiometric imaging, consisting of a new fluorescence resonance energy transfer (FRET) pair mVenus (yellow FP)/mKOκ (orange FP)-based (abbreviated as YO) biosensor and a single FP-based biosensor Grx1-roGFP2. Under our imaging condition, 1.4±0.05% of Grx1-roGFP2 signal contributes to the mVenus channel and 5.2±0.12% of the mVenus signal contributes to the Grx1-roGFP2 channel. We demonstrate that such low degree of cross-talk causes negligible distortion of the ratiometric signal of the YO-based FRET biosensor and Grx1-roGFP2. By using this dual-parameter ratiometric imaging approach, we achieved simultaneous imaging of Src/Ca2+ signaling and glutathione (GSH) redox potential in a single cell, which was previously unattainable. Furthermore, we provided direct evidence that epidermal growth factor (EGF)-induced Src signaling was negatively regulated by H2O2 via its effect on GSH-based redox system, demonstrating the power of this dual-parameter imaging approach for elucidating new connections between different molecular events that occur in a single cell. More importantly, the dual-parameter imaging approach described in this study is highly extendable.

Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA

We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intra-populational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection.

Enhanced dynamic range in a genetically encoded Ca 2+ sensor

Genetically encoded fluorescence resonance energy transfer (FRET) indicators are powerful tools for real-time detection of second messenger molecules and activation of signal proteins. However, these fluorescent protein-based sensors typically display marginal FRET efficiency. To improve their FRET efficiency for optical imaging and screening, we developed a number of fluorescent protein mutants based on cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). To improve FRET ratios, which were initially within a narrow dynamic range, we used DNA shuffling to develop a new FRET pair called 3xCFP/Venus. The optimized 3xCFP/Venus pair exhibited higher FRET ratios than CyPet/YPet, which has one of the greatest dynamic ranges of protein-based FRET pairs. We converted this FRET pair to a Ca 2+ FRET indicators using circular permutation Venus (cpVenus) linked with 3xCFP to form 3xCFP/cpVenus, which displayed an ∼11-fold change in dynamic range in response to Ca 2+ binding. The enhanced dynamic range for Ca 2+ concentration detection using 3xCFP/cpVenus was confirmed in PC12 cells using previously established indicators (TN-XXL, ECFP/cpCitrine). To our knowledge, this FRET pair displays the largest dynamic range so far among genetically-encoded sensors, and can be used for sensitive FRET detection.

Abstract 1381: A high-throughput assay for tumor endothelial marker-8 (TEM8/ANTXR1) inhibitors

Abstract TEM8 was first described as a gene upregulated in colorectal tumor endothelium compared to quiescent endothelium. It is also a receptor for anthrax toxin. It is expressed in the endothelium of bladder, esophageal and lung tumors. Mouse knockout studies have shown that TEM8 expression, within the host, promotes tumor growth. Endothelial TEM8 expression can control cell shape, migration, and adhesion to extracellular matrix (ECM) proteins in vitro, supporting a role for TEM8 in tumor angiogenesis. TEM8 interacts with collagen I, collagen VI and gelatin. Protective antigen is the component of anthrax toxin that binds to its cell surface receptors. We have demonstrated that a mutated form of protective antigen (PASSSR) inhibits endothelial cell migration, corneal angiogenesis, and tumor growth. We hypothesize that PASSSR binds to TEM8 and inhibits its interaction with ECM proteins, thereby inhibiting angiogenesis and tumor growth. As a large immunogenic protein, PASSSR would have significant challenges as a potential therapeutic molecule. Thus our objective is to establish a high-throughput assay to identify small molecule inhibitors of TEM8, which can serve as lead molecules in the identification of novel antiangiogenic agents. Fluorescence resonance energy transfer (FRET) is a sensitive method well suited to high-throughput screening. We successfully labeled recombinant PAE733C with AlexaFluor546 maleimide. However, identifying a TEM8 labeling strategy proved more challenging. Maleimide or FlAsH labeling of native protein TEM8 extracellular domain, truncations, point mutations or tetracysteine motif extensions resulted in insoluble or inactive protein, as did NHS labeling. We thus investigated GFP labeling, and found that the resulting protein was stable and interacted with PA in the expected manner. When formatted for high-throughput screening, the PA*546 and TEM8-GFP FRET pair gave a Z’ score (a measure of assay performance) of >0.8. We discovered that alterations to the TEM8 molecule, that disrupt either of two pairs of disulfide bonds on cysteine residues, prevent its ability to interact with PASSSR. The FRET assay was used to identify inhibitors, from a library of over 5,000 known bioactive compounds. Strong hits (>50% inhibition of the PASSSR/TEM8 interaction) included ebselen, and phenylmercuricacetate. Ebselen, a selenium-containing glutathione peroxidase mimic, is known to disrupt disulfide bonds and is likely to have inhibited FRET via binding important cysteine containing residues of TEM8. We will assess anti-angiogenic effects of identified known bioactive molecules. We expect that screening larger small molecule libraries of over 250,000 compounds available at ICCB, Longwood, HMS will result in the identification of a number of candidate TEM8 inhibitors. These molecules are likely to have anti-angiogenic and anti-tumorigenic activities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1381. doi:10.1158/1538-7445.AM2011-1381

