Direct detection of caspase-3 activation in single live cells by cross-correlation analysis

Dual color fluorescence cross-correlation spectroscopy (FCCS) provides information about the coincidence of spectrally well-defined two fluorescent molecules in a small observation area at the single-molecule level. To evaluate the activity of caspase-3 in vivo directly, FCCS was applied to single live cells. We constructed chimeric proteins that consisted of tandemly fused enhanced green FP (EGFP) and monomeric red FP (mRFP). In control experiments, the protease reaction was monitored in solution, where a decrease in cross-correlation amplitude was observed due to specific cleavage of the amino acid sequence between EGFP and mRFP. Moreover, a decrease in cross-correlation amplitude could be detected in a live cell, where caspase-3 activation was induced by apoptosis. This is the first report of FP-based in vivo cross-correlation analysis. FP-based FCCS may become the most versatile method for analysis of protein-protein interactions in live cells.     

Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Förster radius

Enhanced cyan and yellow fluorescent proteins are widely used for dual color imaging and protein-protein interaction studies based on fluorescence resonance energy transfer. Use of these fluorescent proteins can be limited by their thermosensitivity, dim fluorescence, and tendency for aggregation. Here we report the results of a site-directed mutagenesis approach to improve these fluorescent proteins. We created monomeric optimized variants of ECFP and EYFP, which fold faster and more efficiently at 37 degrees C and have superior solubility and brightness. Bacteria expressing SCFP3A were 9-fold brighter than those expressing ECFP and 1.2-fold brighter than bacteria expressing Cerulean. SCFP3A has an increased quantum yield (0.56) and fluorescence lifetime. Bacteria expressing SYFP2 were 12 times brighter than those expressing EYFP(Q69K) and almost 2-fold brighter than bacteria expressing Venus. In HeLa cells, the improvements were less pronounced; nonetheless, cells expressing SCFP3A and SYFP2 were both 1.5-fold brighter than cells expressing ECFP and EYFP(Q69K), respectively. The enhancements of SCFP3A and SYFP2 are most probably due to an increased intrinsic brightness (1.7-fold and 1.3-fold for purified recombinant proteins, compared to ECFP & EYFP(Q69K), respectively) and due to enhanced protein folding and maturation. The latter enhancements most significantly contribute to the increased fluorescent yield in bacteria whereas they appear less significant for mammalian cell systems. SCFP3A and SYFP2 make a superior donor-acceptor pair for fluorescence resonance energy transfer, because of the high quantum yield and increased lifetime of SCFP3A and the high extinction coefficient of SYFP2. Furthermore, SCFP1, a CFP variant with a short fluorescence lifetime but identical spectra compared to ECFP and SCFP3A, was characterized. Using the large lifetime difference between SCFP1 and SCFP3A enabled us to perform for the first time dual-lifetime imaging of spectrally identical fluorescent species in living cells.     

Fluorescence imaging using a fluorescent protein with a large Stokes shift

Keima is a far-red fluorescent protein endowed with a large Stokes shift. It absorbs light maximally at around 440 nm and emits maximally at around 620 nm. While the original Keima is obligately tetrameric (tKeima), the dimeric and monomeric versions (mKeima and dKeima, respectively) have been generated. More recently, a tandem dimer of Keima (tdKeima) has been developed as the brightest version. Here we describe examples, which show the usefulness of Keima for dual-color fluorescence imaging technologies, such as fluorescence cross-correlation spectroscopy (FCCS) and two-photon laser scanning microscopy (TPLSM). Keima can be used in conjunction with existing fluorescent proteins in which the Stokes shift is much smaller, with the idea that while two fluorescent proteins are excited by a single laser each will fluoresce a different color.     

