International Journal of Molecular Sciences

Article Functioning of Fluorescent Proteins in Aggregates in Species and in Recombinant Artificial Models

Natalia V. Povarova 1,2, Natalia D. Petri 1, Anna E. Blokhina 1, Alexey M. Bogdanov 1,2, Nadya G. Gurskaya 1,2 and Konstantin A. Lukyanov 1,2,* 1 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia; [email protected] (N.V.P.); [email protected] (N.D.P.); [email protected] (A.E.B.); [email protected] (A.M.B.); [email protected] (N.G.G.) 2 Nizhny Novgorod State Medical Academy, 10/1 Minin and Pozharsky Sq., 603005 Nizhny Novgorod, Russia * Correspondence: [email protected]; Tel.: +7-916-561-6221

Received: 31 May 2017; Accepted: 10 July 2017; Published: 12 July 2017

Abstract: Despite great advances in practical applications of fluorescent proteins (FPs), their natural function is poorly understood. FPs display complex spatio-temporal expression patterns in living Anthozoa coral polyps. Here we applied confocal microscopy, specifically, the fluorescence recovery after photobleaching (FRAP) technique to analyze intracellular localization and mobility of endogenous FPs in live tissues. We observed three distinct types of protein distributions in living tissues. One type of distribution, characteristic for Anemonia, Discosoma and Zoanthus, is free, highly mobile cytoplasmic localization. Another pattern is seen in FPs localized to numerous intracellular vesicles, observed in . The third most intriguing type of intracellular localization is with respect to the spindle-shaped aggregates and lozenge crystals several micrometers in size observed in Zoanthus samples. No protein mobility within those structures was detected by FRAP. This finding encouraged us to develop artificial aggregating FPs. We constructed “trio-FPs” consisting of three tandem copies of tetrameric FPs and demonstrated that they form multiple bright foci upon expression in mammalian cells. High brightness of the aggregates is advantageous for early detection of weak promoter activities. Simultaneously, larger aggregates can induce significant cytostatic and cytotoxic effects and thus such tags are not suitable for long-term and high-level expression.

Keywords: green fluorescent protein (GFP); red fluorescent protein (RFP); FRAP; aggregation; gene expression marker

1. Introduction To date, proteins of the green fluorescent protein (GFP) family have been found in species of Hydrozoa, Anthozoa, Copepoda, and Cephalochordata [1–4]. Such dispersed distribution throughout distant taxa suggests that GFP-like proteins originated at very early stages of evolution of kingdom [3]. GFP-like proteins display different colors, the most common among the natural proteins being green, but also cyan, yellow, orange-red fluorescent, as well as non-fluorescent colors of various hues. GFP-like proteins from Anthozoa species possess the widest color diversity compared to other groups; a particular specimen typically expresses several proteins of different colors [2,5,6]. Fluorescent proteins (FPs) represent an indispensable tool in modern biology, enabling protein and cell labeling in live models [7]. Practical interest in development of new improved FPs and FP-based methods has stimulated extensive research on FP structure, biochemistry, photochemistry, and photophysics. At the same time, the biological role of FPs remains less well understood. Recent experimental studies have provided support for a sunscreening function of red fluorescent proteins and nonfluorescent purple-blue chromoproteins in symbiotic shallow water corals [8,9]. However, the high-level expression of fluorescent proteins in corals from mesophotic habitats in which

Int. J. Mol. Sci. 2017, 18, 1503; doi:10.3390/ijms18071503 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017, 18, 1503 2 of 13 an undersupply rather than an oversupply of light challenges the performance of symbiotic corals suggests that the biological functions of FPs are more diverse [10,11]. Using laser scanning confocal microscopy approach, spectral properties and localization of FPs in some reef-building corals (Faviina and Meandriina) were studied [12]. It was found that proteins of different colors, including green-to-red photoconvertible FPs, are localized to different tissues. Unexpectedly complex spatio-temporal patterns of distribution of a red FP in the tissues of the sea anemone Nematostella vectensis were revealed [13]. Interesting correlations between higher brightness of red fluorescence and greater spreading distance were revealed for Acropora millepora larvae [14]. These studies show complex FP functioning with still unclear molecular mechanisms of regulation and biological significance. Here, we use confocal microscopy to study distribution and molecular mobility of endogenous FPs in live tissues of four species from Anthozoa. We demonstrate that FPs can function as solid aggregates. Inspired by this finding, we designed strongly aggregating FP variants, advantageous for some practical applications as bright and early detectable labels.

2. Results

2.1. Confocal Microscopy of Coral Polyps For our work, we chose four species from Anthozoa (available from a local aquarium store) that represent different taxonomic orders and display bright fluorescence of different colors (Figure1): (1) sea anemone Anemonia majano (, Actiniaria, Actiniidae); (2) mushroom coral Discosoma sp. (Hexacorallia, , Discosomatidae); (3) star polyp Clavularia viridis (Octocorallia, , ); and (4) button polyp Zoanthus sp. (Hexacorallia, Zoanthidea, ). Upon inspection with fluorescence stereomicroscope, single small polyps or fluorescent body parts (e.g., a tentacle) were placed in seawater onto a cover slip and immediately examined by high-resolution confocal microscopy. This enabled us to observe live coral tissues albeit not too deep from the surface. At the same time, a high thickness of the samples made it impossible to use transmitted light images for visualization of general morphology, cell nuclei and borders. Thus, in our analysis we relied on fluorescence of endogenous FPs and zooxanthellae alga (which are easily identified due to far-red chlorophyll fluorescence) only. In the Anemonia specimen under study, green fluorescence was concentrated at the tentacle tips, oral disc and strips along the body. Confocal microscopy of the tentacles showed an evenly distributed green signal in the ectoderm cells (Figure2A,B). Emission spectrum measured by the “lambda scan” regime of the microscope had a maximum at about 520 nm. In the Discosoma specimen nearly all ectoderm cells showed bright uniformly distributed red fluorescence with emission max at about 590 nm (Figure2C,D). Clavularia polyps fluoresced cyan (emission max at ~485 nm) at the oral disc and tentacle tips. Detailed inspection by confocal microscopy demonstrated that in this organism fluorescence is characteristic for endoderm cells located next to zooxanthellae alga, the latter easily identifiable due to far-red chlorophyll fluorescence (Figure3). Intracellular distribution of the cyan signal was unusual: most of the cell volume was occupied by numerous small vesicles of about 0.7–1 µm in diameter. Int. J. Mol. Sci. 2017, 18, 1503 3 of 13 Int. J. Mol. Sci. 2017, 18, 1503 3 of 13 Int. J. Mol. Sci. 2017, 18, 1503 3 of 13

Figure 1. Anthozoa specimen used in this work. Left-general view, right-view of individual polyps Figure 1. Anthozoa specimen used in this work. Left-general view, right-view of individual polyps or Figureor tentacles 1. Anthozoa under a specimenfluorescence used stereomicroscope. in this work. Left-g eneral view, right-view of individual polyps tentacles under a fluorescence stereomicroscope. or tentacles under a fluorescence stereomicroscope.

