Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

A Green Fluorescent Protein with Photoswitchable Emission from the Deep Sea

  • Alexander Vogt,

    Affiliation Institute of General Zoology and Endocrinology, University of Ulm, Ulm, Germany

  • Cecilia D'Angelo,

    Affiliation Institute of General Zoology and Endocrinology, University of Ulm, Ulm, Germany

  • Franz Oswald,

    Affiliation Department of Internal Medicine I, University of Ulm, Ulm, Germany

  • Andrea Denzel,

    Affiliation Institute of General Zoology and Endocrinology, University of Ulm, Ulm, Germany

  • Charles H. Mazel,

    Affiliation NightSea, Andover, Massachusetts, United States of America

  • Mikhail V. Matz,

    Affiliation Integrative Biology, University of Texas in Austin, Austin, Texas, United States of America

  • Sergey Ivanchenko,

    Affiliation Institute of Biophysics, University of Ulm, Ulm, Germany

  • G. Ulrich Nienhaus,

    Affiliations Institute of Biophysics, University of Ulm, Ulm, Germany, Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America

  • Jörg Wiedenmann

    J.Wiedenmann@soton.ac.uk

    Affiliations Institute of General Zoology and Endocrinology, University of Ulm, Ulm, Germany, National Oceanography Centre, University of Southampton, Southampton, United Kingdom

A Green Fluorescent Protein with Photoswitchable Emission from the Deep Sea

  • Alexander Vogt, 
  • Cecilia D'Angelo, 
  • Franz Oswald, 
  • Andrea Denzel, 
  • Charles H. Mazel, 
  • Mikhail V. Matz, 
  • Sergey Ivanchenko, 
  • G. Ulrich Nienhaus, 
  • Jörg Wiedenmann
PLOS
x

Abstract

A colorful variety of fluorescent proteins (FPs) from marine invertebrates are utilized as genetically encoded markers for live cell imaging. The increased demand for advanced imaging techniques drives a continuous search for FPs with new and improved properties. Many useful FPs have been isolated from species adapted to sun-flooded habitats such as tropical coral reefs. It has yet remained unknown if species expressing green fluorescent protein (GFP)-like proteins also exist in the darkness of the deep sea. Using a submarine-based and -operated fluorescence detection system in the Gulf of Mexico, we discovered ceriantharians emitting bright green fluorescence in depths between 500 and 600 m and identified a GFP, named cerFP505, with bright fluorescence emission peaking at 505 nm. Spectroscopic studies showed that ∼15% of the protein bulk feature reversible ON/OFF photoswitching that can be induced by alternating irradiation with blue und near-UV light. Despite being derived from an animal adapted to essentially complete darkness and low temperatures, cerFP505 maturation in living mammalian cells at 37°C, its brightness and photostability are comparable to those of EGFP and cmFP512 from shallow water species. Therefore, our findings disclose the deep sea as a potential source of GFP-like molecular marker proteins.

Introduction

In recent years, GFP-like proteins from marine invertebrates have emerged as indispensable tools for tracking of proteins or cells, sensing of intracellular conditions such as Ca2+ or pH variations and for studies of protein interactions and gene activity, opening several stimulating perspectives in live cell imaging [1][9]. The success of these pigments as molecular markers results mainly from their unique capability to form a chromophore autocatalytically; no additional cofactors are required, only molecular oxygen [10], [11]. Recently, the group of photoactivatable GFP-like proteins attracted particular attention. The fluorescence of these proteins can either be switched on or off (paGFP, KFP1, Dronpa, mTFP0.7) or the emission color can be shifted towards longer wavelengths (Kaede, KikGR, EosFP, Dendra, mcavRFP, cjarRFP, lhemOFP, PS-CFP) by irradiation with light of specific wavelengths [3], [12][21]. The possibility to control the fluorescence properties enabled numerous innovative applications, for instance, regional optical marking and tracking of cells, subcellular organelles or proteins [3], [15], [21]. Additionally, photoswitchable or photoconvertible proteins can be employed in novel microscopy concepts that allow imaging of subcellular structures at a resolution beyond the diffraction barrier of optical microscopy [22][24]. The swift evolution of super-resolution microscopy techniques calls for novel and optimized photoswitchable FPs [6], [23], [25].

The application of FPs derived from marine species has been further extended by protein engineering. Optimized variants were created that feature faster and more complete maturation of the chromophore, shifted excitation and emission wavelengths, reduced aggregation and oligomerization, brighter fluorescence and increased photostability [15], [17], [26][34]. However, the development of marker proteins with entirely novel spectral properties greatly relies on the discovery of natural lead structures. To date, GFP-like proteins have been detected in different taxa of marine invertebrates that inhabit the photic zone of the ocean [35], [36]. Most members of this protein family were isolated from cnidarians, mainly from the taxon anthozoa, but GFPs were also found in copepods (crustacea) and amphioxus (chordata) [2], [7], [21], [36][44].

The expression of GFP-like proteins in marine invertebrates has been linked with the photophysiology of the animals in various ways. Potential functions of the pigments include photoprotection, optimization of symbionts photosynthesis, photoreception, or inter- and intra- species signaling [36], [38], [45][49]. In a large number of scleractinian corals, the expression of GFP-like proteins is tightly regulated on the transcriptional level by the intensity of blue light [50]. However, color morphs of the Mediterranean sea anemone Anemonia spp. and the Caribbean coral Montastrea cavernosa are defined by different expression levels of distinct sets of GFP-like proteins that appear to be fixed rather than regulated by light [18], [51][53]. Moreover, the recent discovery of FPs in non-zooxanthellate anthozoans suggests putative functions of these pigments that are independent of the light requirements from symbiotic associations with zooxanthellae [37], [43], [48]. The possibility of light-independent expression of FPs encouraged us to search the dark depths of the ocean as a potential new source of GFP-like proteins.

