9/30/12
1
Jessie Mathews* and Kimberly Wong* *Boston University Marine Program
FPs & Coral Resilience? Healthy Coral Bleached Coral
•30% increase in ocean acidity since preindustrial times (Hall-
Spencer, J.M. et al. 2008) •2-3˚C increase in SST by 2050 or 2100 (Hoegh-Guldberg, O. et al. 2007)
•60% mortality expected in the next few decades (Grottoli, A.G et al. 2006)
Fluorescent proteins may help corals survive.
[1] [2]
9/30/12
2
Discovery of FPs
Green Fluorescent Protein first found the jellyfish Aequorea victoria (Shimomura, O. 2005)
Shimomura et al. Nobel Prize in Chemistry 2008 (Zimmer, M. 2009)
[3]
Widely used in research Useful as reporter in transgenic cells (Carter et al. 2004)
GFP can be fused to another protein allowing that protein to be visualized in situ.
[3]
[32]
9/30/12
3
[31]
Fluorescent Proteins • All FP have similar
structure • Beta-barrel with 11
anti-parallel strands, caped at the ends, to create internal space distinguishable from environment
• Chromophore is inside internal cavity
[4]
9/30/12
4
Fluorescent Colors • GFP are primary
color determinants in corals (Alieva and Konzen 2008)
• Four basic colors - cyan, green, red, and non-fluorescent purple-blue (Alieva and Konzen 2008)
Figure from Technau, Ulrich and Steele, Robert E. (2011)
Fluorescent Colors
• FP expression does not always correlate with the fluorescence color observed (Kao et al. 2007)
Kao et al. 2007
9/30/12
5
Evolution of GFP • One amino acid
substitution can change what color is expressed
• Red FP evolved through several transitions from ancestral GFP (Kao et al. 2007)
• Green FP was derived from a red FP (Alieva et al. 2008)
[30]
FP Concentrations Vary By Environment • Temporal and
spatial patterns in FP concentration may exist (Palmer et al. 2009)
• Possible trend of greater FP expression at shallower depths (Kao et al. 2007)
Figure from Kao et al. (2007)
9/30/12
6
Possible Function
• Wound repair • Photoprotection • Enhance photosynthesis • Visual stimuli (Palmer et al. 2009)
[26] [28]
Wound Repair • Upregulation of FP in injured coral
tissues (Palmer et al. 2009)
• Inflammatory-like response - anti-oxidant activity (Ikmi and Gibson 2010)
(Palmer et al. 2009)
(Palmer et al. 2009)
9/30/12
7
Photoprotection • UV resistance, by converting short wavelengths
into longer, less harmful, wavelengths (Dove et al. 2000). • Coral skeletons convert harmful UV to yellow
fluorescence (Kaniewska and Hoegh-Guldberg 2009)
• DNA lesions
Kaniewska and Hoegh-Guldberg 2009
Enhance Photosynthesis • Symbiotic relationship between corals and
zooxanthellae (Kaniewska and Hoegh-Guldberg 2009)
• More efficient photosynthesis than terrestrial plants (Kaniewska and Hoegh-Guldberg 2009)
• Shade zooxanthellae in high light, or enhance light in low light conditions (Dove et al. 2000)
• Longer wavelengths are more efficient for photosynthesis (Dove et al. 2000)
[26]
[27]
9/30/12
8
Visual Stimuli
• Warn predators; attract other organisms • Effects of coloration on reef ecology
[28] [29]
Fluorescence in Nematostella vectensis
• Bi-fluorescence (Ikmi and Gibson 2010)
• Spatial and temporal variation (Ikmi and Gibson 2010)
• Weak green fluorescence along length of body column, and along lengths of tentacles (Ikmi and Gibson 2010)
• Red fluorescence in oral pole; no red fluorescence in tentacles (Ikmi and Gibson 2010)
Image from Ikmi and Gibson (2010)
9/30/12
9
Species with Fluorescent Proteins
Echinopora forskaliana
Discosoma sp.
Entacmaea quadricolor
Nematostella vectensis
Goniopora djiboutiensis
[5]
[6]
[7]
[8]
[9]
It’s hard for the audience to keep track of so many different species, so it would have been good to organize them into subsets somehow, e.g., by taxonomic group or by those for which FP sequences have already been published and those for which you had to identify putative FP sequences by BLAST.
Acropora hyacinthus
Pocillopora damicornis
Astrangia lajollaensis
Montastrea cavernosa
Platygra lamellina
Stylophora pistillata
Porites porites
Scleractinia sp.
Acropora millepora
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
9/30/12
10
Stylocoenilla sp.