Figure 2. Confocal microscopy of live coral tissues. (A,B) Tip of the tentacle of Anemonia majano, Figureexcitation 2. Confocal488 nm, detection microscopy 500–540 of live nm coral (green tissues. FP); (C (,AD,)B Surface) Tip of of the Discosoma tentacle sp of., excitationAnemonia 543majano nm,, Figure 2. Confocal microscopy of live coral tissues. (A,B) Tip of the tentacle of Anemonia majano, excitationdetection 560–620488 nm, nmdetection (red FP). 500–540 Scale nmbars: (green (A,C ),FP); −50 ( Cµm;,D) (SurfaceB), −5 µm; of Discosoma (D), −10 µm. sp., excitation 543 nm, excitationdetection 488 560–620 nm, detection nm (red 500–540 FP). Scale nm bars: (green (A,C), FP); −50 ( µm;C,D )(B Surface), −5 µm; of (DDiscosoma), −10 µm. sp., excitation 543 nm, detection 560–620 nm (red FP). Scale bars: (A,C), −50 µm; (B), −5 µm; (D), −10 µm.

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Figure 3. Confocal microscopy of live Clavularia tentacle tissues. (A) Cyan fluorescence (excitation 458 Figure 3. Confocal microscopy of live Clavularia tentacle tissues. Left, Cyan fluorescence (excitation Figurenm, 3. detectionConfocal 470–535 microscopy nm); ( Bof) redlive fluorescence Clavularia tentacle(excitation tissues. 543 nm, (A detection) Cyan fluorescence 570–670 nm). (excitationScale bars: 458 458 nm, detection 470–535 nm); middle, red fluorescence (excitation 543 nm, detection 570–670 nm). upper row, −50 µm; bottom row, −10 µm. Rightnm, detection panel—overlay; 470–535 Scalenm); ( bars:B) red upper fluorescence row, −50 (excitationµm; bottom 543 row, nm, −detection10 µm. 570–670 nm). Scale bars: upper row, −50 µm; bottom row, −10 µm. Zoanthus polyp demonstrated bright green tentacle tips and a red oral disc. We studied tentacles with confocal microscopy and found green cells with long processes sparsely distributed in ectoderm Zoanthus polyp demonstrated bright green tentacle tipstips and a red oral disc. We studied tentacles (Figure 4A). Interestingly, we also observed spindle-shaped 5–10-µm-long green fluorescent granules withwith confocalconfocal microscopymicroscopy andand foundfound greengreen cellscells with long processes sparsely distributed in ectoderm (Figure(Figure4A). Interestingly,4B). The signal we from also the observed granules spindle-shapedwas extremely bright, 5–10- roughlyµm-long two green orders fluorescent of magnitude granules (Figurehigher 4A). than Interestingly, that in a regular we also transient observed transfecti spindle-shapedon of mammalian 5–10-µ cellsm-long with green e.g., an fluorescent enhanced GFPgranules (Figure4B). The signal from the granules was extremely bright, roughly two orders of magnitude (Figure(EGFP-C1) 4B). The vector. signal In fromsome cases,the granules lozenge-shaped was ex sttremelyructures bright,were detected, roughly su ggestingtwo orders crystallization of magnitude higherof thanthe green that FP in within aa regularregular the coral transienttransient cells (Figure transfectiontransfecti 4C,D).on of mammalianmammalian cells with e.g., an enhancedenhanced GFP (EGFP-C1)(EGFP-C1) vector.vector. InIn somesome cases, lozenge-shaped structuresstructures were detected, suggestingsuggesting crystallization of the green FPFP withinwithin thethe coralcoral cellscells (Figure(Figure4 4C,D).C,D).

Figure 4. Confocal microscopy of live Zoanthus tentacle tissues. Green fluorescence images (excitation 488 nm, detection 500–535 nm) of representative structures (cells with diffuse signal, aggregates and crystals) at different zoom magnification are shown. Scale bars: (A), −50 µm; (B) and (C), −10 µm; (D), −2 µm. Figure 4. Confocal microscopy of live Zoanthus tentacle tissues. Green fluorescence images (excitation Figure 4. Confocal microscopy of live Zoanthus tentacle tissues. Green fluorescence images (excitation 488 nm, detection 500–535 nm) of representative structures (cells with diffuse signal, aggregates and 488 nm, detection 500–535 nm) of representative structures (cells with diffuse signal, aggregates and crystals) at different zoom magnification are shown. Scale bars: (A), −50 µm; (B) and (C), −10 µm; (D), crystals) at different zoom magnification are shown. Scale bars: (A), −50 µm; (B) and (C), −10 µm; (−D2 ),µm.−2 µm.

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2.2. Fluorescence Recovery Recovery after after Photobleaching Photobleaching (FRAP) (FRAP) of ofEndogenous Endogenous of ofFluorescent Fluorescent Proteins Proteins (FPs) (FPs) Next, we studiedstudied dynamicsdynamics ofof endogenousendogenous FPsFPs usingusing thethe standardstandard fluorescencefluorescence recoveryrecovery afterafter photobleaching technique. technique. For For FPs FPs with with a d aiffuse diffuse intracellular intracellular distribution—green distribution—green FP in Anemonia FP in Anemonia, red FP, redin Discosoma FP in Discosoma, and green, and FP green in Zoanthus FP in Zoanthus—a fast and—a fastfull recovery and full recoverywithin the within bleached the area bleached was observed. area was observed.Notably, all Notably, three FPs all showed three FPs practically showed in practicallydistinguishable indistinguishable FRAP curves FRAP(Figure curves 5). (Figure5).