Results and Discussion

Identification of a GFP-like protein in a deep sea ceriantharian

As part of the project “Operation Deep Scope” (NOAA Ocean Explorer), we screened benthic habitats of the Gulf of Mexico for fluorescent organisms. For this purpose, the Johnson-Sea-Link II submersible (Harbor Branch) was equipped with four lights, two of which (400 W HMI lights, Deepsea Power & Light, San Diego, USA) were mounted on the front edge of the work basket so that they could be positioned close to the sea floor. They were fitted with custom blue short-pass (cutoff at approximately 460 nm) interference filters (NightSea, Andover, MA) mounted in custom filter holders that minimized stray light while allowing water flow for cooling the lamp. The science observer in the sphere wore barrier filter glasses (NightSea, Andover, MA) that blocked the reflected blue light so that any fluorescence could be seen with high contrast, and the observer would call the pilot's attention to features of interest on the sea floor. A fluorescence barrier filter (long-pass acrylic absorption filter, 50% transmission at approximately 500 nm) (NightSea, Andover, MA) was positioned in front of the submersible's video camera using a hinged mount, so that the pilot could use the submersible's manipulator arm to pivot the filter away from the camera to acquire conventional white light images to contrast with the fluorescence images collected with the filter in place. The special detection equipment allowed to document the fluorescence of several fluorescent deep sea organisms. Specimens showing particularly striking green fluorescence were found among the taxon ceriantharia (Figure 1A). The presence of this taxon in the West Atlantic had previously been reported off the coast of New England down to 1600 m depth [54]. To date, two species (Ceriantheopsis americanus, Cerianthus borealis) are known from Nova Scotia to Cape Hatteras [54], and three species (Pachycerianthus curacaoensis, Ceriantheopsis americanus, Isarachnanthus maderensis) from the Caribbean and the Gulf of Mexico, all from shallow water down to a depth of 150 m [55][57]. We collected tentacle samples from an individual displaying green fluorescence found in 530 m depth at the “Green Canyon” site. Using white light illumination, the view of the tentacles under the stereomicroscope is dominated by a pattern of dark pigment rings (Figure 1B). The lighter regions show a brownish color with a yellow-green tint. These regions emit intense green fluorescence peaking at ∼504 nm, if excited with blue light (Figure 1C). In contrast, the dark rings are non-fluorescent. The tentacles were preserved in RNAlater for subsequent analysis. Examination of the cnidom of marginal tentacles revealed the absence of p-mastigophores, thus the animal can be considered to belong to the taxon Spirularia [58]. Spirularia encompasses two families, Botrucnidiferidae and Cerianthidae. The cnidoms of known members of these two families from the region could not be matched with the deep-sea ceriantharian of the present study. Therefore, the specimen might represent a new species (T. Molodtsova, personal communication). Future work should clarify the taxonomic status of the green fluorescent deep sea ceriantharian.

thumbnail
Figure 1. Identification of a GFP-like protein from a deep sea ceriantharian.

A) Fluorescence image of a ceriantharian acquired in 530 m depth in the Gulf of Mexico. The depth (ft), temperature (Temp, °C) and the salinity (Salin) measured during image acquisition are displayed in the upper part of the panel. B–C) Microscopic image of two tentacles photographed under white light (B) or blue light excitation using a GFP-filter set (C). D) Absorption, excitation and emission spectra of purified recombinant cerFP505. Fluorescence spectra were recorded using 450 nm light for excitation, whereas the emission was collected at 550 nm. E) Multiple alignment of amino acid sequences from ceriantharian fluorescent proteins and EGFP. The chromophore – forming tripeptide is highlighted in green. Residues interacting directly with, or found in the proximity of, the chromophore in cmFP512 are labeled in blue or red, respectively. Amino acids underlayed in grey or yellow are involved in A/B or A/C interface interactions in cmFP512, respectively [59]. The numberings for cerFP505 and EGFP are given above and below the sequences, respectively. Regions forming ß-sheets in EGFP are underlined.

https://doi.org/10.1371/journal.pone.0003766.g001

We isolated total RNA from the preserved tentacle tissue and synthesized cDNA. Fragments coding for a GFP-like protein were amplified by PCR using internal primers designed to be complementary to the sequence of cmFP512 in combination with cDNA adapter primers. Subsequently, primers specific for the 5′ and 3′ termini of the open reading frames were synthesized and the full length cDNA was amplified, cloned and sequenced. The coding sequence was deposited at GenBank under the accession number EU927368. The amino acid sequence displays 83% sequence identity with cmFP512 and 78% with an orange fluorescent protein from Cerianthus sp. (OFP) (Figure 1E). Like cmFP512 and OFP, the GFP-like protein from the deep sea ceriantharian shows a remarkable difference from other representatives of the protein family, for instance, it shares only about 40% identical amino acids with anthozoan FPs such as Kaede, EosFP, eqFP611 and Dronpa [12], [15], [21], [44]. The protein was expressed in E. coli and a GFP was purified that had excitation and emission maxima at 494 and 505 nm, respectively (Figure 1D). Therefore, the GFP was named cerFP505 (ceriantharian fluorescent protein with an emission maximum at 505 nm). The elution profile of cerFP505 obtained from size exclusion chromatography evinced a tetrameric structure typical for many anthozoan GFP-like proteins including cmFP512 [43], [59][62]. Analysis by SDS-PAGE showed that the subunits form an unfragmented, single band associated with a molecular mass of ∼26 kDa (data not shown).

The chromophore in cerFP505 is formed by the tripeptide Gln62-Tyr63-Gly64 (Figure 1E). As obvious from the excitation maximum at 494 nm, the side chain of Tyr63 exists predominately in the deprotonated state. In contrast, GFP from Aequorea victoria required the Ser65Thr mutation to transfer the neutral chromophore to the deprotonated state. Glutamine in the first position of the chromophore is common for both green and red fluorescent proteins from anthozoans such cmFP512, asFP499, OFP from Cerianthus sp. or dsRed [41], [59], [63]. All residues homologous to amino acids interacting with the chromophore in cmFP512 are conserved in cerFP505 [59]. Only one amino acid of cmFP512 with a side chain localized in the proximity of the chromophore, Met59, is exchanged by Thr in cerFP505. This alteration seems to be quite common among anthozoan FPs, since threonine is also found in the corresponding position in OFP from Cerianthus sp., EosFP and Dronpa [15], [21], [63]. Further differences between cerFP505 and cmFP512 are three conservative substitutions of residues involved in the formation of the A/B (Leu15Arg) and A/C interfaces (Lys149Gln and Lys193Gln) within the tetrameric assembly. Interestingly, the emission maximum of OFP from Cerianthus sp. is shifted to 573 nm, although the chromophore and its environment appear to be highly conserved in comparison to ceriantharian GFPs. A potential key residue that might be responsible for the red shift of OFP is Thr142. In cmFP512 and in cerFP505, this position is occupied by Val. In contrast to Ser142 in other FPs, this residue is not directly involved in positioning the phenolate group of the chromophore in cmFP512 [60], [62], [64]. Instead, the sidechain of Tyr63 interacts with Lys159 and surrounding water molecules [59]. The introduction of the hydroxyl group of Thr142 will most likely interfere with this hydrogen bonding network and result in a repositioning of the phenolate of the chromophore. The slightly altered conformation of the chromophore might facilitate reactions that extend the conjugated π-sytem, for instance, by formation of a third heterocycle as in the dsRed derivative mOrange [33], [65].

Reversible photoswitching properties of cerFP505

In the spectral range beyond 400 nm, the absorption spectrum of the native protein overlapped completely with the excitation spectrum, while that of the alkali-denatured protein (measured at pH 13) displayed a maximum at 448 nm, typical of the GFP-type chromophore [66], [67]. An extinction coefficient of ε494 nm = 54000 M−1 cm−1 and a fluorescence quantum yield of 0.55 was calculated for cerFP505. These values are comparable to those obtained for cmFP512 and place the pigment in the group of highly fluorescent proteins (Table 1) [43].

thumbnail
Table 1. Photophysical properties of ceriantharian fluorescent proteins.

https://doi.org/10.1371/journal.pone.0003766.t001

While studying the photobleaching behavior of cerFP505, we found that blue-light irradiation (∼450 nm) resulted in a fast initial decrease of the green fluorescence emission. Full fluorescence intensity could be recovered by illumination of the sample with light of ∼400 nm. To analyze this effect in detail, absorption spectra of purified protein samples were collected after 5 min illumination with 400 nm (on-state) or with 450 nm (off-state) light (Figure 2A). Both spectra displayed maxima at 494 nm, however the amplitude of the peak was ∼15% higher for the sample in the on-state. The deactivation of the chromophore was reflected in the lower extinction coefficient (ε494 nm≈46,000 M−1 cm−1) calculated for the protein in the off-state at 494 nm, whereas the quantum yield remained unaltered. A second peak at ∼390 nm appeared at the expense of the decreasing 494 nm absorption band. As the absorption at 390 nm is characteristic for the neutral GFP-type chromophore [68], this finding suggests that a reversible protonation of the chromophore is involved in the photoswitching process [68]. Reversible protonation and cistrans isomerization of the chromophore were recently found to be responsible for the on/off switching of the GFPs Dronpa and mTeal0.7 [13], [15], [69], [70]. However, in contrast to Dronpa and mTeal0.7 that can be switched off essentially completely, the cerFP505 fluorescence intensity can be reduced only by ∼15%. This percentage shows slight variations among different protein preparations, but is stable within a defined sample.

thumbnail
Figure 2. Photoswitching of the chromophore in cerFP505.