Stimuli-Responsive Fluorescent Poly(N-isopropylacrylamide) Microgels Labeled with Phenylboronic Acid Moieties as Multifunctional Ratiometric Probes for Glucose and Temperatures

We report on the fabrication of multifunctional ratiometric probes for glucose and temperatures based on thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) microgels covalently incorporated with glucose-recognizing moieties, N-acryloyl-3-aminophenylboronic acid (APBA), fluorescence resonance energy transfer (FRET) donor dyes, 4-(2-acryloyloxyethylamino)-7-nitro-2,1,3-benzoxadiazole (NBDAE), and rhodamine B-based FRET acceptors (RhBEA). P(NIPAM-APBA-NBDAE-RhBEA) microgels containing FRET pairs and APBA were synthesized via free radical emulsion copolymerization. The spatial proximity of FRET donors and acceptors within microgels can be tuned via thermo-induced microgel collapse or glucose-induced microgel swelling at appropriate pH and temperatures, leading to the facile modulation of FRET efficiencies. APBA moieties within P(NIPAM-APBA-NBDAE-RhBEA) microgels can bind with glucose at appropriate pH to form cyclic boronate moieties, which can decrease the pKa of APBA residues and increase the volume phas...

Characterization of microfluidic mixing and reaction in microchannels via analysis of cross-sectional patterns

For the diagnosis of biochemical reactions, the investigation of microflow behavior, and the confirmation of simulation results in microfluidics, experimentally quantitative measurements are indispensable. To characterize the mixing and reaction of fluids in microchannel devices, we propose a mixing quality index (M(qi)) to quantify the cross-sectional patterns (also called mixing patterns) of fluids, captured with a confocal-fluorescence microscope (CFM). The operating parameters of the CFM for quantification were carefully tested. We analyzed mixing patterns, flow advection, and mass exchange of fluids in the devices with overlapping channels of two kinds. The mixing length of the two devices derived from the analysis of M(qi) is demonstrated to be more precise than that estimated with a commonly applied method of blending dye liquors. By means of fluorescence resonance-energy transfer (FRET), we monitored the hybridization of two complementary oligonucleotides (a FRET pair) in the devices. The captured patterns reveal that hybridization is a progressive process along the downstream channel. The FRET reaction and the hybridization period were characterized through quantification of the reaction patterns. This analytical approach is a promising diagnostic tool that is applicable to the real-time analysis of biochemical and chemical reactions such as polymerase chain reaction (PCR), catalytic, or synthetic processes in microfluidic devices.

Synaptotagmin 1 drives Trans SNARE Zippering with Ca2+

In neurons, Synaptotagmin 1 (Syt1) is a major Ca2++-sensor that evokes vesicle fusion, a necessary event for the neurotransmitter release, in response to the Ca2++ signal. Although Syt1 is known to bind to the core fusion machinery SNAREpin the mechanism by which Syt1 regulates vesicle fusion is not known. Here we used site-specific fluorescence resonance energy transfer (FRET) to investigate the effects of Syt1 and Ca2+ on formation of trans SNAREpin between the complimentary SNAREs reconstituted to proteoliposomes. When the FRET pairs were attached at the N-termini of SNAREs C2AB, a soluble version of Syt1, had virtually no effect on the rate of the FRET change, indicating Syt1 does not interfere with the initial stage of SNARE complex formation that happens at the membrane-distal region. However, when the FRET pairs were placed at the membrane proximal region of the SNAREs C2AB accelerated the FRET change significantly in the presence of Ca2++, indicating C2AB/Ca2+ promotes the transition from the partially assembled SNARE complex to the fusion-competent SNAREpin. Thus, the results suggest that Syt1 and Ca2++ may drive SNARE zippering to facilitate membrane fusion.