Figure 5. Fluorescence recovery after photobleaching (FRAP) analysis of soluble fluorescent proteins Figure 5. Fluorescence recovery after photobleaching (FRAP) analysis of soluble fluorescent proteins (FPs) in Anemonia, Discosoma and Zoanthus tissues. Graphs (mean and s.d., n = 30–50 cells) show (FPs) in Anemonia, Discosoma and Zoanthus tissues. Graphs (mean and s.d., n = 30–50 cells) show recovery of fluorescence after photobleaching at a single point within cytoplasm. A full recovery of recovery ofof fluorescencefluorescence after after photobleaching photobleaching at at a singlea single point point within within cytoplasm. cytoplasm. A fullA full recovery recovery of the of the fluorescence signal (with correction to overall partial bleaching) was observed in all these fluorescencethe fluorescence signal signal (with correction(with correction to overall to partialoverall bleaching) partial bleaching) was observed was in observed all these experiments.in all these experiments.

For the Clavularia cyan FP, neitherneither exchangeexchange betweenbetween vesiclesvesicles nornor long-rangelong-range movement of the vesicles withinwithin thethe cytoplasmcytoplasm was was observed observed during during the the 20-min 20-min period period after after photobleaching photobleaching (Figure (Figure6). At6). theAt the same same time, time, small small size size of theof the vesicles vesicles precluded precluded measurements measurements of theof the intra-vesicle intra-vesicle mobility mobility of theof the protein. protein.

Figure 6. Low mobility of cyan FP-containing vesicles in Clavularia tissues. Cyan fluorescence before Figure 6. Low mobility of cyan FP-containing vesicles in Clavularia tissues. Cyan fluorescence before photobleaching (A); just after photobleaching in a square region (B); and 20 min after photobleaching photobleaching (A); just after photobleaching in a square region (B); and 20 min after photobleaching (C). Scale bar, −10 µm. (C). Scale bar, −10 µm. FRAP experiments on the green granules and crystals in Zoanthus demonstrated complete immobilityFRAP experimentsof FPs in these on structures. the green Even granules after and30 min crystals after inbleaching,Zoanthus thedemonstrated shape and intensity complete of immobilitythe bleached of region FPs in remained these structures. unchanged Even (Figure after 7). 30 min after bleaching, the shape and intensity of the bleached region remained unchanged (Figure7).

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FigureFigure 7.7. FRAPFRAP analysisanalysis ofof aa greengreen FPFP granulegranule inin ZoanthusZoanthus tissues.tissues. ((AA)) BeforeBefore bleaching;bleaching; ((BB)) Figure 7. FRAP analysis of a green FP granule in Zoanthus tissues. (A) Before bleaching; (B) immediately immediatelyimmediately afterafter bleachingbleaching byby 488488 nmnm laserlaser atat thethe twotwo points;points; ((CC)) inin 3030 minmin afterafter bleaching.bleaching. ScaleScale barbar after bleaching by 488 nm laser at the two points; (C) in 30 min after bleaching. Scale bar −10 µm. −−1010 µm.µm. 2.3. Construction of Strongly Aggregating FPs 2.3.2.3. ConstructionConstruction ofof StronglyStrongly AggregatingAggregating FPsFPs Our finding that FPs can function in nature as micron-sized granules encouraged us to develop OurOur findingfinding thatthat FPsFPs cancan functionfunction inin naturenature asas micron-sizedmicron-sized granulesgranules encouragedencouraged usus toto developdevelop artificial FP constructs with a strong tendency to aggregate. We reasoned that bright aggregates might artificialartificial FPFP constructsconstructs withwith aa strostrongng tendencytendency toto aggregate.aggregate. WeWe reasonedreasoned thatthat brightbright aggregatesaggregates mightmight be advantageous for tasks such as early detection of weak FP expression and visualization of local bebe advantageousadvantageous forfor taskstasks suchsuch asas earlyearly detectiondetection ofof weakweak FPFP expressionexpression andand visualizationvisualization ofof locallocal translation foci. translationtranslation foci.foci. We hypothesized that a fusion consisting of three tandem copies of a tetrameric FP should WeWe hypothesizedhypothesized thatthat aa fusionfusion consistingconsisting ofof threethree tandemtandem copiescopies ofof aa tetramerictetrameric FPFP shouldshould strongly aggregate due to formation of intermolecular tetramers connected into an intricate network. stronglystrongly aggregateaggregate duedue toto formationformation ofof intermoleculintermolecularar tetramerstetramers connectedconnected intointo anan intricateintricate network.network. We constructed such “trio” variants for two variants of tetrameric DsRed from Discosoma: red E57 WeWe constructedconstructed suchsuch “trio”“trio” variantsvariants forfor twotwo variantsvariants ofof tetramerictetrameric DsRedDsRed fromfrom Discosoma:Discosoma: redred E57E57 and green AG4 [15]. Each vector encoded three identical copies of a complete FP sequence connected andand greengreen AG4AG4 [15].[15]. EachEach vectorvector encodedencoded threethree identiidenticalcal copiescopies ofof aa completecomplete FPFP sequencesequence connectedconnected by short amino acid linkers under control of a cytomegalovirus (CMV) promoter. These vectors byby shortshort aminoamino acidacid linkerslinkers underunder controlcontrol ofof aa cycytomegalovirustomegalovirus (CMV)(CMV) promoter.promoter. TheseThese vectorsvectors werewere were transiently transfected in HEK293T cells and analyzed by fluorescence microscopy 24–48 h after transientlytransiently transfectedtransfected inin HEK293THEK293T cellscells andand analanalyzedyzed byby fluorescencefluorescence microscopymicroscopy 24–4824–48 hh afterafter transfection. Trio-E57 and Trio-AG4 formed multiple very bright spots with almost no signal between transfection.transfection. Trio-E57Trio-E57 andand Trio-AG4Trio-AG4 formedformed multiplemultiple veryvery brightbright spotsspots withwith almostalmost nono signalsignal betweenbetween them (Figure8). We also analyzed regular “single-unit” AG4 as well as four tandem copies of E57. themthem (Figure(Figure 8).8). WeWe alsoalso analyzedanalyzed regularregular “single-un“single-unit”it” AG4AG4 asas wellwell asas fourfour tandemtandem copiescopies ofof E57.E57. Both proteins showed diffuse cytoplasmic staining with no aggregates (Figure9). BothBoth proteinsproteins showedshowed diffusediffuse cytoplasmiccytoplasmic ststainingaining withwith nono aggregatesaggregates (Figure(Figure 9).9). FRAP analysis showed no detectable exchange of Trio-AG4 between granules during 30-min FRAPFRAP analysisanalysis showedshowed nono detectabledetectable exchangeexchange ofof Trio-AG4Trio-AG4 betweenbetween granulesgranules duringduring 30-min30-min observation (Figure 10). Thus, as expected, it represents immobile aggregates in a solid-like state. observationobservation (Figure(Figure 10).10). Thus,Thus, asas expected,expected, itit represrepresentsents immobileimmobile aggregatesaggregates inin aa solid-likesolid-like state.state.