A) Absorption spectra after irradiation with 400 nm (on-state) or with 450 nm light (off-state). Deactivation gives rise to a second peak around 390 nm. B) Ground state equilibrium. Irradiation at 400 nm drives the protein to the on-state, 450 nm light turns off the “activatable” fraction of the protein. Bars above the x-axis specify the light treatment, 400 nm light (white bar), 450 nm light (grey bar) or no light (black bar). C) Fluorescence emission of the protein in the on- or off-state was recorded for ≥30 min in the dark after 5 min irradiation with 400 or 450 nm light. Bars above the x-axis specify the light treatment as described above. D) The reversibility of the reaction is shown for eight cycles of activation/deactivation. Bars above the x-axis specify the light treatment as described in (B).

https://doi.org/10.1371/journal.pone.0003766.g002

In order to determine the ground state equilibrium of the fluorescent and the non-fluorescent states of cerFP505, the protein was expressed in E. coli in darkness. Purification was performed under minimal illumination and samples were stored in the dark for two weeks afterwards. Subsequent irradiation of cerFP505 with 400 nm light resulted in a full activation of the sample as deduced from the 2% increase of the fluorescence emission (Figure 2B). The emission intensity decreased by ∼15% in response to illumination with 450 nm light but could be fully recovered under 400 nm light irradiation. The intensity differences of the untreated protein, the activated and deactivated states, imply that ∼98% of all chromophores in the ground state equilibrium are found in the active conformation, whereas ∼80% of the photoswitchable fraction adopts a fluorescent state.

After activation, fluorescence emission was recorded in 30 s intervals in the absence of external illumination for 30 min. The intensity did not decrease within this period. Likewise, no increase in emission intensity was recorded for >30 min after deactivation, indicating that the on-switching of cerFP505 is an exclusively light-driven process on the observed minute-time scale (Figure 2C). A bi-stability of the on/off-states for more than half an hour as shown by cerFP505 was recently also observed for Dronpa [71]. In contrast, the photoswitchable proteins KFP and rsFastLime reach the ground state equilibrium by thermal relaxation within a few minutes [16], [71]. The photoswitching of cerFP505 was reversible over numerous cycles of alternating 400/450 nm irradiation (Figure 2D). Under the light conditions used in the experiment (450 nm; ∼80 mW cm−2), we determined a half-life of 30 s for off switching. An identical value was calculated for rsFastLime, the fast-switching derivative of Dronpa under similar blue light intensities (∼300 mW cm−2) [71]. However, within the time required to switch off rsFastLime completely, only 15% of all cerFP505 molecules can be converted in the dark state. The absence of measurable fluorescence recovery of cerFP505 by thermal relaxation within the time scale of this experiment (Figure 2C) indicates that the off-switching reaction is saturated. This observation suggests the existence of two different conformations of the protein, one that is stably fluorescent and another one that allows photoswitching. Such a durable coexistence of two spectroscopically distinct states has been noticed only for GFP-like red fluorescent proteins (RFPs) with a mixture of green or red fluorescent chromophores [44], [72][74].

Most recently, a collection of photoswitchable/photoconvertible FPs including KFP, Dronpa, Kaede, Dendra and EosFP and their biotechnologically engineered variants have enabled imaging of protein localization with a resolution beyond the diffraction barrier of optical microscopy [22], [24], [25], [75], [76]. Microscopy concepts such as RESOLFT (Reversible Saturable Optical Fluorescence Transitions), Photoactivated Localization Microscopy (PALM) [22], [77], Fluorescence Photoactivation Localization Microscopy (FPALM) [78], Stochastic Optical Reconstruction Microscopy (STORM) [79], [80], or PALM with Independently Running Acquisition (PALMIRA) [25] offer great application potential for photoswitchable marker proteins with novel properties. Interestingly, cerFP505 combines the bistability of the on/off-states of Dronpa with the increased switching speed of rsFastLime [71]. These properties allow a tightly controlled switching using comparably low irradiation energy, and are beneficial for local optical marking and super-resolution microscopy. Therefore, cerFP505 appears as important lead structure for the development of a variant in which the stable green fluorescent conformation is converted to the photoswitchable state. Moreover, the discovery of photoswitchable cerFP505 from a deep sea animal reveals the lightless depths of the oceans as a new reservoir of GFP-like proteins with novel and highly desirable properties for imaging applications.

Eukaryotic expression and imaging of cerFP505

Deep sea organisms are adapted to a life in constant darkness at temperatures below 10°C. This realization raises the question if their GFP-like pigments would meet the demands on a reporter in mammalian cells, such as functional folding at 37°C and sufficient photostability. Moreover, in cerFP505 as well as in GFP from Aequorea victoria, the residue preceding the chromophore is formed by phenylalanine and the exchange of phenylalanine in this position by leucine (Figure 1E) was found necessary to promote functional folding of avGFP at 37°C [9]. The performance of cerFP505 as marker in mammalian cells was tested by transfecting HEK293 cells with an eukaryotic expression vector carrying the cDNA sequence of cerFP505. Transient expression in cells at 37°C showed a uniform distribution without signs of aggregation (Figure 3A). The intensity of green fluorescence was comparable to cells expressing cmFP512 or EGFP under the same conditions. This observation was particularly surprising considering the low-temperature adaptation of the animal that yielded cerFP505. Moreover, eqFP611 from a tropical sea anemone required extensive engineering to create a variant folding efficiently at 37°C [32], [44]. These findings show that the folding of fluorescent proteins is not necessarily correlated with the temperature adaptations of the animals they originated from. It is, furthermore, obvious that a phenylalanine in front of the chromophore does not always impair folding of GFPs at higher temperatures. The fluorescence of cerFP505 in mammalian cells could be already detected ∼6 h after transfection (Figure 3B). The same time period is required by EGFP and cmFP512, indicating that cerFP505 has comparable folding and maturation rates, making the protein suitable for most cell biology applications [43].

thumbnail
Figure 3. Expression of cerFP505 in mammalian cells.

