Contributions of host and symbiont pigments to the coloration of reef corals

For a variety of coral species, we have studied the molecular origin of their coloration to assess the contributions of host and symbiont pigments. For the corals Catalaphyllia jardinei and an orange-emitting color morph of Lobophyllia hemprichii, the pigments belong to a particular class of green fluorescent protein-like proteins that change their color from green to red upon irradiation with approximately 400 nm light. The optical absorption and emission properties of these proteins were characterized in detail. Their spectra were found to be similar to those of phycoerythrin from cyanobacterial symbionts. To unambiguously determine the molecular origin of the coloration, we performed immunochemical studies using double diffusion in gel analysis on tissue extracts, including also a third coral species, Montastrea cavernosa, which allowed us to attribute the red fluorescent coloration to green-to-red photoconvertible fluorescent proteins. The red fluorescent proteins are localized mainly in the ectodermal tissue and contribute up to 7.0% of the total soluble cellular proteins in these species. Distinct spatial distributions of green and cyan fluorescent proteins were observed for the tissues of M. cavernosa. This observation may suggest that differently colored green fluorescent protein-like proteins have different, specific functions. In addition to green fluorescent protein-like proteins, the pigments of zooxanthellae have a strong effect on the visual appearance of the latter species.     

Elementary research of the formation mechanism of sex-related fluorescent cocoon of silkworm, Bombyx mori

To understand mechanisms for the difference of uptaking and transporting the pigments between the male and female in the silkworm, Bombyx mori strain of sex-related fluorescent cocoon, the fluorescent pigments in the midgut lumen, midgut, blood, silk glands and cocoon were analyzed with thin-layer chromatography, and showed that fluorescent colors of cocoons consisted with that of blood and silk glands. The different fluorescent colors of cocoons between the male and female may be mainly caused by the difference of accumulation and transportation for fluorescent pigments in the midgut and in the silk glands. Furthermore the midgut proteins were separated with Native-PAGE, and the proteins respectively recovered from three fluorescent regions presenting on a Native-PAGE gel for the female silkworms were determined using shotgun proteomics and mass spectrometry sequencing, of which 60, 40 and 18 proteins respectively from the region 1, 2 and 3 were identified. It was found that the several kinds of low molecular mass 30 kDa lipoproteins and the actins could be detected in all three regions, troponin, 30 kDa lipoprotein and 27 kDa glycoprotein precursor could be detected in the region 2 and 3, suggesting these proteins may be fluorescent pigments binding candidates proteins. Analysis of gene ontology indicated that the identified proteins in the three regions linked to the cellular component, molecular function, and biological process categories. These results provide a new clew to understand the formation mechanism of sex-related fluorescent cocoon of silkworm.     

Flow cytometric screening of cDNA expression libraries for fluorescent proteins

Fluorescence-activated cell sorting (FACS) was applied for quantitative screening of cDNA expression libraries in bacteria for rare fluorescent protein encoding cDNAs. Rare fluorescent cells, observed at a frequency of 1 in 200,000 bacteria in a cDNA expression library constructed from Astrangia lajollaensis, were detected, enriched, and purified by sorting, yielding three distinct green fluorescent proteins. Two of the isolated fluorescent proteins were found to be 2.5-fold brighter in whole cell fluorescence than the widely used and already optimized EGFP variant and possessed a novel cysteine-containing chromophore. FACS can possess significant advantages in the screening of cDNA libraries in bacteria, since desired genes may occur at low frequencies and possess unexpected properties. This strategy provides a high-throughput, quantitative approach for isolating fluorescent proteins from a more diverse range of organisms and should be extendable to proteins that are not intrinsically fluorescent with the use of available fluorescent indicators.     

Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits

Formation of fluorescent proteins was explored after incubation of recombinant apo-subunits of phycobiliprotein R-phycoerythrin with phycoerythrobilin chromophore. Alpha and beta apo-subunit genes of R-phycoerythrin from red algae Polisiphonia boldii were cloned in plasmid pET-21d(+). Hexahistidine-tagged alpha and beta apo-subunits were expressed in Escherichia coli. Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were constituted after incubation of E. coli cells with phycoerythrobilin. Holo-subunits contained both phycoerythrobilin and urobilin chromophores. Fluorescence and differential interference contrast microscopy showed polar location of holo-subunit inclusion bodies in bacterial cells. Cells containing fluorescent holo-subunits were several times brighter than control cells as found by fluorescence microscopy and flow cytometry. The addition of phycoerythrobilin to cells did not show cytotoxic effects, in contrast to expression of proteins in inclusion bodies. In an attempt to improve solubility, R-phycoerythrin apo-subunits were fused to maltose-binding protein and incubated with phycoerythrobilin both in vitro and in vivo. Highly fluorescent soluble fusion proteins containing phycoerythrobilin as the sole chromophore were formed. Fusion proteins were localized by fluorescence microscopy either throughout E. coli cells or at cell poles. Flow cytometry showed that cells containing fluorescent fusion proteins were up to 10 times brighter than control cells. Results indicate that fluorescent proteins formed by attachment of phycoerythrobilin to expressed apo-subunits of phycobiliproteins can be used as fluorescent probes for analysis of cells by microscopy and flow cytometry. A unique property of these fluorescent reporters is their utility in both properly folded (soluble) subunits and subunits aggregated in inclusion bodies.     

Dynamic Regulation of Fluorescent Proteins from a Single Species of Coral

To gain a better understanding of the natural function of fluorescent proteins, we have undertaken quantitative analyses of these proteins in a single species of coral, Montastraea cavernosa, residing around Turneffe atoll, on the Belizean Barrier Reef. We identified at least 10 members of a fluorescent protein family in this species, which consist of 4 distinct spectral classes. As much as a 10-fold change in the overall expression of fluorescent proteins was observed from specimen to specimen, suggesting that fluorescent proteins are dynamically regulated in response to environmental or physiological conditions. We found that the expression of some proteins was inversely correlated with depth, and that groups of proteins were coordinately expressed. There was no relationship between the expression of fluorescent proteins and the natural coloration of the Montastraea cavernosa specimens in this study. These findings have implications for current hypotheses regarding the properties and natural function of fluorescent proteins.     

Patterns of fluorescent protein expression in Scleractinian corals

Biofluorescence exists in only a few classes of organisms, with Anthozoa possessing the majority of species known to express fluorescent proteins. Most species within the Anthozoan subgroup Scleractinia (reef-building corals) not only express green fluorescent proteins, they also localize the proteins in distinct anatomical patterns.We examined the distribution of biofluorescence in 33 coral species, representing 8 families, from study sites on Australia's Great Barrier Reef. For 28 of these species, we report the presence of biofluorescence for the first time. The dominant fluorescent emissions observed were green (480-520 nm) and red (580-600 nm). Fluorescent proteins were expressed in three distinct patterns (highlighted, uniform, and complementary) among specific anatomical structures of corals across a variety of families. We report no significant overlap between the distribution of fluorescent proteins and the distribution of zooxanthellae. Analysis of the patterns of fluorescent protein distribution provides evidence that the scheme in which fluorescent proteins are distributed among the anatomical structures of corals is nonrandom. This targeted expression of fluorescent proteins in corals produces contrast and may function as a signaling mechanism to organisms with sensitivity to specific wavelengths of light.     

Structural basis for the fast maturation of Arthropoda green fluorescent protein

Since the cloning of Aequorea victoria green fluorescent protein (GFP) in 1992, a family of known GFP-like proteins has been growing rapidly. Today, it includes more than a hundred proteins with different spectral characteristics cloned from Cnidaria species. For some of these proteins, crystal structures have been solved, showing diversity in chromophore modifications and conformational states. However, we are still far from a complete understanding of the origin, functions and evolution of the GFP family. Novel proteins of the family were recently cloned from evolutionarily distant marine Copepoda species, phylum Arthropoda, demonstrating an extremely rapid generation of fluorescent signal. Here, we have generated a non-aggregating mutant of Copepoda fluorescent protein and solved its high-resolution crystal structure. It was found that the protein beta-barrel contains a pore, leading to the chromophore. Using site-directed mutagenesis, we showed that this feature is critical for the fast maturation of the chromophore.     

