Reef-building corals farm and feed on their photosynthetic symbionts.

Coral reefs are highly diverse ecosystems that thrive in nutrient-poor waters, a phenomenon frequently referred to as the Darwin paradox1. The energy demand of coral animal hosts can often be fully met by the excess production of carbon-rich photosynthates by their algal symbionts2,3. However, the understanding of mechanisms that enable corals to acquire the vital nutrients nitrogen and phosphorus from their symbionts is incomplete4-9. Here we show, through a series of long-term experiments, that the uptake of dissolved inorganic nitrogen and phosphorus by the symbionts alone is sufficient to sustain rapid coral growth. Next, considering the nitrogen and phosphorus budgets of host and symbionts, we identify that these nutrients are gathered through symbiont 'farming' and are translocated to the host by digestion of excess symbiont cells. Finally, we use a large-scale natural experiment in which seabirds fertilize some reefs but not others, to show that the efficient utilization of dissolved inorganic nutrients by symbiotic corals established in our laboratory experiments has the potential to enhance coral growth in the wild at the ecosystem level. Feeding on symbionts enables coral animals to tap into an important nutrient pool and helps to explain the evolutionary and ecological success of symbiotic corals in nutrient-limited waters.

Ancestral genetic diversity associated with the rapid spread of stress-tolerant coral symbionts in response to Holocene climate change.

Coral communities in the Persian/Arabian Gulf (PAG) withstand unusually high salinity levels and regular summer temperature maxima of up to ∼35 °C that kill conspecifics elsewhere. Due to the recent formation of the PAG and its subsequent shift to a hot climate, these corals have had only <6,000 y to adapt to these extreme conditions and can therefore inform on how coral reefs may respond to global warming. One key to coral survival in the world's warmest reefs are symbioses with a newly discovered alga,Symbiodinium thermophilum Currently, it is unknown whether this symbiont originated elsewhere or emerged from unexpectedly fast evolution catalyzed by the extreme environment. Analyzing genetic diversity of symbiotic algae across >5,000 km of the PAG, the Gulf of Oman, and the Red Sea coastline, we show thatS. thermophilumis a member of a highly diverse, ancient group of symbionts cryptically distributed outside the PAG. We argue that the adjustment to temperature extremes by PAG corals was facilitated by the positive selection of preadapted symbionts. Our findings suggest that maintaining the largest possible pool of potentially stress-tolerant genotypes by protecting existing biodiversity is crucial to promote rapid adaptation to present-day climate change, not only for coral reefs, but for ecosystems in general.

Validation of the binary designation Symbiodinium thermophilum (Dinophyceae).

The binary designation Symbiodinium thermophilum was invalid due to the absence of an illustration as required by Article 44.2 of the ICN. Herein, it is validated. This species is the most common symbiont in reef corals in the southern Persian/Arabian Gulf, the world's hottest body of water sustaining reef coral growth.

FRET-Mediated Long-Range Wavelength Transformation by Photoconvertible Fluorescent Proteins as an Efficient Mechanism to Generate Orange-Red Light in Symbiotic Deep Water Corals.

Photoconvertible fluorescent proteins (pcRFPs) are a group of fluorophores that undergo an irreversible green-to-red shift in emission colour upon irradiation with near-ultraviolet (near-UV) light. Despite their wide application in biotechnology, the high-level expression of pcRFPs in mesophotic and depth-generalist coral species currently lacks a biological explanation. Additionally, reduced penetration of near-UV wavelengths in water poses the question whether light-driven photoconversion is relevant in the mesophotic zone, or whether a different mechanism is involved in the post-translational pigment modification in vivo. Here, we show in a long-term mesocosm experiment that photoconversion in vivo is entirely dependent on near-UV wavelengths. However, a near-UV intensity equivalent to the mesophotic underwater light field at 80 m depth is sufficient to drive the process in vitro, suggesting that photoconversion can occur near the lower distribution limits of these corals. Furthermore, live coral colonies showed evidence of efficient Förster Resonance Energy Transfer (FRET). Our simulated mesophotic light field maintained the pcRFP pool in a partially photoconverted state in vivo, maximising intra-tetrameric FRET and creating a long-range wavelength conversion system with higher quantum yield than other native RFPs. We hypothesise that efficient conversion of blue wavelengths, abundant at depth, into orange-red light could constitute an adaptation of corals to life in light-limited environments.

RITA, a novel modulator of Notch signalling, acts via nuclear export of RBP-J.