A) cerFP505, cmFP512 and EGFP were expressed in HEK293 cells. Photographs were taken 12 h after transfection. B) The upper panels show the expression and maturation of cerFP505 in vivo in HEK293 cells. Images were excised from a time-lapse movie. The time-points of image acquisition are indicated in the panels as minutes after transfection. The increase in fluorescence intensity of cells in the green channel was analyzed for each image of the time-lapse movie. The graph displays the mean of the individual measurement normalized to 1.0, error bars denote standard deviations. C) Photobleaching of cerFP505, cmFP512 and EGFP in HEK293 cells under continuous irradiation with blue light under the fluorescence microscope. The upper panels are images from a time-lapse movie showing the photobleaching of cells expressing cerFP505. Imaging time-points are given and correspond to minutes of irradiation. The graph displays the mean values of the green fluorescence emission of blue-light irradiated cells expressing cerFP505, cmFP512 and EGFP. Error bars denote standard deviations. Inset: Photobleaching measured in vitro using purified recombinant protein samples.

https://doi.org/10.1371/journal.pone.0003766.g003

Under constant irradiation with blue light (480/40 nm; 1.2 W cm−2) under the microscope, cerFP505 displayed faster photobleaching in cells compared to EGFP and cmFP512 (Figure 3C, Table 1). The difference in photobleaching rates of cerFP505 and cmFP512 could also be detected in purified protein solutions at low energy irradiation (450/40; 80 mW cm−2) (Figure 3C Inset). However, the bleaching rate of cerFP505 is still within the range of values determined for FPs commonly used in imaging applications [27]. Taken together, these results evidence the suitability of GFP-like proteins from deep sea organisms as fluorescent marker for live cell imaging.

Concluding remarks

We discovered fluorescent deep sea organisms as a new source of GFP-like proteins with highly desirable properties such as reversible photoswitching. In spite of its origin in an animal adapted to conditions hostile for mammalian cells, cerFP505 isolated from a deep sea ceriantharian performed well as a marker protein in a human cell line at 37°C. Furthermore, the identification of an FP from an animal living in an essentially lightless habitat reinforces the emerging idea that proteins belonging to the GFP-like family can exert functions in anthozoans that are not related to photoprotection or the photosynthetic productivity of algal symbionts.

Materials and Methods

Animal collection

Using the Johnson-Sea-Link submersible (Harbor Branch), tentacle samples were obtained from a green fluorescent ceriantharian found in the Gulf of Mexico at a depth of 530 m. The tentacles were conserved in RNAlater (Ambion, Foster City, USA) and stored at 4°C for later analysis.

cDNA library construction and cloning

Total RNA from the deep sea ceriantharian was extracted from tentacle tissue preserved in RNAlater (Ambion, Foster City, USA) using RNAqueous® (Ambion, Foster City, USA) according to the instructions of the manufacturer. The cDNA library was constructed as described [18] using the SMART cDNA Library Construction Kit (Clontech, Mountain View, USA) and 5′/3′ RACE was performed using the adapter primers in combination with primers designed against cmFP512 (5′ gcagtgatatcacatataaagacaaagttctgcatgg 3′). After sequencing the amplicons, primers were synthesized that bind to the 5′ and 3′ termini of the open reading frame. The full length coding sequence was amplified by PCR and ligated in pQE32 (Qiagen, Hilden, Germany).

Spectroscopic characterization

Tentacle fluorescence was photographed with a Leica MZ FL III dissecting microscope (Leica Microsystems, Wetzlar, Germany) equipped with a mercury lamp, a standard epifluorescence filter set and a Spot Insight QE digital camera (Diagnostic Instruments, Sterling Heights, USA), whereas spectra of the tentacles were recorded using a USB2000 spectrometer (Ocean Optics, Dunedin, USA) equipped with a fiber optic probe coupled to the eyepiece of the microscope.

The novel GFP-like protein was expressed in fusion with an N-terminal 6× histidine tag in E. coli BL21 cells. The protein was purified from the bacterial culture using TALON metal affinity resin (BD Biosciences, Clontech) and size exclusion chromatography as described [43], [44]. Absorption spectra were measured with a Varian Cary 50 Scan UV-Vis Spectrophotometer (Varian, Palo Alto, USA). Fluorescence and photoswitching properties were determined with a Varian Cary Eclipse fluorescence spectrophotometer (Varian, Palo Alto, USA). The molar extinction coefficient was calculated as described for cmFP512 [43]. The fluorescence quantum yield was determined using purified solutions of cmFP512 as reference samples [43]. Photoswitching was accomplished by irradiating the protein sample with a 150 W Xe lamp (LOT-Oriel, Leatherhead, UK), equipped with band pass filters (Schott, Mainz, Germany) with transmission at 400/40 or 450/40 nm (center/bandwidth). The protein solution in the cuvette experienced an energy density of ∼80 mW cm−2.

Eukaryotic expression and imaging

The cerFP505 cDNA was isolated after digestion of pQE32-cerFP505 with Acc65I and BamHI and ligated into the corresponding sites of pcDNA3.1- (Invitrogen, Carlsbad, CA, USA), resulting in pcDNA3.1-cerFP505. HEK-293 cells (ATCC CRL 1573) were grown at 37°C under 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS, Biochrom, Berlin, Germany). For live cell imaging, cells were plated on chambered cover glasses (Nunc, Rochester, NY, USA) at a density of 80,000 cells per chamber in 2 ml medium. After 16 h incubation at 37°C, cells were transfected with 500 ng of pcDNA3.1-cerFP505, pcDNA3-cmFP512 or pcDNA3-EGFP expression plasmid using the Nanofectine transfection reagent (PAA, Pasching, Austria) according to the manufacturer's instructions. 24 h after transfection the living cells were analysed using a fluorescence microscope (DM IRB, Leica Microsystems, Wetzlar, Germany) equipped with a 100-W Hg light source (HO 103W/2, Osram, Munich, Germany) and a standard FITC filterset. Images were taken with a digital camera (C4742, Hamamatsu Photonics, Hamamatsu City, Japan). To study expression and maturation of cerFP505 within transfected cells, time-lapse movies were recorded. Starting 180 min after transfection, frames were acquired in 10 min intervals for a period of 10 h. The fluorescence intensity of four individual cells was analyzed using the ImageJ software (http://rsb.info.nih.gov/ij/). Photobleaching of cerFP505, cmFP512 and EGFP within living cells was analyzed 24 h after transfection. Cells were continuously irradiated with blue light (Excitation filter: HQ480/40, AHF, Tübingen, Germany) with an energy density of 1.2 W cm−2. Images were taken every minute for 2 hours. Fluorescence intensity was analyzed as described above, based on five individual image regions each containing ∼6 cells.

Acknowledgments

The authors are grateful to Tina Molodtsova for cnidom analysis and expert advice on taxonomy.

Author Contributions

Conceived and designed the experiments: FO CHM MVM JW. Performed the experiments: AV CD FO AD CHM MVM SI JW. Analyzed the data: AV CD FO SI GUN JW. Contributed reagents/materials/analysis tools: CHM MVM JW. Wrote the paper: CD GUN JW.