Bidirectional Fluorescence Resonance Energy Transfer (FRET) in Mutated and Chemically Modified Yellow Fluorescent Protein (YFP)

Fluorescence resonance energy transfer (FRET) using fluorescent protein variants are used for studying the associations and biomolecular motions of macromolecules inside the cell. Intramolecular FRET utilizing fluorescent chemical labels has been applied in nucleic acid chemistry for detection of specific sequence. However, the biotechnological applications of intramolecular FRET in fluorescent proteins have not been exploited. This study demonstrates the intramolecular FRET between fluorescent protein and conjugated chemical label whereby FRET occurs from inside to outside and vice versa for fluorescent protein. The fluorescent protein is modified for the attachment of chemical fluorophores and the novel FRET pairs created by conjugation are MDCC (435/475)-Citrine (516/529) and Citrine-Alexa fluor (568/603). These protein-label pairs exhibited strong intramolecular FRET and the energy transfer efficiency was determined based on the time evolution of the ratio of emission intensities of labeled and unlabeled proteins. The efficiency was found to be 0.79 and 0.89 for MDCC-Citrine and 0.24 and 0.65 for Citrine-Alexa Fluor pairs when the label is conjugated at different sites in the protein. Förster distance and the average distance between the fluorophores were also determined. The bidirectional approach described here can provide new insights into designing FRET-based sensors.

The photoluminescent graphene oxide serves as an acceptor rather than a donor in the fluorescence resonance energy transfer pair of Cy3.5–graphene oxide

We have systematically studied the fluorescence resonance energy transfer (FRET) efficiency between the photoluminescent graphene oxide (GO) and Cy3.5 dye by controlling the donor-acceptor distance with a double stranded DNA and demonstrated that the GO serves as an acceptor rather than a donor in this FRET system.

Thermo- and light-regulated fluorescence resonance energy transfer processes within dually responsive microgels

We report on the fabrication of thermo- and light-responsive P(NIPAM-DMNA-NBDAE-RhBEA) microgels consisting of N-isopropylacrylamide (NIPAM), photocleavable moieties, 5-(2′-(dimethylamino)ethoxy)-2-nitrobenzyl acrylate (DMNA), fluorescence resonance energy transfer (FRET) donors, 4-(2-acryloyloxyethylamino)-7-nitro-2,1,3-benzoxadiazole (NBDAE), and rhodamine B-based FRET acceptors (RhBEA) via the free radical emulsion polymerization technique. Thermo-induced collapse and swelling of responsive microgels above and below the lower critical solution temperatures (LCSTs), respectively, can finely tune the spatial proximity between NBDAE and RhBEA dyes, leading to the facile modulation of FRET efficiencies. Upon UV irradiation (pH 8.5), DMNA moieties within microgels undergo photolysis reactions and the formation of sodium carboxylate residues on microgel scaffolds results in elevated LCST. Thus, UV irradiation of microgel dispersions in the intermediate temperature range (between the LCST of original microgels and that of UV-irradiated microgels) can directly lead to the re-swelling of initially collapsed microgels. The incorporation of FRET pairs (NBDAE and RhBEA dyes) allow for the in situ monitoring of thermo- and UV irradiation-induced volume phase transitions (VPTs) of the reported dually responsive microgels. This work represents the first report of thermoresponsive microgels with VPTs tunable by photolabile 2-nitrobenzyl ester moieties.