Circularly permuted monomeric red fluorescent proteins with new termini in the ��-sheet

Circularly permuted fluorescent proteins (FPs) have a growing number of uses in live cell fluorescence biosensing applications. Most notably, they enable the construction of single fluorescent protein‐based biosensors for Ca²⁺ and other analytes of interest. Circularly permuted FPs are also of great utility in the optimization of fluorescence resonance energy transfer (FRET)‐based biosensors by providing a means for varying the critical dipole–dipole orientation. We have previously reported on our efforts to create circularly permuted variants of a monomeric red FP (RFP) known as mCherry. In our previous work, we had identified six distinct locations within mCherry that tolerated the insertion of a short peptide sequence. Creation of circularly permuted variants with new termini at the locations corresponding to the sites of insertion led to the discovery of three permuted variants that retained no more than 18% of the brightness of mCherry. We now report the extensive directed evolution of the variant with new termini at position 193 of the protein sequence for improved fluorescent brightness. The resulting variant, known as cp193g7, has 61% of the intrinsic brightness of mCherry and was found to be highly tolerant of circular permutation at other locations within the sequence. We have exploited this property to engineer an expanded series of circularly permuted variants with new termini located along the length of the 10th β‐strand of mCherry. These new variants may ultimately prove useful for the creation of single FP‐based Ca²⁺ biosensors.     

Induced heterodimerization and purification of two target proteins by a synthetic coiled-coil tag

A synthetic de novo designed heterodimeric coiled-coil was used to copurify two target fluorescent proteins, Venus and enhanced cyan fluorescent protein (ECFP). The coiled-coil consists of two 21-amino acid repetitive sequences, (EIAALEK)(3) and (KIAALKE)(3), named E3 and K3, respectively. These sequences were fused to the C-termini of ECFP or Venus followed by either a strep- or a his-tag, respectively, for affinity purification. Mixed lysates of Venus-K3 and ECFP-E3 were subjected to consecutive affinity purification and showed highly specific association between the coiled-coil pair by SDS-PAGE, gel filtration, isothermal titration calorimetry (ITC), and fluorescence resonance energy transfer (FRET). The tagged proteins eluted as heterodimers at the concentrations tested. FRET analysis further showed that the coiled-coil pair was stable in buffers commonly used for protein purification, including those containing high salt concentration and detergent. This study shows that the E3/K3 pair is very well suited for the copurification of two target proteins expressed in vivo because of its high specificity: it forms exclusively heterodimers in solution, it does not interact with any cellular proteins and it is stable under different buffer conditions.     

Afp::mCherry, a red fluorescent transgenic reporter of the mouse visceral endoderm

Live imaging of genetically encoded fluorescent protein reporters is increasingly being used to investigate details of the cellular behaviors that underlie the large-scale tissue rearrangements that shape the embryo. However, the majority of mouse fluorescent reporter strains are based on the green fluorescent protein (GFP). Mouse reporter strains expressing fluorescent colors other than GFP are therefore valuable for co-visualization studies with GFP, where relative positioning and relationship between two different tissues or compartments within cells are being investigated. Here, we report the generation and characterization of a transgenic Afp::mCherry mouse strain in which cis-regulatory elements from the Alpha-fetoprotein (Afp) locus were used to drive expression of the monomeric Cherry red fluorescent protein. The Afp::mCherry transgene is based on and recapitulates reporter expression of a previously described Afp::GFP strain. However, we note that perdurance of mCherry protein is not as prolonged as GFP, making the Afp::mCherry line a more faithful reporter of endogenous Afp expression. Afp::mCherry transgenic mice expressed mCherry specifically in the visceral endoderm and its derivatives, including the visceral yolk sac, gut endoderm, fetal liver, and pancreas of the embryo. The Afp::mCherry reporter was also noted to be expressed in other documented sites of Afp expression including hepatocytes as well as in pancreas, digestive tract, and brain of postnatal mice.     