Involvement of lipid oxidation products in the formation of fluorescent and cross-linked proteins

Age-related fluorescent and cross-linked proteins increase with lipid oxidation of tissues. The fluorophores and cross-links have been considered to be conjugated Schiff bases between amino groups of proteins and malonaldehyde. Our recent studies showed that the fluorophores produced in the in vitro reaction of proteins with malonaldehyde are 1,4-dihydropyridine-3,5-dicarbaldehydes, whose fluorescence characteristics are similar to but not always the same as those of the age-related fluorescent substances, and that the cross-linking is due to less fluorescent conjugated Schiff bases. The in vitro reaction of proteins with oxidized lipids produces fluorescent and cross-linked proteins similar to those in the aging cells or tissues. Monofunctional aldehydes such as alkanals, alk-2-enals and alka-2,4-dienals can also participate in the formation of the fluorophores and cross-links. The fluorescent substances produced from the reaction of primary amines or proteins with these aldehydes showed spectra close to those of the age-related fluorescent substances.     

Mass spectrometry based approach for identification and characterisation of fluorescent proteins from marine organisms

We present here a new analytical strategy for identification and characterisation of fluorescent proteins from marine organisms. By applying basic proteomics tools it is possible to screen large sample collections for fluorescent proteins of desired characteristics prior to gene cloning. Our methodology which includes isolation, spectral characterisation, stability testing, gel-based separation and mass spectrometric identification was optimised on samples collected during the Danish Galathea 3 expedition. Four corals of the Fungia, Sarcophyton and Acropora species emitting green fluorescence were tested. Each of the fluorescent extracts behaves differently under denaturing conditions but complete fluorescence loss was not observed. Optimised electrophoretic conditions yielded effective separation of active fluorescent proteins in both 1DE and 2DE. Mass spectrometric analysis of the proteins in the fluorescent spots excised directly from unstained 2DE gels provides sequence information that might be sufficient to design degenerate primers for gene cloning. Identified fluorescent proteins are in agreement with the coral species determined by visual examination of the samples. The presented methodology is a viable alternative to direct gene cloning for the discovery of novel fluorescent proteins and will be further validated on other samples collected during the Galathea 3 expedition.     

A conserved interaction with the chromophore of fluorescent proteins

The chromophore of fluorescent proteins, including the green fluorescent protein (GFP), contains a highly conjugated imidazolidinone ring. In many fluorescent proteins, the carbonyl group of the imidazolidinone ring engages in a hydrogen bond with the side chain of an arginine residue. Prior studies have indicated that such an electrophilic carbonyl group in a protein often accepts electron density from a main-chain oxygen. A survey of high-resolution structures of fluorescent proteins indicates that electron lone pairs of a main-chain oxygen-Thr62 in GFP-donate electron density into an antibonding orbital of the imidazolidinone carbonyl group. This n→π* electron delocalization prevents structural distortion during chromophore excitation that could otherwise lead to fluorescence quenching. In addition, this interaction is present in on-pathway intermediates leading to the chromophore, and thus could direct its biogenesis. Accordingly, this n→π* interaction merits inclusion in computational and photophysical analyses of the chromophore, and in speculations about the molecular evolution of fluorescent proteins.     

Expression, purification and some properties of fluorescent chimeras of human small heat shock proteins