References

  1. 1. Griesbeck O (2004) Fluorescent proteins as sensors for cellular functions. Curr Opin Neurobiol 14: 636–641.O. Griesbeck2004Fluorescent proteins as sensors for cellular functions.Curr Opin Neurobiol14636641
  2. 2. Verkhusha VV, Lukyanov KA (2004) The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins. Nat Biotechnol 22: 289–296.VV VerkhushaKA Lukyanov2004The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins.Nat Biotechnol22289296
  3. 3. Wiedenmann J, Nienhaus GU (2006) Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family. Expert Rev Proteomics 3: 361–374.J. WiedenmannGU Nienhaus2006Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family.Expert Rev Proteomics3361374
  4. 4. Hanson GT, Aggeler R, Oglesbee D, Cannon M, Capaldi RA, et al. (2004) Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J Biol Chem 279: 13044–13053.GT HansonR. AggelerD. OglesbeeM. CannonRA Capaldi2004Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators.J Biol Chem2791304413053
  5. 5. Mocz G (2007) Fluorescent proteins and their use in marine biosciences, biotechnology, and proteomics. Mar Biotechnol 9: 305–328.G. Mocz2007Fluorescent proteins and their use in marine biosciences, biotechnology, and proteomics.Mar Biotechnol9305328
  6. 6. Shaner NC, Patterson GH, Davidson MW (2007) Advances in fluorescent protein technology. J Cell Sci 120: 4247–4260.NC ShanerGH PattersonMW Davidson2007Advances in fluorescent protein technology.J Cell Sci12042474260
  7. 7. Miyawaki A (2002) Green fluorescent protein-like proteins in reef Anthozoa animals. Cell Struct Funct 27: 343–347.A. Miyawaki2002Green fluorescent protein-like proteins in reef Anthozoa animals.Cell Struct Funct27343347
  8. 8. Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, et al. (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388: 882–887.A. MiyawakiJ. LlopisR. HeimJM McCafferyJA Adams1997Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.Nature388882887
  9. 9. Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67: 509–544.RY Tsien1998The green fluorescent protein.Annu Rev Biochem67509544
  10. 10. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263: 802–805.M. ChalfieY. TuG. EuskirchenWW WardDC Prasher1994Green fluorescent protein as a marker for gene expression.Science263802805
  11. 11. Heim R, Prasher DC, Tsien RY (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci U S A 91: 12501–12504.R. HeimDC PrasherRY Tsien1994Wavelength mutations and posttranslational autoxidation of green fluorescent protein.Proc Natl Acad Sci U S A911250112504
  12. 12. Ando R, Hama H, Yamamoto-Hino M, Mizuno H, Miyawaki A (2002) An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc Natl Acad Sci U S A 99: 12651–12656.R. AndoH. HamaM. Yamamoto-HinoH. MizunoA. Miyawaki2002An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.Proc Natl Acad Sci U S A991265112656
  13. 13. Henderson JN, Ai HW, Campbell RE, Remington SJ (2007) Structural basis for reversible photobleaching of a green fluorescent protein homologue. Proc Natl Acad Sci U S A 104: 6672–6677.JN HendersonHW AiRE CampbellSJ Remington2007Structural basis for reversible photobleaching of a green fluorescent protein homologue.Proc Natl Acad Sci U S A10466726677
  14. 14. Chudakov DM, Verkhusha VV, Staroverov DB, Souslova EA, Lukyanov S, et al. (2004) Photoswitchable cyan fluorescent protein for protein tracking. Nat Biotechnol 22: 1435–1439.DM ChudakovVV VerkhushaDB StaroverovEA SouslovaS. Lukyanov2004Photoswitchable cyan fluorescent protein for protein tracking.Nat Biotechnol2214351439
  15. 15. Ando R, Mizuno H, Miyawaki A (2004) Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306: 1370–1373.R. AndoH. MizunoA. Miyawaki2004Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting.Science30613701373
  16. 16. Chudakov DM, Belousov VV, Zaraisky AG, Novoselov VV, Staroverov DB, et al. (2003) Kindling fluorescent proteins for precise in vivo photolabeling. Nat Biotechnol 21: 191–194.DM ChudakovVV BelousovAG ZaraiskyVV NovoselovDB Staroverov2003Kindling fluorescent proteins for precise in vivo photolabeling.Nat Biotechnol21191194
  17. 17. Gurskaya NG, Verkhusha VV, Shcheglov AS, Staroverov DB, Chepurnykh TV, et al. (2006) Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat Biotechnol 24: 461–465.NG GurskayaVV VerkhushaAS ShcheglovDB StaroverovTV Chepurnykh2006Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light.Nat Biotechnol24461465
  18. 18. Oswald F, Schmitt F, Leutenegger A, Ivanchenko S, D'Angelo C, et al. (2007) Contributions of host and symbiont pigments to the coloration of reef corals. FEBS J 274: 1102–1109.F. OswaldF. SchmittA. LeuteneggerS. IvanchenkoC. D'Angelo2007Contributions of host and symbiont pigments to the coloration of reef corals.FEBS J27411021109
  19. 19. Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297: 1873–1877.GH PattersonJ. Lippincott-Schwartz2002A photoactivatable GFP for selective photolabeling of proteins and cells.Science29718731877
  20. 20. Tsutsui H, Karasawa S, Shimizu H, Nukina N, Miyawaki A (2005) Semi-rational engineering of a coral fluorescent protein into an efficient highlighter. EMBO Rep 6: 233–238.H. TsutsuiS. KarasawaH. ShimizuN. NukinaA. Miyawaki2005Semi-rational engineering of a coral fluorescent protein into an efficient highlighter.EMBO Rep6233238
  21. 21. Wiedenmann J, Ivanchenko S, Oswald F, Schmitt F, Rocker C, et al. (2004) EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc Natl Acad Sci U S A 101: 15905–15910.J. WiedenmannS. IvanchenkoF. OswaldF. SchmittC. Rocker2004EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.Proc Natl Acad Sci U S A1011590515910
  22. 22. Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, et al. (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313: 1642–1645.E. BetzigGH PattersonR. SougratOW LindwasserS. Olenych2006Imaging intracellular fluorescent proteins at nanometer resolution.Science31316421645
  23. 23. Hell SW (2003) Toward fluorescence nanoscopy. Nat Biotechnol 21: 1347–1355.SW Hell2003Toward fluorescence nanoscopy.Nat Biotechnol2113471355
  24. 24. Hofmann M, Eggeling C, Jakobs S, Hell SW (2005) Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc Natl Acad Sci U S A 102: 17565–17569.M. HofmannC. EggelingS. JakobsSW Hell2005Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins.Proc Natl Acad Sci U S A1021756517569