Gauging the Flexibility of Fluorescent Markers for the Interpretation of Fluorescence Resonance Energy Transfer

Intramolecular distances in proteins and other biomolecules can be studied in living cells by means of fluorescence resonance energy transfer (FRET) in steady-state or pulsed-excitation experiments. The major uncertainty originates from the unknown orientation between the optical dipole moments of the fluorescent markers, especially when the molecule undergoes thermal fluctuations in physiological conditions. We introduce a statistical method based on the von Mises-Fisher distribution for the interpretation of fluorescence decay dynamics in donor-acceptor FRET pairs that allows us to retrieve both the orientation and the extent of directional fluctuations of the involved dipole moments. We verify the method by applying it to donor-acceptor pairs controllably attached to DNA helices and find that common assumptions such as complete rotational freedom or fully hindered rotation of the dipoles fail a physical interpretation of the fluorescence decay dynamics. This methodology is applicable in single-molecule and ensemble measurements of FRET to derive more accurate distance estimates from optical experiments, without the need for more complex and expensive NMR studies.

Ligase detection reaction generation of reverse molecular beacons for near real-time analysis of bacterial pathogens using single-pair fluorescence resonance energy transfer and a cyclic olefin copolymer microfluidic chip.

Detection of pathogenic bacteria and viruses require strategies that can signal the presence of these targets in near real-time due to the potential threats created by rapid dissemination into water and/or food supplies. In this paper, we report an innovative strategy that can rapidly detect bacterial pathogens using reporter sequences found in their genome without requiring polymerase chain reaction (PCR). A pair of strain-specific primers was designed based on the 16S rRNA gene and were end-labeled with a donor (Cy5) or acceptor (Cy5.5) dye. In the presence of the target bacterium, the primers were joined using a ligase detection reaction (LDR) only when the primers were completely complementary to the target sequence to form a reverse molecular beacon (rMB), thus bringing Cy5 (donor) and Cy5.5 (acceptor) into close proximity to allow fluorescence resonance energy transfer (FRET) to occur. These rMBs were subsequently analyzed using single-molecule detection of the FRET pairs (single-pair FRET; spFRET). The LDR was performed using a continuous flow thermal cycling process configured in a cyclic olefin copolymer (COC) microfluidic device using either 2 or 20 thermal cycles. Single-molecule photon bursts from the resulting rMBs were detected on-chip and registered using a simple laser-induced fluorescence (LIF) instrument. The spFRET signatures from the target pathogens were reported in as little as 2.6 min using spFRET.

Development of an Image-Based System for Measurement of Membrane Potential, Intracellular Ca2+ and Contraction in Arteriolar Smooth Muscle Cells

Changes in smooth muscle cell (SMC) membrane potential (Em) are critical to vasomotor responses. As a fluorescent indicator approach would lessen limitations of glass electrodes in contracting preparations, we aimed to develop a Forster (or fluorescence) resonance energy transfer (FRET)-based measurement for Em. The FRET pair used in this study (donor CC2-DMPE [excitation 405 nm] and acceptor DisBAC(4) (3)) provide rapid measurements at a sensitivity not achievable with many ratiometric indicators. The method also combined measurement of changes in Ca(2+) (i) using fluo-4 and excitation at 490 nm. After establishing loading conditions, a linear relationship was demonstrated between Em and fluorescence signal in FRET dye-loaded HEK cells held under voltage clamp. Over the voltage range from -70 to +30 mV, slope (of FRET signal vs. voltage, m) = 0.49 ± 0.07, r(2) = 0.96 ± 0.025. Similar data were obtained in cerebral artery SMCs, slope (m) = 0.30 ± 0.02, r(2) = 0.98 ± 0.02. Change in FRET emission ratio over the holding potential of -70 to +30 mV was 41.7 ± 4.9% for HEK cells and 30.0 ± 2.3% for arterial SMCs. The FRET signal was also shown to be modulated by KCl-induced depolarization in a concentration-dependent manner. Further, in isolated arterial SMCs, KCl-induced depolarization (60 mM) measurements occurred with increased fluo-4 fluorescence emission (62 ± 9%) and contraction (-27 ± 4.2%). The data support the FRET-based approach for measuring changes in Em in arterial SMCs. Further, image-based measurements of Em can be combined with analysis of temporal changes in Ca(2+) (i) and contraction.