Small heat shock proteins (sHsp) are ubiquitously expressed in all human tissues and have an important housekeeping role in preventing the accumulation of aggregates of improperly folded or denatured proteins. They also participate in the regulation of the cytoskeleton, proliferation, apoptosis and many other vital processes. Fluorescent chimeras composed of sHsp and enhanced fluorescent proteins have been used to determine the intracellular locations of small heat shock proteins and to analyse the hetero-oligomeric complexes formed by different sHsp. However, the biochemical properties and chaperone-like activities of these chimeras have not been investigated. To determine the properties of these chimeras, we fused enhanced yellow and cyan fluorescent proteins (EYFP and ECFP) to the N-termini of four ubiquitously expressed human small heat shock proteins: HspB1, HspB5, HspB6, and HspB8. The eight fluorescent chimeras of small heat shock proteins and isolated fluorescent proteins were expressed in Escherichia coli. The chimeric proteins were isolated and purified via ammonium sulphate fractionation, ion exchange and size-exclusion chromatography. This method provided 20-100 mg of fluorescent chimeras from 1 L of bacterial culture. The spectral properties of the chimeras were similar to those of the isolated fluorescent proteins. The fusion of fluorescent proteins to HspB6 and HspB8, which typically form dimers, did not affect their quaternary structures. Oligomers of the fluorescent chimeras of HspB1 and HspB5 were less stable and contained fewer subunits than oligomers formed by the wild-type proteins. Fusion with EYFP decreased the chaperone-like activity of HspB5 and HspB6 whereas fusion with ECFP increased chaperone-like activity. All fluorescent chimeras of HspB1 and HspB8 had higher chaperone-like activity than the wild-type proteins. Thus, although fluorescent chimeras are useful for many purposes, the fluorescent proteins used to form these chimeras may affect certain important properties of sHsp.     

Differential fluorescence labelling with 5-dimethyl-aminonaphthalene-1-sulfonyl chloride of intact cells and isolated membranes in Salmonella typhimurium and Acholeplasma laidlawii

For fluorescence labelling intact cells and isolated cell envelopes (membranes) from Salmonella typhimurium and Acholeplasma laidlawii were treated with mixed dansylchloride-lecithin-cholesterol vesicles. This kind of dansylation, which has been supposed to be specific for cell surface proteins, produced fluorescent protein pattern after SDS-polyacrylamide gel electrophoresis only when isolated envelopes were dansylated. Acid hydrolysis of fluorescent cell envelopes of Salmonella typhimurium yielded O-dansyltryosine and epsilon-N-dansyl-lysine besides the free sulfonic acid and unidentified compounds. However, no fluorescent proteins were detectable in cell envelopes isolated from dansylated intact bacteria from Salmonella typhimurium. In accord Acholeplasma laidlawii showed only fluorescence from proteins with a molecular weight higher than 100000.     

Novel synthetic signal peptides for the periplasmic secretion of green fluorescent protein in Escherichia coli

New secretion vectors containing synthetic signal peptides were constructed to study the periplasmic translocation of green fluorescent protein (GFP) in Escherichia coli. These constructs encode synthetic signal peptides spA and spD fused to the amino terminal end of GFP, and expressed from T7/lac promoter in the BL21DE3 strain by induction with IPTG. The recombinant protein was detected in both the cytoplasmic and periplasmic fractions. Fluorescence analysis revealed that recombinant proteins with signal peptides were not fluorescent, indicating translocation to periplasmic space. In contrast, recombinant proteins without signal peptide were fluorescent. These results indicate that the expressed recombinant proteins were translocated into the periplasm. Therefore, the synthetic signal peptides derived from signal peptides of Bacillus sp. could efficiently secrete the heterologous proteins to the periplasmic space of E. coli.     