Figure 8. Aggregation of Trio-E57 (upper row) and Trio-AG4 (bottom row) in HeLa Kyoto cells. Left— Figure 8. 8. AggregationAggregation of ofTrio-E57 Trio-E57 (upper (upper row) row) and andTrio-AG4 Trio-AG4 (bottom (bottom row) row)in HeLa in Kyoto HeLa Kyotocells. Left— cells. fluorescence in red or green channels, middle—transmitted light, right—overlay. Scale bars, −20 µm. Left—fluorescencefluorescence in red in or red green or green channels, channels, middle—trans middle—transmittedmitted light, light, right—overlay. right—overlay. Scale Scale bars, −−2020 µm.µm.

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Figure 9. Even cytoplasmic distribution of expressing single-copy green FP AG4 (A) and four tandem Figure 9. Even cytoplasmic distribution of expressing single-copy green FP AG4 (A) and four tandem copiesFigure of 9. redEven FP cytoplasmic E57 in HeLa distribution Kyoto cells of (B ex). pressingScale bar, single-copy −20 µm. green FP AG4 (A) and four tandem copies of red FP E57 in HeLa Kyoto cells (B). Scale bar, −20 µm. copiesFigure of red9. Even FP E57 cytoplasmic in HeLa Kyoto distribution cells (B of). exScalepressing bar, − single-copy20 µm. green FP AG4 (A) and four tandem copies of red FP E57 in HeLa Kyoto cells (B). Scale bar, −20 µm.

Figure 10. FRAP analysis of Trio-AG4 aggregates in HeLa Kyoto cells. Fluorescence was bleached by 488Figure nm 10.laser FRAP at the analysis point designated of Trio-AG4 by aggregates arrow. Numbers in HeLa indicate Kyoto cells.time inFluorescence minutes. Scale was bar, bleached −5 µm. by Figure 10. FRAP analysis of Trio-AG4 aggregates in HeLa Kyoto cells. Fluorescence was bleached by 488Figure nm laser 10. atFRAP the pointanalysis designated of Trio-AG4 by arrow. aggregates Numbers in HeLa indicate Kyoto time cells. in Fluorescenceminutes. Scale was bar, bleached −5 µm. by 488 nm laser at the point designated by arrow. Numbers indicate time in minutes. Scale bar, −5 µm. Next488 nmwe lasertested at the whether point designated said aggregates by arrow. Numbersare associated indicate timewith in minutes.lysosomes Scale as bar, previously −5 µm. demonstratedNext we fortested some whether FPs [16]. said To aggregatesthis end, wear e coexpressedassociated withthe aggregating lysosomes Trio-E57as previously with lysosomaldemonstratedNextNext marker wewe for tested testedlysosomal-associatedsome whether whetherFPs said[16]. aggregatessaidTo this membraneaggregates end, are associatedwe protein arcoexpressede associated with 1 (LAMP1-EGFP) lysosomes the with aggregating aslysosomes previously [17]. NoTrio-E57 as demonstratedsignificant previously with colocalizationlysosomalfordemonstrated some marker FPs of [the16for lysosomal-associated]. greensome To this and FPs end, red [16]. wesignals To coexpressed membrane thiswas end,observed theweprotein aggregating coexpressed(Figure 1 (LAMP1-EGFP) 11). Trio-E57Moreover,the aggregating with [17].time-lapse lysosomal No Trio-E57 significant imaging marker with showedcolocalizationlysosomal-associatedlysosomal that marker greenof the greenlysosomeslysosomal-associated membrane and redundergo signals protein fast wasmembrane 1 (LAMP1-EGFP) moobservedvement, protein (Figure whereas [117 11).(LAMP1-EGFP)]. NoMoreover,red significantTrio-E57 time-lapse [17]. aggregates colocalization No imagingsignificant are of practicallyshowedthecolocalization green that immobile and green of red the (Supplementary signalslysosomes green wasand undergo red observed Moviesignals fast (FigureS1). was mo obvement, 11served). Moreover, (Figurewhereas 11). time-lapsered Moreover, Trio-E57 imaging time-lapse aggregates showed imaging are that practicallygreenshowed lysosomes immobilethat green undergo (Supplementary lysosomes fast movement, undergo Movie whereasS1).fast mo redvement, Trio-E57 whereas aggregates red Trio-E57 are practically aggregates immobile are (Supplementarypractically immobile Movie (Supplementary S1). Movie S1).