The evolutionarily conserved Notch signal transduction pathway regulates fundamental cellular processes during embryonic development and in the adult. Ligand binding induces presenilin-dependent cleavage of the receptor and a subsequent nuclear translocation of the Notch intracellular domain (NICD). In the nucleus, NICD binds to the recombination signal sequence-binding protein J (RBP-J)/CBF-1 transcription factor to induce expression of Notch target genes. Here, we report the identification and functional characterization of RBP-J interacting and tubulin associated (RITA) (C12ORF52) as a novel RBP-J/CBF-1-interacting protein. RITA is a highly conserved 36 kDa protein that, most interestingly, binds to tubulin in the cytoplasm and shuttles rapidly between cytoplasm and nucleus. This shuttling RITA exports RBP-J/CBF-1 from the nucleus. Functionally, we show that RITA can reverse a Notch-induced loss of primary neurogenesis in Xenopus laevis. Furthermore, RITA is able to downregulate Notch-mediated transcription. Thus, we propose that RITA acts as a negative modulator of the Notch signalling pathway, controlling the level of nuclear RBP-J/CBF-1, where its amounts are limiting.

Improving FRET dynamic range with bright green and red fluorescent proteins.

A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action-potential firing and RhoA activation in growth cones.

Fluorescent protein-mediated colour polymorphism in reef corals: multicopy genes extend the adaptation/acclimatization potential to variable light environments.

The genomic framework that enables corals to adjust to unfavourable conditions is crucial for coral reef survival in a rapidly changing climate. We have explored the striking intraspecific variability in the expression of coral pigments from the green fluorescent protein (GFP) family to elucidate the genomic basis for the plasticity of stress responses among reef corals. We show that multicopy genes can greatly increase the dynamic range over which corals can modulate transcript levels in response to the light environment. Using the red fluorescent protein amilFP597 in the coral Acropora millepora as a model, we demonstrate that its expression increases with light intensity, but both the minimal and maximal gene transcript levels vary markedly among colour morphs. The pigment concentration in the tissue of different morphs is strongly correlated with the number of gene copies with a particular promoter type. These findings indicate that colour polymorphism in reef corals can be caused by the environmentally regulated expression of multicopy genes. High-level expression of amilFP597 is correlated with reduced photodamage of zooxanthellae under acute light stress, supporting a photoprotective function of this pigment. The cluster of light-regulated pigment genes can enable corals to invest either in expensive high-level pigmentation, offering benefits under light stress, or to rely on low tissue pigment concentrations and use the conserved resources for other purposes, which is preferable in less light-exposed environments. The genomic framework described here allows corals to pursue different strategies to succeed in habitats with highly variable light stress levels. In summary, our results suggest that the intraspecific plasticity of reef corals' stress responses is larger than previously thought.

Polymersomes prepared from thermoresponsive fluorescent protein-polymer bioconjugates: capture of and report on drug and protein payloads.

Polymersomes provide a good platform for targeted drug delivery and the creation of complex (bio)catalytically active systems for research in synthetic biology. To realize these applications requires both spatial control over the encapsulation components in these polymersomes and a means to report where the components are in the polymersomes. To address these twin challenges, we synthesized the protein-polymer bioconjugate PNIPAM-b-amilFP497 composed of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and a green-fluorescent protein variant (amilFP497). Above 37 °C, this bioconjugate forms polymersomes that can (co-)encapsulate the fluorescent drug doxorubicin and the fluorescent light-harvesting protein phycoerythrin 545 (PE545). Using fluorescence lifetime imaging microscopy and Förster resonance energy transfer (FLIM-FRET), we can distinguish the co-encapsulated PE545 protein inside the polymersome membrane while doxorubicin is found both in the polymersome core and membrane.

One-pot synthesis of high molecular weight synthetic heteroprotein dimers driven by charge complementarity electrostatic interactions.

Despite the importance of protein dimers and dimerization in biology, the formation of protein dimers through synthetic covalent chemistry has not found widespread use. In the case of maleimide-cysteine-based dimerization of proteins, we show here that when the proteins have the same charge, dimerization appears to be inherently difficult with yields around 1% or less, regardless of the nature of the spacer used or whether homo- or heteroprotein dimers are targeted. In contrast, if the proteins have opposing (complementary) charges, the formation of heteroprotein dimers proceeds much more readily, and in the case of one high molecular weight (>80 kDa) synthetic dimer between cytochrome c and bovine serum albumin, a 30% yield of the purified, isolated dimer was achieved. This represents at least a 30-fold increase in yield for protein dimers formed from proteins with complementary charges, compared to when the proteins have the same charge, under otherwise similar conditions. These results illustrate the role of ionic supramolecular interactions in controlling the reactivity of proteins toward bis-functionalized spacers. The strategy here for effective synthetic dimerization of proteins could be very useful for developing novel approaches to study the important role of protein-protein interactions in chemical biology.