Stable expression of Anthozoa fluorescent proteins in mammalian cells

BACKGROUND: Fluorescent proteins have become invaluable reporters in many areas of cellular and developmental biology. An enhanced version of the Aequorea victoria green fluorescent protein (AvEGFP) is the most widely used fluorescent protein. For a variety of reasons, it is useful to have alternative fluorescent proteins to AvEGFP. METHODS: The cDNA sequences for enhanced variants of the Anemonia cyan fluorescent protein (AmCyan1), as well as the Zoanthus green (ZsGreen1) and yellow (ZsYellow1) fluorescent proteins, were cloned downstream of a constitutive cytomegalovirus (CMV) promoter within a retroviral expression vector. NIH3T3, HEK293, SW620, and WM35 cells were transduced with recombinant retroviruses at a low multiplicity of infection (MOI) to bias for single-copy integration. Both unselected and stably selected cells transduced with the retroviral expression constructs were characterized. Expression of each fluorescent protein in cells was detected using flow cytometry and fluorescence microscopy with filter sets typically used for AvEGFP/fluorescein isothiocyanate (FITC) detection and was compared with the expression of AvEGFP. In addition, a fluorescence plate reader with several excitation and emission filter sets was used for detection. RESULTS: Expression of each protein was observable by fluorescence microscopy. Under given conditions of flow cytometry, the ZsGreen1 mean fluorescence was approximately 3-fold, 10-fold, and 50-fold greater than that of AvEGFP, ZsYellow1, and AmCyan1, respectively. AmCyan1, ZsGreen1, and AvEGFP were detected by a fluorescence plate reader. CONCLUSION: We determined that fluorescent proteins from Anthozoa species are detectable using a standard flow cytometer and fluorescence microscope. All of the mammalian cell lines tested expressed detectable levels of fluorescent proteins from stable integrated provirus. In cell lines where the AvEGFP protein is toxic or poorly expressed, these Anthozoa fluorescent proteins may serve as alternative fluorescent reporters.     

Development of cysteine-free fluorescent proteins for the oxidative environment

Molecular imaging employing fluorescent proteins has been widely used to highlight specific reactions or processes in various fields of the life sciences. Despite extensive improvements of the fluorescent tag, this technology is still limited in the study of molecular events in the extracellular milieu. This is partly due to the presence of cysteine in the fluorescent proteins. These proteins almost cotranslationally form disulfide bonded oligomers when expressed in the endoplasmic reticulum (ER). Although single molecule photobleaching analysis showed that these oligomers were not fluorescent, the fluorescent monomer form often showed aberrant behavior in folding and motion, particularly when fused to cysteine-containing cargo. Therefore we investigated whether it was possible to eliminate the cysteine without losing the brightness. By site-saturated mutagenesis, we found that the cysteine residues in fluorescent proteins could be replaced with specific alternatives while still retaining their brightness. cf(cysteine-free)SGFP2 showed significantly reduced restriction of free diffusion in the ER and marked improvement of maturation when fused to the prion protein. We further applied this approach to TagRFP family proteins and found a set of mutations that obtains the same level of brightness as the cysteine-containing proteins. The approach used in this study to generate new cysteine-free fluorescent tags should expand the application of molecular imaging to the extracellular milieu and facilitate its usage in medicine and biotechnology.     

Assessing the tendency of fluorescent proteins to oligomerize under physiologic conditions

Several fluorescent proteins (FPs) are prone to forming low-affinity oligomers. This undesirable tendency is exacerbated when FPs are confined to membranes or when fused to naturally oligomeric proteins. Oligomerization of FPs limits their suitability for creating fusions with proteins of interest. Unfortunately, no standardized method evaluates the biologically relevant oligomeric state of FPs. Here, we describe a quantitative visual assay for assessing whether FPs are sufficiently monomeric under physiologic conditions. Membrane-associated FP-fusion proteins, by virtue of their constrained planar geometry, achieve high effective concentrations. We exploited this propensity to develop an assay to measure FP tendencies to oligomerize in cells. FPs were fused on the cytoplasmic end of an endoplasmic reticulum (ER) signal-anchor membrane protein (CytERM) and expressed in cells. Cells were scored based on the ability of CytERM to homo-oligomerize with proteins on apposing membranes and restructure the ER from a tubular network into organized smooth ER (OSER) whorl structures. The ratio of nuclear envelope and OSER structures mean fluorescent intensities for cells expressing enhanced green fluorescent protein (EGFP) or monomeric green fluorescent protein (mGFP) CytERM established standards for comparison of uncharacterized FPs. We tested three FPs and identified two as sufficiently monomeric, while a third previously reported as monomeric was found to strongly oligomerize.     