Figure 11. Trio-E57 aggregates and lysosomes. Wide-field fluorescence microscopy of HeLa Kyoto cellsFigure coexpressing 11. Trio-E57 Trio-E57 aggregates and and lysosomal lysosomes. marker Wide-field LAMP1-EGFP fluorescence in red microscopy (left) and greenof HeLa (middle) Kyoto channels.cellsFigure coexpressing Right11. Trio-E57 panel—overlay; Trio-E57 aggregates and scale lysosomal and bar, lysosomes. −20 marker µm. Wide-field LAMP1-EGFP fluorescence in red (left)microscopy and green of HeLa (middle) Kyoto Figure 11. Trio-E57 aggregates and lysosomes. Wide-field fluorescence microscopy of HeLa Kyoto cells channels.cells coexpressing Right panel—overlay; Trio-E57 and scale lysosomal bar, −20 µm.marker LAMP1-EGFP in red (left) and green (middle) coexpressing Trio-E57 and lysosomal marker LAMP1-EGFP in red (left) and green (middle) channels. Wechannels. further Righttested panel—overlay; possible toxicity scale of bar, aggregat −20 µm.es for mammalian cells by time-lapse imaging to Right panel—overlay; scale bar, −20 µm. followWe events further of testedcell division possible and toxicity death of in aggregat the Trio-AG4-expressinges for mammalian cells. cells Weby time-lapsefound that imaging cells with to smallfollow aggregates Weevents further of undergocell tested division possible mitosis and normally toxicitydeath in of (Figurethe aggregat Trio-AG4-expressing 12A).es for At mammalianthe same cells.time, cells largeWe by found aggregatestime-lapse that cells imaging(usually with to formedsmallfollow aggregates as events a fusion of undergo ofcell several division mitosis small and normally foci) death can inresult(Figure the inTrio-AG4-expressing 12 abortiveA). At the mitosis same and time, cells. cell large deathWe aggregatesfound (Figure that 12B). (usually cells We with formedsmall asaggregates a fusion of undergo several mitosissmall foci) normally can result (Figure in abortive 12A). At mitosis the same and time, cell deathlarge aggregates(Figure 12B). (usually We formed as a fusion of several small foci) can result in abortive mitosis and cell death (Figure 12B). We

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We further tested possible toxicity of aggregates for mammalian cells by time-lapse imaging to follow events of cell division and death in the Trio-AG4-expressing cells. We found that cells with small aggregatesInt. J. Mol. Sci. undergo 2017, 18, 1503 mitosis normally (Figure 12A). At the same time, large aggregates (usually formed8 of 13 as a fusion of several small foci) can result in abortive mitosis and cell death (Figure 12B). We concluded thatconcluded Trio-AG4 that is suitableTrio-AG4 for is short-term suitable for expression short-term at low expression level, but at can low produce level, significantbut can produce toxicity whensignificant abundant. toxicity when abundant.

Figure 12. Mitosis in HEK293T cells expressing Trio-AG4. Typical examples of normal (A) and Figure 12. Mitosis in HEK293T cells expressing Trio-AG4. Typical examples of normal (A) and abortive abortive (B) mitosis are shown. Numbers above the images designate relative time in hours. Upper (B) mitosis are shown. Numbers above the images designate relative time in hours. Upper rows—green fluorescence,rows—green middlefluorescence, rows—transmitted middle rows—transmitted light images, bottom light rows—overlay images, bottom (brightness rows—overlay of green signal(brightness was enhanced of green signal on overlay was enhanced for clarity). on Formation overlay for of clarity). a rounded Formation mitotic of cell a rounded carrying fluorescentmitotic cell aggregatescarrying fluorescent is pointed aggregates by arrowheads is pointed (time by 0 h).arrowheads Note that (time in (A 0) h). the Note cell divided that in (A into) the two cell daughter divided cellsinto two (two daughter arrows, timecells 2.0(two h), arrows, whereas time in (2.0B) theh), whereas cell remained in (B) roundedthe cell remained for a long rounded time (arrow, for a timelong 5.0time h) (arrow, and did time not 5.0 undergo h) and division. did not undergo Scale bars, division.−30 µm. Scale bars, −30 µm.

To check for the potential advantages of aggregating tag for early detection of weak gene To check for the potential advantages of aggregating tag for early detection of weak gene expression, we cloned Trio-AG4 under control of histone H1 promoter. This promoter is active in the expression, we cloned Trio-AG4 under control of histone H1 promoter. This promoter is active in the S-phase of the cell cycle only; it is considered to be a weak promoter (a high expression level of H1 S-phase of the cell cycle only; it is considered to be a weak promoter (a high expression level of H1 and and other histones is achieved due to amplification of the histone genes in genome rather than other histones is achieved due to amplification of the histone genes in genome rather than increased increased promoter efficiency) [18]. As a control, one of the brightest green FPs available, promoter efficiency) [18]. As a control, one of the brightest green FPs available, mNeonGreen, was used mNeonGreen, was used under control of the same promoter. Time lapse imaging showed appearance under control of the same promoter. Time lapse imaging showed appearance of green spots of Trio-AG4 of green spots of Trio-AG4 as early as 4–6 h after transfection (Figure 13A); many brightly fluorescent as early as 4–6 h after transfection (Figure 13A); many brightly fluorescent cells were observed 15 h cells were observed 15 h post transfection (Figure 13B). In contrast, a few mNeonGreen-expressing post transfection (Figure 13B). In contrast, a few mNeonGreen-expressing cells with very weak signal cells with very weak signal (just above background) were detectable 15 h post transfection (Figure (just above background) were detectable 15 h post transfection (Figure 13C). Only additional 2 days of 13C). Only additional 2 days of cell growth ensured bright signal from mNeonGreen under control cell growth ensured bright signal from mNeonGreen under control of H1 promoter (Figure 13D). Thus, of H1 promoter (Figure 13D). Thus, we concluded that Trio-AG4 strongly outperforms mNeonGreen we concluded that Trio-AG4 strongly outperforms mNeonGreen in this model. in this model.