A photoactivatable marker protein for pulse-chase imaging with superresolution.

IrisFP is a photoactivatable fluorescent protein that combines irreversible photoconversion from a green- to a red-emitting form with reversible photoswitching between a fluorescent and a nonfluorescent state in both forms. Here we introduce a monomeric variant, mIrisFP, and demonstrate how its multiple photoactivation modes can be used for pulse-chase experiments combined with subdiffraction-resolution imaging in living cells by using dual-color photoactivation localization microscopy (PALM).

Green fluorescent protein-like pigments optimise the internal light environment in symbiotic reef-building corals.

Pigments homologous to the green fluorescent protein (GFP) have been proposed to fine-tune the internal light microclimate of corals, facilitating photoacclimation of photosynthetic coral symbionts (Symbiodiniaceae) to life in different reef habitats and environmental conditions. However, direct measurements of the in vivo light conditions inside the coral tissue supporting this conclusion are lacking. Here, we quantified the intra-tissue spectral light environment of corals expressing GFP-like proteins from widely different light regimes. We focus on: (1) photoconvertible red fluorescent proteins (pcRFPs), thought to enhance photosynthesis in mesophotic habitats via wavelength conversion, and (2) chromoproteins (CPs), which provide photoprotection to the symbionts in shallow water via light absorption. Optical microsensor measurements indicated that both pigment groups strongly alter the coral intra-tissue light environment. Estimates derived from light spectra measured in pcRFP-containing corals showed that fluorescence emission can contribute to >50% of orange-red light available to the photosynthetic symbionts at mesophotic depths. We further show that upregulation of pink CPs in shallow-water corals during bleaching leads to a reduction of orange light by 10-20% compared to low-CP tissue. Thus, screening by CPs has an important role in mitigating the light-enhancing effect of coral tissue scattering and skeletal reflection during bleaching. Our results provide the first experimental quantification of the importance of GFP-like proteins in fine-tuning the light microclimate of corals during photoacclimation.

Optical Feedback Loop Involving Dinoflagellate Symbiont and Scleractinian Host Drives Colorful Coral Bleaching.

Coral bleaching, caused by the loss of brownish-colored dinoflagellate photosymbionts from the host tissue of reef-building corals, is a major threat to reef survival. Occasionally, bleached corals become exceptionally colorful rather than white. These colors derive from photoprotective green fluorescent protein (GFP)-like pigments produced by the coral host. There is currently no consensus regarding what causes colorful bleaching events and what the consequences for the corals are. Here, we document that colorful bleaching events are a recurring phenomenon in reef regions around the globe. Our analysis of temperature conditions associated with colorful bleaching events suggests that corals develop extreme coloration within 2 to 3 weeks after exposure to mild or temporary heat stress. We demonstrate that the increase of light fluxes in symbiont-depleted tissue promoted by reflection of the incident light from the coral skeleton induces strong expression of the photoprotective coral host pigments. We describe an optical feedback loop involving both partners of the association, discussing that the mitigation of light stress offered by host pigments could facilitate recolonization of bleached tissue by symbionts. Our data indicate that colorful bleaching has the potential to identify local environmental factors, such as nutrient stress, that can exacerbate the impact of elevated temperatures on corals, to indicate the severity of heat stress experienced by corals and to gauge their post-stress recovery potential. VIDEO ABSTRACT.

Fluorescent proteins for live cell imaging: opportunities, limitations, and challenges.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria can be used as a genetically encoded fluorescence marker due to its autocatalytic formation of the chromophore. In recent years, numerous GFP-like proteins with emission colors ranging from cyan to red were discovered in marine organisms. Their diverse molecular properties enabled novel approaches in live cell imaging but also impose certain limitations on their applicability as markers. In this review, we give an overview of key structural and functional properties of fluorescent proteins that should be considered when selecting a marker protein for a particular application and also discuss challenges that lie ahead in the further optimization of the glowing probes.

Structure, dynamics and optical properties of fluorescent proteins: perspectives for marker development.

GFP-like proteins, originally cloned from marine animals, are genetically encoded fluorescence markers that have become indispensable tools for the life sciences. The search for GFP-like proteins with novel and improved properties is still ongoing, however, driven by the persistent need for advanced and specialized fluorescence labels for cellular imaging. Overall, the structures of these proteins are similar, but considerable variations have been found in the covalent structures and stereochemistry of the fluorophore, which govern essential optical properties such as the absorption/emission wavelengths. Moreover, as the fluorophore-enclosing cavity forms its solvation shell, it can also have a significant effect on the absorption/emission wavelengths and the brightness of the fluorophore. Most exciting are recent developments of photoactivatable fluorescence markers which change their color and/or intensity upon irradiation with light of specific wavelengths. A detailed understanding of the structure and dynamics of GFP-like proteins greatly aids in the rational engineering of advanced fluorescence marker proteins. Herein, we review our present knowledge of the structural diversity of GFP-like proteins and discuss how structure and dynamics govern their optical properties, with an emphasis on red fluorescent proteins.