Extremely low polymerizability of a highly-divergent Chlamydomonas actin (NAP)

Novel actin-like protein (NAP) is a highly divergent actin expressed in Chlamydomonas. With its low sequence similarity, it is uncertain whether NAP can polymerize into filaments. Here I assessed it by ectopically expressing enhanced green fluorescent protein-tagged NAP (EGFP-NAP) in cultured cells. EGFP-NAP was excluded from stress fibres but partially co-localized with endogenous actin in the cell periphery. In fluorescence recovery after photobleaching experiment, turnover rate of EGFP-NAP was similar to the estimated diffusion rate of monomeric actin. Therefore, EGFP-NAP likely accumulates by diffusion. These findings suggest that NAP has extremely poor ability to polymerize.     

R26R-GR: A Cre-Activable Dual Fluorescent Protein Reporter Mouse

Green fluorescent protein (GFP) and its derivatives are the most widely used molecular reporters for live cell imagining. The development of organelle-specific fusion fluorescent proteins improves the labeling resolution to a higher level. Here we generate a R26 dual fluorescent protein reporter mouse, activated by Cre-mediated DNA recombination, labeling target cells with a chromatin-specific enhanced green fluorescence protein (EGFP) and a plasma membrane-anchored monomeric cherry fluorescent protein (mCherry). This dual labeling allows the visualization of mitotic events, cell shapes and intracellular vesicle behaviors. We expect this reporter mouse to have a wide application in developmental biology studies, transplantation experiments as well as cancer/stem cell lineage tracing.     

Enhanced dynamic range in a genetically encoded Ca2+ 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.     

Characterization of proteinase activation dynamics by capillary electrophoresis conjugating with fluorescent protein-based probe

In this paper, a novel strategy was reported to characterize dynamics of proteinase activation based on capillary electrophoresis (CE), using caspase-2 as the model system. A fusion protein conjugating an amino acid sequence VDVAD with two fluorescent proteins enhanced cyan fluorescence protein (ECFP) and red fluorescence protein (DsRed), ECFP-VDVAD-DsRed, was specially designed and expressed in HeLa cells as the substrate of proteinase caspase-2. In this substrate, the sequence VDVAD could be specifically recognized and cleaved by caspase-2 as soon as its activation was initiated with treatment of a certain dose of cisplatin to HeLa cells, which led to a break of the substrate into two fragments. Analyses of the cell lysates using CE in a time course of the apoptosis illustrated the dynamics of caspase-2 activation. It showed that the employment of fusion fluorescent protein greatly facilitated both CE separation and identification of the analytes. This result from cell colony by CE was compared with that from single cell achieved by optical imaging.     

A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy

Dual-color fluorescence cross-correlation spectroscopy (FCCS) is a promising technique for quantifying protein-protein interactions. In this technique, two different fluorescent labels are excited and detected simultaneously within a common measurement volume. Difficulties in aligning two laser lines and emission crossover between the two fluorophores, however, make this technique complex. To overcome these limitations, we developed a fluorescent protein with a large Stokes shift. This protein, named Keima, absorbs and emits light maximally at 440 nm and 620 nm, respectively. Combining a monomeric version of Keima with cyan fluorescent protein allowed dual-color FCCS with a single 458-nm laser line and complete separation of the fluorescent protein emissions. This FCCS approach enabled sensitive detection of proteolysis by caspase-3 and the association of calmodulin with calmodulin-dependent enzymes. In addition, Keima and a spectral variant that emits maximally at 570 nm might facilitate simultaneous multicolor imaging with single-wavelength excitation.     