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Figure 13. Expression of fluorescent proteins Trio-AG4 or mNeonGreen under control of H1 promoter Figure 13. Expression of fluorescent proteins Trio-AG4 or mNeonGreen under control of H1 promoter in HEK293T cells. (A) Time-lapse microscopy of cell expressing Trio-AG4. Numbers above the images in HEK293T cells. (A) Time-lapse microscopy of cell expressing Trio-AG4. Numbers above the images designate time (in hours) post transfection. Note that fluorescent spots can be reliably detected 4 h designatepost timetransfection; (in hours) (B) postcells transfection. expressing Trio-AG4 Note that 15 fluorescent h post transfection; spots canbe (C reliably,D) cells detected expressing 4 h post transfection;mNeonGreen (B) cells 15 expressingh (C) and Trio-AG472 h (D) 15post h posttransfection. transfection; In each (C, Dpanel,) cells upper expressing images—green mNeonGreen 15 h (fluorescence,C) and 72 h bottom (D) post images—correspondi transfection. Inng each transmitted panel, upper light photos. images—green In (A), fluorescence fluorescence, images bottom images—correspondingwere taken under the same transmitted settings; lightin (B– photos.D)—under In (differentA), fluorescence settings. Scale images bars, were −30 µm. taken under the same settings; in (B–D)—under different settings. Scale bars, −30 µm. Then, we used flow cytometry to compare Trio-AG4 and mNeonGreen in more detail. HEK293T cells were transiently transfected in parallel with vectors encoding Trio-AG4 or mNeonGreen under Then, we used flow cytometry to compare Trio-AG4 and mNeonGreen in more detail. HEK293T control of either CMV or H1 promoters. This side-by-side comparison showed the following (Table cells were1). For transiently CMV-driven transfected expression, inmNeonGreen parallel with gave vectors a much encoding brighter signal Trio-AG4 than Trio-AG4, or mNeonGreen especially under controlon ofthe either first day CMV after or transfection. H1 promoters. In contrast, This side-by-side in the case comparisonof weak H1 promoter showed theTrio-AG4 following ensured (Table 1). For CMV-drivenabout a two-fold expression, higher level mNeonGreen of fluorescence gave comp a muchared to brighter mNeonGreen. signal As than integral Trio-AG4, signal especiallyintensity on the firstin flow day cytometry after transfection. is practically In contrast, independent in the of case intracellular of weak localization H1 promoter (i.e., Trio-AG4 aggregated ensured FP spots about a two-foldwould higher give the level same of fluorescencesignal as corresponding compared amou to mNeonGreen.nts of soluble FP), As integralwe concluded signal that intensity enhanced in flow cytometrydetection is practically of Trio-AG4 independent in the H1 promoter of intracellular samples is localization probably due (i.e., to increased aggregated stability FP spots of FP would in the give the sameaggregated signal state as corresponding against protein amountsdegradation. of soluble FP), we concluded that enhanced detection of Trio-AG4 in the H1 promoter samples is probably due to increased stability of FP in the aggregated Table 1. Flow cytometry analysis of HEK293T cells transiently expressing Trio-AG4 or mNeonGreen state againstfluorescent protein proteins degradation. under control of CMV or H1 gene promoters.

Mean (Median) Green Fluorescence Intensity, Arbitrary Units (a.u.) TablePromoter 1. Flow cytometryFluorescent analysisProtein of HEK293T cells transiently expressing Trio-AG4 or mNeonGreen 24 h 48 h 72 h fluorescent proteins under control of CMV or H1 gene promoters. CMV Trio-AG4 86 (47) 458 (209) 450 (147) mNeonGreen 1088 (259) 800 (146) 806 (110) H1 Trio-AG4 Mean 57(Median) (20) Green Fluorescence 95 (40) Intensity, Arbitrary 117 (47) Units (a.u.) Promoter Fluorescent Protein mNeonGreen 3224 (20) h 47 48 (26) h 50 (29) 72 h CMV Trio-AG4 86 (47) 458 (209) 450 (147) mNeonGreen 1088 (259) 800 (146) 806 (110) H1 Trio-AG4 57 (20) 95 (40) 117 (47) mNeonGreen 32 (20) 47 (26) 50 (29) Int. J. Mol. Sci. 2017, 18, 1503 10 of 13

3. Discussion Confocal microscopy of endogenous FPs in vivo can help to unravel their diverse biological functions in anthozoans and other organisms. To the best of our knowledge, we for the first time applied FRAP to study the molecular mobility of FPs in live tissues of Anthozoa specimens. We observed essentially identical fast diffusion of soluble FPs in Anemonia, Discosoma and Zoanthus. Considering the rather distant relationship between these species (different orders) and color variations (green and red FPs), our finding suggests that many natural FPs are freely diffusible cytoplasmic proteins, probably with no association with other intracellular structures or proteins. At the same time, we found two cases of strictly immobilized FPs. Although we did not perform additional staining for membranes, we believe that in Clavularia cyan FP is probably localized to vesicles. First, their shape was always rounded (in contrast to aggregates and crystals in Zoanthus cells). Even more importantly, the cloned cyan FP from Clavularia contains an N-terminal signal peptide [2] that probably directs its synthesis into the specialized vesicles observed here. Zoanthus shows another striking example of intracellular FP distribution as aggregates and crystals of several microns in size. These structures contain FP in a “solid” state with no internal mobility of protein molecules. Examples of protein functioning as aggregates or crystals are rare and mostly associated with pathological processes. Here we showed that FPs can naturally occur as large aggregates or crystals. Many wild-type FPs from Anthozoa species were found to strongly aggregate in vitro [19,20]. Also, some engineered variants of FPs that spontaneously form crystals in mammalian cells were described [21]. This suggests that FP aggregation may be a rather general phenomenon characteristic not only for Zoanthus species. Possibly, the presence of FPs in aggregates, crystals or within vesicles provides a way to accumulate a large amount of FPs at the levels unachievable for a soluble protein in cytoplasm. Also, light scattering on FP granulas was proposed to have a role in decrease of heating of deep coral tissues [22]. Compared to the diffuse fluorescent signal, localized aggregates are better detectable and provide additional ways for structure-functional characterization in recombinant models. For example, the nanobody-mediated accumulation of a GFP-tagged protein partner in an intracellular locus (e.g., a spot in the nucleus) provides an opportunity to study its interaction with another red FP (RFP)-tagged partner through colocalization in that spot [23]. Recently, a spectacular method based on reversible formation of liquid-phase FP-containing droplets in the cytoplasm was developed to monitor protein–protein interactions [24]. Here, we designed strongly aggregated versions of tetrameric green and red FPs by concatenating their three tandem copies. The formed intracellular bright foci are excellently visible compared to regular diffusely distributed FP and thus enhance early detection of weak promoters. Increased brightness of Trio-E57 and Trio-AG4 probably results from a combination of several factors: presence of multiple FP copies instead of one, concentration of the fluorescence signal within small foci which are easier to detect against background, and increased stability against degradation by intracellular protein turnover machinery. High brightness and immobility of FP aggregates can be useful for other tasks such as visualization of spatio-temporal patterns of local translation. At the same time, larger aggregates can induce significant cytostatic and cytotoxic effects. Thus, these tags appear to be advantageous only for models with short-term low-level expression.