Mobilization of rubisco and stroma-localized fluorescent proteins of chloroplasts to the vacuole by an ATG gene-dependent autophagic process

During senescence and at times of stress, plants can mobilize needed nitrogen from chloroplasts in leaves to other organs. Much of the total leaf nitrogen is allocated to the most abundant plant protein, Rubisco. While bulk degradation of the cytosol and organelles in plants occurs by autophagy, the role of autophagy in the degradation of chloroplast proteins is still unclear. We have visualized the fate of Rubisco, stroma-targeted green fluorescent protein (GFP) and DsRed, and GFP-labeled Rubisco in order to investigate the involvement of autophagy in the mobilization of stromal proteins to the vacuole. Using immunoelectron microscopy, we previously demonstrated that Rubisco is released from the chloroplast into Rubisco-containing bodies (RCBs) in naturally senescent leaves. When leaves of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing stroma-targeted fluorescent proteins were incubated with concanamycin A to inhibit vacuolar H(+)-ATPase activity, spherical bodies exhibiting GFP or DsRed fluorescence without chlorophyll fluorescence were observed in the vacuolar lumen. Double-labeled immunoelectron microscopy with anti-Rubisco and anti-GFP antibodies confirmed that the fluorescent bodies correspond to RCBs. RCBs could also be visualized using GFP-labeled Rubisco directly. RCBs were not observed in leaves of a T-DNA insertion mutant in ATG5, one of the essential genes for autophagy. Stroma-targeted DsRed and GFP-ATG8 fusion proteins were observed together in autophagic bodies in the vacuole. We conclude that Rubisco and stroma-targeted fluorescent proteins can be mobilized to the vacuole through an ATG gene-dependent autophagic process without prior chloroplast destruction.     

The ability of green fluorescent proteins for photoconversion under oxygen-free conditions is determined by the chromophore structure rather than its amino acid environment

Photoconversion of various green and cyan fluorescent proteins to the red fluorescent state under the oxygen-free conditions was studied. Such photoconversion has earlier been described for the EGFP green fluorescent protein. Phylogenetically distant fluorescent proteins that have a low identity of their amino acid sequences but contain chemically identical chromophores based on a Tyr residue were shown to be susceptible to this type of photoconversion. At the same time, the ECFP protein, which has 92% homology with EGFP but contains a chromophore based on tryptophan did not undergo the photoconversion. Thus, it is precisely the chromophore structure, rather than the amino acid environment that determines the ability of green fluorescent proteins to display photoconversion to the red fluorescent state under anaerobic conditions.     

Fluorogen activating proteins in flow cytometry for the study of surface molecules and receptors

The use of fluorescent proteins, particularly when genetically fused to proteins of biological interest, have greatly advanced many flow cytometry research applications. However, there remains a major limitation to this methodology in that only total cellular fluorescence is measured. Commonly used fluorescent proteins (e.g. EGFP and its variants) are fluorescent whether the fusion protein exists on the surface or in sub-cellular compartments. A flow cytometer cannot distinguish between these separate sources of fluorescence. This can be of great concern when using flow cytometry, plate readers or microscopy to quantify cell surface receptors or other surface proteins genetically fused to fluorescent proteins. Recently developed fluorogen activating proteins (FAPs) solve many of these issues by allowing the selective visualization of only those cell surface proteins that are exposed to the extracellular milieu. FAPs are GFP-sized single chain antibodies that specifically bind to and generate fluorescence from otherwise non-fluorescent dyes ('activate the fluorogen'). Like the fluorescent proteins, FAPs can be genetically fused to proteins of interest. When exogenously added fluorogens bind FAPs, fluorescence immediately increases by as much as 20,000-fold, rendering the FAP fusion proteins highly fluorescent. Moreover, since fluorogens can be made membrane impermeant, fluorescence can be limited to only those receptors expressed on the cell surface. Using cells expressing beta-2 adrenergic receptor (β2AR) fused at its N-terminus to a FAP, flow cytometry based receptor internalization assays have been developed and characterized. The fluorogen/FAP system is ideally suited to the study of cell surface proteins by fluorescence and avoids drawbacks of using receptor/fluorescent protein fusions, such as internal accumulation. We also briefly comment on extending FAP-based technologies to the study of events occurring inside of the cell as well.