  25. 25. Egner A, Geisler C, von Middendorff C, Bock H, Wenzel D, et al. (2007) Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophys J 93: 3285–3290.A. EgnerC. GeislerC. von MiddendorffH. BockD. Wenzel2007Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters.Biophys J9332853290
  26. 26. Heim R, Tsien RY (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6: 178–182.R. HeimRY Tsien1996Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer.Curr Biol6178182
  27. 27. Shcherbo D, Merzlyak EM, Chepurnykh TV, Fradkov AF, Ermakova GV, et al. (2007) Bright far-red fluorescent protein for whole-body imaging. Nat Methods 4: 741–746.D. ShcherboEM MerzlyakTV ChepurnykhAF FradkovGV Ermakova2007Bright far-red fluorescent protein for whole-body imaging.Nat Methods4741746
  28. 28. Shaner NC, Lin MZ, McKeown MR, Steinbach PA, Hazelwood KL, et al. (2008) Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods 5: 545–551.NC ShanerMZ LinMR McKeownPA SteinbachKL Hazelwood2008Improving the photostability of bright monomeric orange and red fluorescent proteins.Nat Methods5545551
  29. 29. Bevis BJ, Glick BS (2002) Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat Biotechnol 20: 83–87.BJ BevisBS Glick2002Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed).Nat Biotechnol208387
  30. 30. Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, et al. (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci U S A 99: 7877–7882.RE CampbellO. TourAE PalmerPA SteinbachGS Baird2002A monomeric red fluorescent protein.Proc Natl Acad Sci U S A9978777882
  31. 31. Kogure T, Karasawa S, Araki T, Saito K, Kinjo M, et al. (2006) A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy. Nat Biotechnol 24: 577–581.T. KogureS. KarasawaT. ArakiK. SaitoM. Kinjo2006A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy.Nat Biotechnol24577581
  32. 32. Kredel S, Nienhaus K, Oswald F, Wolff M, Ivanchenko S, et al. (2008) Optimized and Far-Red-Emitting Variants of Fluorescent Protein eqFP611. Chem Biol 15: 224–233.S. KredelK. NienhausF. OswaldM. WolffS. Ivanchenko2008Optimized and Far-Red-Emitting Variants of Fluorescent Protein eqFP611.Chem Biol15224233
  33. 33. Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, et al. (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22: 1567–1572.NC ShanerRE CampbellPA SteinbachBN GiepmansAE Palmer2004Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.Nat Biotechnol2215671572
  34. 34. Yanushevich YG, Staroverov DB, Savitsky AP, Fradkov AF, Gurskaya NG, et al. (2002) A strategy for the generation of non-aggregating mutants of Anthozoa fluorescent proteins. FEBS Lett 511: 11–14.YG YanushevichDB StaroverovAP SavitskyAF FradkovNG Gurskaya2002A strategy for the generation of non-aggregating mutants of Anthozoa fluorescent proteins.FEBS Lett5111114
  35. 35. Alieva NO, Konzen KA, Field SF, Meleshkevitch EA, Hunt ME, et al. (2008) Diversity and evolution of coral fluorescent proteins. PLoS ONE 3: e2680.NO AlievaKA KonzenSF FieldEA MeleshkevitchME Hunt2008Diversity and evolution of coral fluorescent proteins.PLoS ONE3e2680
  36. 36. Shagin DA, Barsova EV, Yanushevich YG, Fradkov AF, Lukyanov KA, et al. (2004) GFP-like proteins as ubiquitous metazoan superfamily: Evolution of functional features and structural complexity. Mol Biol Evol 21: 841–850.DA ShaginEV BarsovaYG YanushevichAF FradkovKA Lukyanov2004GFP-like proteins as ubiquitous metazoan superfamily: Evolution of functional features and structural complexity.Mol Biol Evol21841850
  37. 37. Ip DTM, Chan S-H, Allen MD, Bycroft M, Wana DCC, et al. (2004) Crystallization and preliminary crystallographic analysis of a novel orange fluorescent protein from the Cnidaria tube anemone Cerianthus sp. Acta Crystallogr Sect D - Bio Crystallogr D60: 340–341.DTM IpS-H ChanMD AllenM. BycroftDCC Wana2004Crystallization and preliminary crystallographic analysis of a novel orange fluorescent protein from the Cnidaria tube anemone Cerianthus sp.Acta Crystallogr Sect D - Bio CrystallogrD60340341
  38. 38. Deheyn DD, Kubokawa K, McCarthy JK, Murakami A, Porrachia M, et al. (2007) Endogenous green fluorescent protein (GFP) in amphioxus. Biol Bull 213: 95–100.DD DeheynK. KubokawaJK McCarthyA. MurakamiM. Porrachia2007Endogenous green fluorescent protein (GFP) in amphioxus.Biol Bull21395100
  39. 39. Wiedenmann J (1997) The application of an orange fluorescent protein and further colored proteins and the corresponding genes from the species group Anemonia sp. (sulcata) Pennant, (Cnidaria, Anthozoa, Actinaria) in gene technology and molecular biology. pp. 1–18.J. Wiedenmann1997The application of an orange fluorescent protein and further colored proteins and the corresponding genes from the species group Anemonia sp. (sulcata) Pennant, (Cnidaria, Anthozoa, Actinaria) in gene technology and molecular biology.118(Die Anwendung eines orange fluoreszierenden Proteins und weiterer farbiger Proteine und der zugehörenden Gene aus der Artengruppe Anemonia sp. (sulcata) Pennant, (Cnidaria, Anthozoa, Actinaria) in Gentechnologie und Molekularbiologie.). Patent DE 197 18 640 Deutsches Patent- und Markenamt. Deutschland. (Die Anwendung eines orange fluoreszierenden Proteins und weiterer farbiger Proteine und der zugehörenden Gene aus der Artengruppe Anemonia sp. (sulcata) Pennant, (Cnidaria, Anthozoa, Actinaria) in Gentechnologie und Molekularbiologie.). Patent DE 197 18 640 Deutsches Patent- und Markenamt. Deutschland.
  40. 40. Karasawa S, Araki T, Yamamoto-Hino M, Miyawaki A (2003) A green-emitting fluorescent protein from Galaxeidae coral and its monomeric version for use in fluorescent labeling. J Biol Chem 278: 34167–34171.S. KarasawaT. ArakiM. Yamamoto-HinoA. Miyawaki2003A green-emitting fluorescent protein from Galaxeidae coral and its monomeric version for use in fluorescent labeling.J Biol Chem2783416734171
  41. 41. Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, et al. (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol 17: 969–973.MV MatzAF FradkovYA LabasAP SavitskyAG Zaraisky1999Fluorescent proteins from nonbioluminescent Anthozoa species.Nat Biotechnol17969973
  42. 42. Wiedenmann J, Elke C, Spindler KD, Funke W (2000) Cracks in the beta-can: fluorescent proteins from Anemonia sulcata (Anthozoa, Actinaria). Proc Natl Acad Sci USA 97: 14091–14096.J. WiedenmannC. ElkeKD SpindlerW. Funke2000Cracks in the beta-can: fluorescent proteins from Anemonia sulcata (Anthozoa, Actinaria).Proc Natl Acad Sci USA971409114096