4. Materials and Methods

4.1. Confocal Microscopy Anthozoa specimens were obtained at the local aquarium store (Available online: www.aqualogo.ru). Single polyps or small pieces of tissues were placed on a cover glass in seawater and immediately used for microscopy. Fluorescence stereomicroscope SZX12 (Olympus, Tokyo, Japan) with a color CCD camera was used to get a general view of the specimens and tissues. The following filters were used: excitation 400–410 nm, detection >440 nm (blue-cyan channel); excitation 450–480 nm, detection >520 nm (green channel); excitation 435–455 nm, detection >600 nm (red channel). Int. J. Mol. Sci. 2017, 18, 1503 11 of 13

Laser scanning confocal microscopy was performed using an inverted microscope DMIRE2 TCS SP2 (Leica, Wetzlar, Germany) with an HCX PL APO lbd.BL 63× 1.4NA oil objective. Ar (458 and 488 nm lines) and HeNe (543 nm) lasers were used to excite cyan, green and red fluorescence, respectively. FRAP analysis of fast-diffusing FPs in Anemonia, Discosoma and Zoanthus tissues was performed by taking 40 consecutive images of 25 µm × 25 µm area (64 × 64 pixels, 3.5 fps) immediately after local fluorescence bleaching for 0.25 s with 100% 488 nm (for the green FPs) or 543 nm (for the red FP) at a single point within a cell. FRAP analysis of vesicles and aggregates (in Clavularia and Zoanthus tissues, respectively) was performed by bleaching of a region or a point with 100% 458 or 488 nm laser, followed by a 30-min observation period. Manual correction of focus and position of the area of interest was used to compensate for movements of live tissues.

4.2. Cloning Standard PCR and cloning methods were used to construct “Trio” versions of E57 and AG4 proteins [10]. As a result, each vector encoded three identical tandem copies of either E57 or AG4 connected by linkers RSPG (between first and second FP) and RTRPVAT (between the second and third FP) under control of the CMV promoter in the standard C-vector backbone. Linkers in the variant of four tandem copies of E57 were RTRPVAT and RSPG. A 400-bp fragment of histone H1 promoter [13] was amplified on a template of HeLa genomic DNA using primers H1-for 50-ATATATTAATGATGCCCTCAGCCCAATGGATTC and H1-rev 50-AAATACCGGTGGGGTGGCTGTCTCGCCAGGAGC (Ase I and Age I restriction sites are underlined). This fragment was cloned using Ase I and Age I in Trio-AG4 and mNeonGreen vectors instead of a CMV promoter.

4.3. Mammalian Cells HEK293T and HeLa Kyoto cells were grown under standard conditions and transfected with FuGene 6 reagent (Promega, Fitchburg, WI, USA) in accordance to the manufacturer’s protocol.

4.4. Wide Field Fluorescence Microscopy For wide-field fluorescence microscopy of mammalian cells, an AF6000 LX inverted microscope (Leica, Wetzlar, Germany) with an HCX PL APO lbd. BL 63× 1.4NA oil objective and a CoolSNAP HQ CCD camera (Photometrics, Tucson, AZ, USA) was used. Green and red fluorescence were acquired using standard filter cubes: GFP (excitation 450–490 nm, emission 500–550 nm) and TX2 (excitation 540–580 nm, emission 610–680 nm). Prolonged time-lapse imaging was performed at 37 ◦C in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered imaging medium.

4.5. Flow Cytometry Flow cytometry analysis of live cells was performed using Cytomics FC500 (Beckman Coulter, Indianapolis, IN, USA). Fluorescence was excited with a 488-nm laser and detected at 510–540 nm using the same settings for all cell samples.

Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/18/7/1503/s1. Acknowledgments: This work was supported by the Russian Science Foundation (grant number 14-25-00129). Author Contributions: Konstantin A. Lukyanov, Natalia V. Povarova and Alexey M. Bogdanov conceived and designed the experiments; Natalia V. Povarova, Natalia D. Petri, Anna E. Blokhina, Alexey M. Bogdanov, Nadya G. Gurskaya, and Konstantin A. Lukyanov performed the experiments; Natalia D. Petri, Natalia V. Povarova, Anna E. Blokhina, and Konstantin A. Lukyanov analyzed the data; Konstantin A. Lukyanov, Natalia V. Povarova, Anna E. Blokhina, and Natalia D. Petri wrote the paper. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. Int. J. Mol. Sci. 2017, 18, 1503 12 of 13

Abbreviations

GFP Green fluorescent protein FP Fluorescent protein FRAP Fluorescence recovery after photobleaching RFP Red fluorescent protein