Sertoli cell behaviors in developing testis cords and postnatal seminiferous tubules of the mouse

Sertoli cells are the primary structural component of the fetal testis cords and postnatal seminiferous tubules. Live imaging technologies facilitate the visualization of cell morphologies and behaviors through developmental processes. A transgenic mouse line was generated using a fragment of the rat Gata4 gene to direct the expression of a dual-color fluorescent protein reporter in fetal and adult Sertoli cells. The reporter encoded a red fluorescent protein, monomeric Cherry (mCherry), fused to histone 2B and enhanced green fluorescent protein (EGFP) fused to a glycosylphosphatidylinositol sequence, with a self-cleaving 2A polypeptide separating the two fusion proteins. After translation, the red and green fluorescent proteins translocated to the nucleus and plasma membrane, respectively, of Sertoli cells. Transgene expression in testes was first detected by fluorescent microscopy around Embryonic Day 12.0. Sertoli cell division and migration were visualized during testis cord formation in organ culture. Initially, the Sertoli cells had mesenchyme-like morphologies and behaviors, but later, the cells migrated to the periphery of the testis cords to become epithelialized. In postnatal seminiferous tubules, Sertoli nuclei were evenly spaced when viewed from the external surface of tubules, and Sertoli cytoplasm and membranes were associated with germ cells basally in a rosette pattern. This mouse line was bred to previously described transgenic mouse lines expressing EGFP in Sertoli cytoplasm or a nuclear cyan fluorescent protein (Cerulean) and mCherry in plasma membranes of germ cells. This revealed the physical relationship between Sertoli and germ cells in developing testis cords and provided a novel perspective on Sertoli cell development.     

Enhanced dynamic range in a genetically encoded Ca 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 Ca²⁺ 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 Ca²⁺ binding. The enhanced dynamic range for Ca²⁺ 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.     

Ubiquitous expression of the monomeric red fluorescent protein mcherry in transgenic mice

The use of the green fluorescent protein (GFP) to label specific cell types and track gene expression in animal models, such as mice, has evolved to become an essential tool in biological research. Transgenic animals expressing genes of interest linked to GFP, either as a fusion protein or transcribed from an internal ribosomal entry site (IRES) are widely used. Enhanced GFP (eGFP) is the most common form of GFP used for such applications. However, a red fluorescent protein (RFP) would be highly desirable for use in dual‐labeling applications with GFP derived fluorescent proteins, and for deep in vivo imaging of tissues. Recently, a new generation of monomeric (m)RFPs, such as monomeric (m)Cherry, has been developed that are potentially useful experimentally. mCherry exhibits brighter fluorescence, matures more rapidly, has a higher tolerance for N‐terminal fusion proteins, and is more photostable compared with its predecessor mRFP1. mRFP1 itself was the first true monomer derived from its ancestor DsRed, an obligate tetramer in vivo. Here, we report the successful generation of a transgenic mouse line expressing mCherry as a fluorescent marker, driven by the ubiquitin‐C promoter. mCherry is expressed in almost all tissues analyzed including pre‐ and post‐implantation stage embryos, and white blood cells. No expression was detected in erythrocytes and thrombocytes. Importantly, we did not encounter any changes in normal development, general physiology, or reproduction. mCherry is spectrally and genetically distinct from eGFP and, therefore, serves as an excellent red fluorescent marker alone or in combination with eGFP for labelling transgenic animals. genesis 48:723–729, 2010. © 2010 Wiley‐Liss, Inc.     

Fluorophores for live cell imaging of AGT fusion proteins across the visible spectrum

O6-alkylguanine-DNA alkyltransferase (AGT) fusion proteins can be specifically and covalently labeled with fluorescent O6-benzylguanine (O6-BG) derivatives for multicolor live cell imaging approaches. Here, we characterize several new BG fluorophores suitable for in vivo AGT labeling that display fluorescence emission maxima covering the visible spectrum from 472 to 673 nm, thereby extending the spectral limits set by fluorescent proteins. We show that the photostability of the cell-permeable dyes BG Rhodamine Green (BG505) and CP tetramethylrhodamine (CP-TMR) is in the range of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP), and that BG diethylaminomethyl coumarin (BGDEAC), a derivative of coumarin, is even more stable than enhanced cyan fluorescent protein (ECFP). Due to the increasing number of new BG derivatives with interesting fluorescence properties, such as far-red emission, fluorescence labeling of AGT fusion proteins is becoming a versatile alternative to existing live cell imaging approaches.     