  43. 43. Wiedenmann J, Ivanchenko S, Oswald F, Nienhaus GU (2004) Identification of GFP-like proteins in nonbioluminescent, azooxanthellate anthozoa opens new perspectives for bioprospecting. Mar Biotechnol 6: 270–277.J. WiedenmannS. IvanchenkoF. OswaldGU Nienhaus2004Identification of GFP-like proteins in nonbioluminescent, azooxanthellate anthozoa opens new perspectives for bioprospecting.Mar Biotechnol6270277
  44. 44. Wiedenmann J, Schenk A, Rocker C, Girod A, Spindler KD, et al. (2002) A far-red fluorescent protein with fast maturation and reduced oligomerization tendency from Entacmaea quadricolor (Anthozoa, Actinaria). Proc Natl Acad Sci U S A 99: 11646–11651.J. WiedenmannA. SchenkC. RockerA. GirodKD Spindler2002A far-red fluorescent protein with fast maturation and reduced oligomerization tendency from Entacmaea quadricolor (Anthozoa, Actinaria).Proc Natl Acad Sci U S A991164611651
  45. 45. Kawaguti S (1969) Effect of the green fluorescent pigment on the productivity of the reef corals. Micronesica 5: 313.S. Kawaguti1969Effect of the green fluorescent pigment on the productivity of the reef corals.Micronesica5313
  46. 46. Field SF, Bulina MY, Kelmanson IV, Bielawski JP, Matz MV (2006) Adaptive evolution of multicolored fluorescent proteins in reef-building corals. J Mol Evol 62: 332–U315.SF FieldMY BulinaIV KelmansonJP BielawskiMV Matz2006Adaptive evolution of multicolored fluorescent proteins in reef-building corals.J Mol Evol62332U315
  47. 47. Salih A, Larkum A, Cox G, Kuhl M, Hoegh-Guldberg O (2000) Fluorescent pigments in corals are photoprotective. Nature 408: 850–853.A. SalihA. LarkumG. CoxM. KuhlO. Hoegh-Guldberg2000Fluorescent pigments in corals are photoprotective.Nature408850853
  48. 48. Schnitzler CE, Keenan RJ, McCord R, Matysik A, Christianson LM, et al. (2008) Spectral Diversity of Fluorescent Proteins from the Anthozoan Corynactis californica. Mar Biotechnol. CE SchnitzlerRJ KeenanR. McCordA. MatysikLM Christianson2008Spectral Diversity of Fluorescent Proteins from the Anthozoan Corynactis californica.Mar Biotechnol
  49. 49. Wiedenmann J, Röcker C, Funke W (1999) The morphs of Anemonia aff. sulcata (Cnidaria, Anthozoa) in particular consideration of the ectodermal pigments. Verhandlungen der Gesellschaft für Ökologie 29: 497–503.J. WiedenmannC. RöckerW. Funke1999The morphs of Anemonia aff. sulcata (Cnidaria, Anthozoa) in particular consideration of the ectodermal pigments.Verhandlungen der Gesellschaft für Ökologie29497503
  50. 50. D'Angelo C, Denzel A, Vogt A, Matz MV, Oswald F, et al. (2008) Blue light regulation of host pigment in reef-building corals. Mar Ecol Prog Ser 364: 97–106.C. D'AngeloA. DenzelA. VogtMV MatzF. Oswald2008Blue light regulation of host pigment in reef-building corals.Mar Ecol Prog Ser36497106
  51. 51. Leutenegger A, Kredel S, Gundel S, D'Angelo C, Salih A, et al. (2007) Analysis of fluorescent and non-fluorescent sea anemones from the Mediterranean Sea during a bleaching event. J Exp Mar Biol Ecol 353: 221–234.A. LeuteneggerS. KredelS. GundelC. D'AngeloA. Salih2007Analysis of fluorescent and non-fluorescent sea anemones from the Mediterranean Sea during a bleaching event.J Exp Mar Biol Ecol353221234
  52. 52. Wiedenmann J, Kraus P, Funke W, Vogel W (2000) The relationship between different morphs of Anemonia aff. Sulcata evaluated by DNA fingerprinting (Anthozoa, Actinaria). Ophelia 52: 57–64.J. WiedenmannP. KrausW. FunkeW. Vogel2000The relationship between different morphs of Anemonia aff. Sulcata evaluated by DNA fingerprinting (Anthozoa, Actinaria).Ophelia525764
  53. 53. Kelmanson IV, Matz MV (2003) Molecular basis and evolutionary origins of color diversity in great star coral Montastraea cavernosa (Scleractinia : Faviida). Mol Biol Evol 20: 1125–1133.IV KelmansonMV Matz2003Molecular basis and evolutionary origins of color diversity in great star coral Montastraea cavernosa (Scleractinia : Faviida).Mol Biol Evol2011251133
  54. 54. Shepard AN, Theroux RB, Cooper RA, Uzmann JR (1986) Ecology of Ceriantharia (Colenterata, Anthozoa) of the northwest Atlantic from Cape Hatteras to Nova Scotia. Fish Bull 84: 625–646.AN ShepardRB TherouxRA CooperJR Uzmann1986Ecology of Ceriantharia (Colenterata, Anthozoa) of the northwest Atlantic from Cape Hatteras to Nova Scotia.Fish Bull84625646
  55. 55. Carlgren O, Hedgpeth JW (1952) Actiniaria, Zoantharia and Ceriantharia from Shallow Water in the northwestern Gulf of Mexico. Publications of the Institute of Marine Science (University of Texas) 2: 143–172.O. CarlgrenJW Hedgpeth1952Actiniaria, Zoantharia and Ceriantharia from Shallow Water in the northwestern Gulf of Mexico.Publications of the Institute of Marine Science (University of Texas)2143172
  56. 56. den Hartog JC (1977) Description of two new Ceriantharia from the Caribbean region, Pachycerianthus curacaoensis n. sp. and Arachnanthus nocturnus n.sp., with a discussion of the cnidom and of the classification of the Ceriantharia. Zoologische Mededelingen, Leiden 51: 211–242.JC den Hartog1977Description of two new Ceriantharia from the Caribbean region, Pachycerianthus curacaoensis n. sp. and Arachnanthus nocturnus n.sp., with a discussion of the cnidom and of the classification of the Ceriantharia.Zoologische Mededelingen, Leiden51211242
  57. 57. Molodtsova TN (2003) On “Isarachnanthus” from central Atlantic and Caribbean region with notes on “Isarachnactis lobiancoi” (Carlgren, 1912). Zoologische Verhandelingen 345: 249–255.TN Molodtsova2003On “Isarachnanthus” from central Atlantic and Caribbean region with notes on “Isarachnactis lobiancoi” (Carlgren, 1912).Zoologische Verhandelingen345249255
  58. 58. Molodtsova TN (2004) On the taxonomy and presumable evolutionary pathways of planktonic larvae of Ceriantharia (Anthozoa, Cnidaria). Hydrobiologia 530/531: 261–266.TN Molodtsova2004On the taxonomy and presumable evolutionary pathways of planktonic larvae of Ceriantharia (Anthozoa, Cnidaria).Hydrobiologia530/531261266
  59. 59. Nienhaus K, Renzi F, Vallone B, Wiedenmann J, Nienhaus GU (2006) Exploring chromophore-protein interactions in fluorescent protein cmFP512 from Cerianthus membranaceus: X-ray structure analysis and optical spectroscopy. Biochemistry 45: 12942–12953.K. NienhausF. RenziB. ValloneJ. WiedenmannGU Nienhaus2006Exploring chromophore-protein interactions in fluorescent protein cmFP512 from Cerianthus membranaceus: X-ray structure analysis and optical spectroscopy.Biochemistry451294212953
  60. 60. Nienhaus K, Renzi F, Vallone B, Wiedenmann J, Nienhaus GU (2006) Chromophore-protein interactions in the anthozoan green fluorescent protein asFP499. Biophys J 91: 4210–4220.K. NienhausF. RenziB. ValloneJ. WiedenmannGU Nienhaus2006Chromophore-protein interactions in the anthozoan green fluorescent protein asFP499.Biophys J9142104220