References

1. Shimomura, O.; Johnson, F.H.; Saiga, Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell. Comp. Physiol. 1962, 59, 223–239. [CrossRef] [PubMed] 2. Matz, M.V.; Fradkov, A.F.; Labas, Y.A.; Savitsky, A.P.; Zaraisky, A.G.; Markelov, M.L.; Lukyanov, S.A. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 1999, 17, 969–973. [CrossRef][PubMed] 3. Shagin, D.A.; Barsova, E.V.; Yanushevich, Y.G.; Fradkov, A.F.; Lukyanov, K.A.; Labas, Y.A.; Semenova, T.N.; Ugalde, J.A.; Meyers, A.; Nunez, J.M.; et al. GFP-like proteins as ubiquitous metazoan superfamily: Evolution of functional features and structural complexity. Mol. Biol. Evol. 2004, 21, 841–850. [CrossRef][PubMed] 4. Deheyn, D.D.; Kubokawa, K.; McCarthy, J.K.; Murakami, A.; Porrachia, M.; Rouse, G.W.; Holland, N.D. Endogenous green fluorescent protein (GFP) in amphioxus. Biol. Bull. 2007, 213, 95–100. [CrossRef] [PubMed] 5. Matz, M.V.; Marshall, N.J.; Vorobyev, M. Are corals colorful? Photochem. Photobiol. 2006, 82, 345–350. [CrossRef][PubMed] 6. Alieva, N.O.; Konzen, K.A.; Field, S.F.; Meleshkevitch, E.A.; Hunt, M.E.; Beltran-Ramirez, V.; Miller, D.J.; Wiedenmann, J.; Salih, A.; Matz, M.V. Diversity and evolution of coral fluorescent proteins. PLoS ONE 2008, 3, e2680. [CrossRef][PubMed] 7. Chudakov, D.M.; Matz, M.V.; Lukyanov, S.; Lukyanov, K.A. Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues. Physiol. Rev. 2010, 90, 1103–1163. [CrossRef][PubMed] 8. Smith, E.G.; D’Angelo, C.; Salih, A.; Wiedenmann, J. Screening by coral green fluorescent protein (GFP)-like chromoproteins supports a role in photoprotection of zooxanthellae. Coral Reefs 2013, 32, 463–474. [CrossRef] 9. Gittins, J.R.; D’Angelo, C.; Oswald, F.; Edwards, R.J.; Wiedenmann, J. Fluorescent protein-mediated colour polymorphism in reef corals: Multicopy genes extend the adaptation/acclimatization potential to variable light environments. Mol. Ecol. 2015, 24, 453–465. [CrossRef][PubMed] 10. Eyal, G.; Wiedenmann, J.; Grinblat, M.; D’Angelo, C.; Kramarsky-Winter, E.; Treibitz, T.; Ben-Zvi, O.; Shaked, Y.; Smith, T.B.; Harii, S.; et al. Spectral Diversity and Regulation of Coral Fluorescence in a Mesophotic Reef Habitat in the Red Sea. PLoS ONE 2015, 10, e0128697. [CrossRef][PubMed] 11. Roth, M.S.; Padilla-Gamiño, J.L.; Pochon, X.; Bidigare, R.R.; Gates, R.D.; Smith, C.M.; Spalding, H.L. Fluorescent proteins in dominant mesophotic reef-building corals. Mar. Ecol. Prog. Ser. 2015, 521, 63–79. [CrossRef] 12. Oswald, F.; Schmitt, F.; Leutenegger, A.; Ivanchenko, S.; D’Angelo, C.; Salih, A.; Maslakova, S.; Bulina, M.; Schirmbeck, R.; Nienhaus, G.U.; et al. Contributions of host and symbiont pigments to the coloration of reef corals. FEBS J. 2007, 274, 1102–1109. [CrossRef][PubMed] 13. Ikmi, A.; Gibson, M.C. Identification and in vivo characterization of NvFP-7R, a developmentally regulated red fluorescent protein of Nematostella vectensis. PLoS ONE 2010, 5, e11807. [CrossRef][PubMed] 14. Kenkel, C.D.; Traylor, M.R.; Wiedenmann, J.; Salih, A.; Matz, M.V. Fluorescence of coral larvae predicts their settlement response to crustose coralline algae and reflects stress. Proc. Biol. Sci. 2011, 278, 2691–2697. [CrossRef][PubMed] 15. Terskikh, A.V.; Fradkov, A.F.; Zaraisky, A.G.; Kajava, A.V.; Angres, B. Analysis of DsRed mutants Space around the fluorophore accelerates fluorescence development. J. Biol. Chem. 2002, 277, 7633–7636. [CrossRef] [PubMed] 16. Katayama, H.; Yamamoto, A.; Mizushima, N.; Yoshimori, T.; Miyawaki, A. GFP-like proteins stably accumulate in lysosomes. Cell Struct. Funct. 2008, 33, 1–12. [CrossRef][PubMed] Int. J. Mol. Sci. 2017, 18, 1503 13 of 13

17. Falcón-Pérez, J.M.; Nazarian, R.; Sabatti, C.; Dell’Angelica, E.C. Distribution and dynamics of Lamp1-containing endocytic organelles in fibroblasts deficient in BLOC-3. J. Cell Sci. 2005, 118, 5243–5255. [CrossRef][PubMed] 18. Marzluff, W.F.; Wagner, E.J.; Duronio, R.J. Metabolism and regulation of canonical histone mRNAs: Life without a poly(A) tail. Nat. Rev. Genet. 2008, 9, 843–854. [CrossRef][PubMed] 19. Yanushevich, Y.G.; Staroverov, D.B.; Savitsky, A.P.; Fradkov, A.F.; Gurskaya, N.G.; Bulina, M.E.; Lukyanov, K.A.; Lukyanov, S.A. A strategy for the generation of non-aggregating mutants of Anthozoa fluorescent proteins. FEBS Lett. 2002, 511, 11–14. [CrossRef] 20. Zubova, N.N.; Korolenko, V.A.; Astafyev, A.A.; Petrukhin, A.N.; Vinokurov, L.M.; Sarkisov, O.M.; Savitsky, A.P. Brightness of yellow fluorescent protein from coral (zFP538) depends on aggregation. Biochemistry 2005, 44, 3982–3993. [CrossRef][PubMed] 21. Tsutsui, H.; Jinno, Y.; Shoda, K.; Tomita, A.; Matsuda, M.; Yamashita, E.; Katayama, H.; Nakagawa, A.; Miyawaki, A. A diffraction-quality protein crystal processed as an autophagic cargo. Mol. Cell 2015, 58, 186–193. [CrossRef][PubMed] 22. Lyndby, N.H.; Kühl, M.; Wangpraseurt, D. Heat generation and light scattering of green fluorescent protein-like pigments in coral tissue. Sci. Rep. 2016, 6, 26599. [CrossRef][PubMed] 23. Herce, H.D.; Deng, W.; Helma, J.; Leonhardt, H.; Cardoso, M.C. Visualization and targeted disruption of protein interactions in living cells. Nat. Commun. 2013, 4, 2660. [CrossRef][PubMed] 24. Watanabe, T.; Seki, T.; Fukano, T.; Sakaue-Sawano, A.; Karasawa, S.; Kubota, M.; Kurokawa, H.; Inoue, K.; Akatsuka, J.; Miyawaki, A. Genetic visualization of protein interactions harnessing liquid phase transitions. Sci. Rep. 2017, 7, 46380. [CrossRef][PubMed]

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