Fluorescence Fluctuation Spectroscopy of mCherry in Living Cells

The red fluorescent protein mCherry is of considerable interest for fluorescence fluctuation spectroscopy (FFS), because the wide separation in color between mCherry and green fluorescent protein provides excellent conditions for identifying protein interactions inside cells. This two-photon study reveals that mCherry exists in more than a single brightness state. Unbiased analysis of the data needs to account for the presence of multiple states. We introduce a two-state model that successfully describes the brightness and fluctuation amplitude of mCherry. The properties of the two states are characterized by FFS and fluorescence lifetime experiments. No interconversion between the two states was observed over the experimentally probed timescales. The effect of fluorescence resonance energy transfer between enhanced green fluorescent protein (EGFP) and mCherry is incorporated into the two-state model to describe protein hetero-oligomerization. The model is verified by comparing the predicted and measured brightness and fluctuation amplitude of several fusion proteins that contain mCherry and EGFP. In addition, hetero-fluorescence resonance energy transfer between mCherry molecules in different states is detected, but its influence on FFS parameters is small enough to be negligible. Finally, the two-state model is applied to study protein oligomerization in living cells. We demonstrate that the model successfully describes the homodimerization of nuclear receptors. In addition, we resolved a mixture of interacting and noninteracting proteins labeled with EGFP and mCherry. These results provide the foundation for quantitative applications of mCherry in FFS studies.     

Controlled ablation of microtubules using a picosecond laser

The use of focused high-intensity light sources for ablative perturbation has been an important technique for cell biological and developmental studies. In targeting subcellular structures many studies have to deal with the inability to target, with certainty, an organelle or large macromolecular complex. Here we demonstrate the ability to selectively target microtubule-based structures with a laser microbeam through the use of enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) variants of green fluorescent protein fusions of tubule. Potorous tridactylus (PTK2) cell lines were generated that stably express EYFP and ECFP tagged to the α-subunit of tubulin. Using microtubule fluorescence as a guide, cells were irradiated with picosecond laser pulses at discrete microtubule sites in the cytoplasm and the mitotic spindle. Correlative thin-section transmission electron micrographs of cells fixed one second after irradiation demonstrated that the nature of the ultrastructural damage appeared to be different between the EYFP and the ECFP constructs suggesting different photon interaction mechanisms. We conclude that focal disruption of single cytoplasmic and spindle microtubules can be precisely controlled by combining laser microbeam irradiation with different fluorescent fusion constructs. The possible photon interaction mechanisms are discussed in detail.     

Imaging the cell entry of the anthrax oedema and lethal toxins with fluorescent protein chimeras

To investigate the cell entry and intracellular trafficking of anthrax oedema factor (EF) and lethal factor (LF), they were C‐terminally fused to the enhanced green fluorescent protein (EGFP) and monomeric Cherry (mCherry) fluorescent proteins. Both chimeras bound to the surface of BHK cells treated with protective antigen (PA) in a patchy mode. Binding was followed by rapid internalization, and the two anthrax factors were found to traffic along the same endocytic route and with identical kinetics, indicating that their intracellular path is essentially dictated by PA. Colocalization studies indicated that anthrax toxins enter caveolin‐1 containing compartments and then endosomes marked by phoshatidylinositol 3‐phoshate and Rab5, but not by early endosome antigen 1 and transferrin. After 40 min, both EF and LF chimeras were observed to localize within late compartments. Eventually, LF and EF appeared in the cytosol with a time‐course consistent with translocation from late endosomes. Only the EGFP derivatives reached the cytosol because they are translocated by the PA channel, while the mCherry derivatives are not. This difference is attributed to a higher resistance of mCherry to unfolding. After translocation, LF disperses in the cytosol, while EF localizes on the cytosolic face of late endosomes.