  61. 61. Wiedenmann J, Vallone B, Renzi F, Nienhaus K, Ivanchenko S, et al. (2005) Red fluorescent protein eqFP611 and its genetically engineered dimeric variants. J Biomed Opt 10: 14003.J. WiedenmannB. ValloneF. RenziK. NienhausS. Ivanchenko2005Red fluorescent protein eqFP611 and its genetically engineered dimeric variants.J Biomed Opt1014003
  62. 62. Yarbrough D, Wachter RM, Kallio K, Matz MV, Remington SJ (2001) Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-A resolution. Proc Natl Acad Sci U S A 98: 462–467.D. YarbroughRM WachterK. KallioMV MatzSJ Remington2001Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-A resolution.Proc Natl Acad Sci U S A98462467
  63. 63. Ip DT, Wong KB, Wan DC (2007) Characterization of novel orange fluorescent protein cloned from cnidarian tube anemone Cerianthus sp. Mar Biotechnol 9: 469–478.DT IpKB WongDC Wan2007Characterization of novel orange fluorescent protein cloned from cnidarian tube anemone Cerianthus sp.Mar Biotechnol9469478
  64. 64. Petersen J, Wilmann PG, Beddoe T, Oakley AJ, Devenish RJ, et al. (2003) The 2.0-angstrom crystal structure of eqFP611, a far red fluorescent protein from the sea anemone Entacmaea quadricolor. J Biol Chem 278: 44626–44631.J. PetersenPG WilmannT. BeddoeAJ OakleyRJ Devenish2003The 2.0-angstrom crystal structure of eqFP611, a far red fluorescent protein from the sea anemone Entacmaea quadricolor.J Biol Chem2784462644631
  65. 65. Shu X, Shaner NC, Yarbrough CA, Tsien RY, Remington SJ (2006) Novel chromophores and buried charges control color in mFruits. Biochemistry 45: 9639–9647.X. ShuNC ShanerCA YarbroughRY TsienSJ Remington2006Novel chromophores and buried charges control color in mFruits.Biochemistry4596399647
  66. 66. Niwa H, Inouye S, Hirano T, Matsuno T, Kojima S, et al. (1996) Chemical nature of the light emitter of the Aequorea green fluorescent protein. Proc Natl Acad Sci U S A 93: 13617–13622.H. NiwaS. InouyeT. HiranoT. MatsunoS. Kojima1996Chemical nature of the light emitter of the Aequorea green fluorescent protein.Proc Natl Acad Sci U S A931361713622
  67. 67. Ward WW (2005) Biochemical and Physical Properties of Green Fluorescent Protein. In: Chalfie M, Kain SR, editors. Green fluorescent protein: properties, applications and protocols. 2nd ed. Hoboken, USA: Wiley and Sons. pp. 39–65.WW Ward2005Biochemical and Physical Properties of Green Fluorescent Protein.M. ChalfieSR KainGreen fluorescent protein: properties, applications and protocols. 2nd edHoboken, USAWiley and Sons3965
  68. 68. Chattoraj M, King BA, Bublitz GU, Boxer SG (1996) Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer. Proc Natl Acad Sci U S A 93: 8362–8367.M. ChattorajBA KingGU BublitzSG Boxer1996Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer.Proc Natl Acad Sci U S A9383628367
  69. 69. Andresen M, Stiel AC, Trowitzsch S, Weber G, Eggeling C, et al. (2007) Structural basis for reversible photoswitching in Dronpa. Proc Natl Acad Sci U S A 104: 13005–13009.M. AndresenAC StielS. TrowitzschG. WeberC. Eggeling2007Structural basis for reversible photoswitching in Dronpa.Proc Natl Acad Sci U S A1041300513009
  70. 70. Habuchi S, Dedecker P, Hotta J, Flors C, Ando R, et al. (2006) Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching. Photochem Photobiol Sci 5: 567–576.S. HabuchiP. DedeckerJ. HottaC. FlorsR. Ando2006Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching.Photochem Photobiol Sci5567576
  71. 71. Stiel AC, Trowitzsch S, Weber G, Andresen M, Eggeling C, et al. (2007) 1.8 A bright-state structure of the reversibly switchable fluorescent protein Dronpa guides the generation of fast switching variants. Biochem J 402: 35–42.AC StielS. TrowitzschG. WeberM. AndresenC. Eggeling20071.8 A bright-state structure of the reversibly switchable fluorescent protein Dronpa guides the generation of fast switching variants.Biochem J4023542
  72. 72. Baird GS, Zacharias DA, Tsien RY (2000) Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci U S A 97: 11984–11989.GS BairdDA ZachariasRY Tsien2000Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral.Proc Natl Acad Sci U S A971198411989
  73. 73. Remington SJ (2006) Fluorescent proteins: maturation, photochemistry and photophysics. Curr Opin Struct Biol 16: 714–721.SJ Remington2006Fluorescent proteins: maturation, photochemistry and photophysics.Curr Opin Struct Biol16714721
  74. 74. Verkhusha VV, Chudakov DM, Gurskaya NG, Lukyanov S, Lukyanov KA (2004) Common pathway for the red chromophore formation in fluorescent proteins and chromoproteins. Chem Biol 11: 845–854.VV VerkhushaDM ChudakovNG GurskayaS. LukyanovKA Lukyanov2004Common pathway for the red chromophore formation in fluorescent proteins and chromoproteins.Chem Biol11845854
  75. 75. Hell SW (2007) Far-field optical nanoscopy. Science 316: 1153–1158.SW Hell2007Far-field optical nanoscopy.Science31611531158
  76. 76. Shroff H, Galbraith CG, Galbraith JA, White H, Gillette J, et al. (2007) Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. Proc Natl Acad Sci U S A 104: 20308–20313.H. ShroffCG GalbraithJA GalbraithH. WhiteJ. Gillette2007Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes.Proc Natl Acad Sci U S A1042030820313
  77. 77. Flors C, Hotta J, Uji-i H, Dedecker P, Ando R, et al. (2007) A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants. J Am Chem Soc 129: 13970–13977.C. FlorsJ. HottaH. Uji-iP. DedeckerR. Ando2007A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants.J Am Chem Soc1291397013977
  78. 78. Hess ST, Girirajan TP, Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91: 4258–4272.ST HessTP GirirajanMD Mason2006Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.Biophys J9142584272
  79. 79. Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3: 793–795.MJ RustM. BatesX. Zhuang2006Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).Nat Methods3793795
  80. 80. Juette MF, Gould TJ, Lessard MD, Mlodzianoski MJ, Nagpure BS, et al. (2008) Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat Methods 5: 527–529.MF JuetteTJ GouldMD LessardMJ MlodzianoskiBS Nagpure2008Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples.Nat Methods5527529
  81. 81. Patterson GH, Knobel SM, Sharif WD, Kain SR, Piston DW (1997) Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys J 73: 2782–2790.GH PattersonSM KnobelWD SharifSR KainDW Piston1997Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy.Biophys J7327822790