Optimized and far-red-emitting variants of fluorescent protein eqFP611.

Fluorescent proteins (FPs) emitting in the far-red region of the spectrum are highly advantageous for whole-body imaging applications because scattering and absorption of long-wavelength light is markedly reduced in tissue. We characterized variants of the red fluorescent protein eqFP611 with bright fluorescence emission shifted up to 639 nm. The additional red shift is caused by a trans-cis isomerization of the chromophore. The equilibrium between the trans and cis conformations is strongly influenced by amino acid residues 143 and 158. Pseudo monomeric tags were obtained by further genetic engineering. For the red chromophores of eqFP611 variants, molar extinction coefficients of up to approximately 150,000 were determined by an approach that is not affected by the presence of molecules with nonfunctional red chromophores. The bright fluorescence makes the red-shifted eqFP611 variants promising lead structures for the development of near-infrared fluorescent markers. The red fluorescent proteins performed well in cell biological applications, including two-photon imaging.

Trans-cis isomerization is responsible for the red-shifted fluorescence in variants of the red fluorescent protein eqFP611.

An important class of red fluorescent proteins (RFPs) feature a 2-iminomethyl-5-(4-hydroxybenzylidene)imidazolinone chromophore. Among these proteins, eqFP611 has the chromophore in a coplanar trans orientation, whereas the cis isomer is preferred by other RFPs such as DsRed and its variants. In the photoactivatable protein asFP595, the chromophore can even be switched from the nonfluorescent trans to the fluorescent cis state by light. By using X-ray crystallography, we have determined the structure of dimeric eqFP611 at high resolution (up to 1.1 A). In the far-red emitting eqFP611 variant d2RFP630, which carries an additional Asn143Ser mutation, the chromophore resides predominantly (approximately 80%) in the cis isomeric state, and in RFP639, which has Asn143Ser and Ser158Cys mutations, the chromophore is found completely in the cis form. The pronounced red shift of excitation and emission maxima of RFP639 can thus unambiguously be assigned to trans-cis isomerization of the chromophore. Among RFPs, eqFP611 is thus unique because its chromophore is highly fluorescent in both the cis and trans isomeric forms.

Cell-based assays in practice: cell markers from autofluorescent proteins of the GFP-family.

The more recently discovered anthozoan fluorescent proteins (FPs) and the classic Aequorea victoria Green Fluorescent Protein (avGFP) as well as their derivatives have become versatile tools as live cell markers in fluorescence microscopy. In this review, we show the use of these FPs in drug discovery assays. Assay examples are given for the application of FPs in multiplexed imaging, as photosensitizers, as fluorescent timers, as pulse-chase labels and for robotically integrated compound testing. The development of fast microscopic imaging devices has enabled the application of automated fluorescence microscopy combined with image analysis to pharmaceutical high throughput drug discovery assays, generally referred to as High Content Screening (HCS).

Symbiosis: High-Carb Diet of Reef Corals as Seen from Space.

High levels of phytoplankton visible in satellite imagery are correlated with an increased uptake of carbon compounds by corals. This suggests that corals rely less on carbon production by photosynthetic symbionts when other resources are plentiful, and that the changes in the acquisition mode of carbon can be inferred by remote-sensing techniques.

It's cheap to be colorful. Anthozoans show a slow turnover of GFP-like proteins.

Pigments homologous to the green fluorescent protein (GFP) contribute up to approximately 14% of the soluble protein content of many anthozoans. Maintenance of such high tissue levels poses a severe energetic penalty to the animals if protein turnover is fast. To address this as yet unexplored issue, we established that the irreversible green-to-red conversion of the GFP-like pigments from the reef corals Montastrea cavernosa (mcavRFP) and Lobophyllia hemprichii (EosFP) is driven by violet-blue radiation in vivo and in situ. In the absence of photoconverting light, we subsequently tracked degradation of the red-converted forms of the two proteins in coral tissue using in vivo spectroscopy and immunochemical detection of the post-translational peptide backbone modification. The pigments displayed surprisingly slow decay rates, characterized by half-lives of approximately 20 days. The slow turnover of GFP-like proteins implies that the associated energetic costs for being colorful are comparatively low. Moreover, high in vivo stability makes GFP-like proteins suitable for functions requiring high pigment concentrations, such as photoprotection.