Lifetime imaging of FRET between red fluorescent proteins

Numerous processes in cells can be traced by using fluorescence resonance energy transfer (FRET) between two fluorescent proteins. The novel FRET pair including the red fluorescent protein TagRFP and kindling fluorescent protein KFP for sensing caspase-3 activity is developed. The lifetime mode of FRET measurements with a nonfluorescent protein KFP as an acceptor is used to minimize crosstalk due to its direct excitation. The red fluorescence is characterized by a better penetrability through the tissues and minimizes the cell autofluorescence signal. The effective transfection and expression of the FRET sensor in eukaryotic cells is shown by FLIM. The induction of apoptosis by camptothecine increases the fluorescence lifetime, which means effective cleavage of the FRET sensor by caspase-3. The instruments for detecting whole-body fluorescent lifetime imaging are described. Experiments on animals show distinct fluorescence lifetimes for the red fluorescent proteins possessing similar spectral properties.

FRET Studies on a Series of BODIPY‐Dye‐Labeled Switchable Resorcin[4]arene Cavitands

A series of borondipyrromethane (BODIPY)-dye-labeled resorcin[4]arene cavitands 1-4 with different lengths of oligo(phenylene-ethynylene) spacers between the dyes and the macrocyclic rim has been synthesized. Their switching behavior from the "vase" to "kite" conformations in bulk solution was examined by both variable-temperature (VT) NMR and fluorescence spectroscopy. Both VT-NMR and VT fluorescence resonance energy transfer (FRET) experiments showed that cavitands 1-4 undergo vase-to-kite switching at low temperatures. Acid-triggered switching to the kite conformation was observed by fluorescence spectroscopy. Quantitative evaluation of the FRET data led to the determination of the Förster radius R(0)=37 Å for the BODIPY-dye FRET pair and an average cavitand opening angle α=16° in the vase conformation.

FRET-Derived Ratiometric Fluorescent K+ Sensors Fabricated from Thermoresponsive Poly(N-isopropylacrylamide) Microgels Labeled with Crown Ether Moieties

We report on the fabrication of ratiometric fluorescent K(+) sensors based on thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) microgels covalently incorporated with K(+)-recognizing 4-acrylamidobenzo-18-crown-6 residues (B18C6Am), fluorescence resonance energy transfer (FRET) donor dyes, 4-(2-acryloyloxyethylamino)-7-nitro-2,1,3-benzoxadiazole (NBDAE), and rhodamine-B-based FRET acceptors (RhBEA) by utilizing K(+)-induced changes in microgel volume phase transition (VPT) temperatures. P(NIPAM-B18C6Am-NBDAE-RhBEA) microgels were synthesized via the free radical emulsion copolymerization technique. The spatial proximity between FRET pairs (NBDAE and RhBEA dyes) within microgels can be tuned via thermoinduced collapse and swelling of thermoresponsive microgels above and below VPT temperatures, leading to the facile modulation of FRET efficiencies. B18C6Am moieties within P(NIPAM-B18C6Am-NBDAE-RhBEA) microgels can preferentially capture K(+) via the formation of 1:1 molecular recognition complexes, resulting in the enhancement of microgel hydrophilicity and elevated VPT temperatures. Thus, the gradual addition of K(+) into microgel dispersions at intermediate temperatures, i.e., between VPT temperatures of P(NIPAM-B18C6Am-NBDAE-RhBEA) microgels in the absence and presence of K(+) ions, respectively, can directly lead to the reswelling of initially collapsed microgels. This process can be monitored by changes in fluorescence intensity ratios, i.e., FRET efficiencies. The presence of FRET pairs within P(NIPAM-B18C6Am-NBDAE-RhBEA) microgels allows for the facile in situ monitoring of thermoinduced and K(+)-induced VPTs of dually responsive microgels. The response time for fluorescent K(+)-sensing was further investigated via the stopped-flow technique, which reveals that the process completes within ∼4 s. This work represents the first report of thermoresponsive microgel-based ratiometric fluorescent sensors for both K(+) ions and temperatures.

APPL proteins FRET at the BAR: direct observation of APPL1 and APPL2 BAR domain-mediated interactions on cell membranes using FRET microscopy.

BackgroundHuman APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR) domain, a central pleckstrin homology (PH) domain, and a C-terminal phosphotyrosine binding (PTB) domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported.MethodologyHerein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET) experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP) FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species.ConclusionsAll three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2) and heterotypic (i.e., APPL1-APPL2) manner on curved cell membranes. Furthermore, the results of our experiments did not show photoconversion of YFP into a CFP-like species following photobleaching, supporting the use of CFP donor/YFP acceptor FRET pairs in acceptor photobleaching studies.