Acropora digitifera
Galaxea fascicularis
Acropora pulchra
Edwardsiella lineata
Corallimorph
Echinophyllia echinata
[19]
[20]
[21]
[22]
[23]
[24]
[25]
Conserved motifs in FPs
9/30/12
11
Phylogeny of FPs
Conserved Motifs
Acropora pulcra Galaxea fascicularis Acropora hyacinthus Acropora millepora Echinopora forskaliana Goniopora dijiboutiensis Montastrea cavernosa
[18]
[10]
[13]
[23]
[22] [5]
[9]
Because you make reference to which taxa are related to each other, it would have been useful to provide background on relationships among the major groups of anthozoans, and indicate whereyour model systems fit.
I really like how you show individual motif arrangements with the species that exhibit them. You might have taken it one step farther by showing the sequence alignment of motifs of interest. Also, how do the motifs related to the structure of FPs that you discussed in the background?
9/30/12
12
Conserved Motifs
Porites porites Stylocoeniella sp. Stylocoeniella sp. 2 Pocillopora damicornis
[11]
[16]
[19]
Conserved Motifs
Echinophyllia echinata Scleractinia sp. Platygra lamellina
[21] [17] [14]
9/30/12
13
Missing Motifs
Corallimorph Corallimorph 2 Edwardsiella lineata Astrangia lajollaensis
[25]
[24] [12]
Conclusions
• FPs identified in next-generation sequencing data from 22 species.
• Cannot predict color from sequence or phylogenetic position
• All 23 proteins are clearly related and similar in structure
9/30/12
14
Future Directions • “Knock out” fluorescent proteins to
determine if • UV resistance diminishes • Wound healing is compromised
• Investigate how environment (e.g., depth) correlates with FP expression.
• Express “novel” FPs in cell culture to investigate their color.
[29]
Acknowledgements
We would like to thank Dr. John Finnerty, Liz Burmester, and the entire Boston University Marine Program for their dedication, funding, and support
9/30/12
15
References • Alieva NO, Konzen KA, Field SF, Meleshkevitch EA, Hunt ME, Beltran-Ramirez V, Miller DJ, Wiedenmann J, Salih A, and Matz MV. 2008. Diversity and
Evolution of Coral Fluorescent Proteins. PLoS ONE 3(7): e2680. doi:10.1371/journal.pone.0002680. • Bailey TL. et al. 2009. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Research 37:W202-W208. • Carter RW, Schmale MC, and Gibbs PDL. 2004. Cloning of anthozoan fluorescent protein genes. Comparative Biochemistry and Physiology, Part C 138:
259-270. • Felsenstein J. 1993. PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the author. Department of Genetics, University of Washington,
Seattle. • Dove SG, Hoegh-Guldberg O, and Ranganathan S. 2000. Major colour patterns of reef-building corals are due to a family of GFP-like proteins. Coral Reefs
19: 197-202. • Field SF, Bulina MY, Kelmanson IV, Bielawski JP, and Matz MV. 2005. Adaptive Evolution of Multicolored Fluorescent Proteins in Reef-Building Corals.
Journal of Molecular Evolution 62: 332-339. doi:10.1007/s00239-005-0129-9. • Goodsell DS. 2003. Green Fluorescent Protein (GFP) Molecule of the Month. RCSB Protein Data Bank. http://www.rcsb.org/pdb/education_discussion/
molecule_of_the_month/download/GFP.pdf • Grottoli AG, Rodrigues LJ, and Palardy JE.. 2006. Heterotrophic plasticity and resilience in bleached corals. Letters to Nature 440(27):1186-1189. • Granger et al. unpublished. EdwardBase and CorallimorphBase. Finnerty Lab server. • Hall-Spencer JM, Rodolfo-Metalpa R, Martin S, Ransome E, Fine M, Turner SM, Rowley SJ, Tedesco D, and Buia MC.. 2008. Volcanic carbon dioxide vents
show ecosystem effects of ocean acidification. Nature 454: 96-99. • Hoegh-Guldberg O, Mumby PJ, Hooten AJ, Steneck RS, Greenfield P, Gomez E, Harvell CD, Sale PF, Edwards AJ, Caldeira K, Knowlton N, Eakin CM,
Iglesias-Prieto R, Muthiga N, Bradbury RH, Dubi A, and Hatziolos Me. 2007. Coral Reefs Under Rapid Climate Change and Ocean Acidification. Science 318(5857): 1737-1742.
• Ikmi A and Gibson MC. 2010. Developmentally regulated red fluorescent protein of Nematostella vectensis. PLoS ONE 5(7): e11807. doi:10.1371/journal.pone.0011807.
• Kaniewska P and Hoegh-Guldberg O. 2009. Coral Skeletons Defend against Ultraviolet Radiation. PLoS ONE 4(7): e7995. doi:10.1371/journal.pone.0007995.
• Kao H, Sturgis S, DeSalle R, Tsai J, Davis D, Gruber DF, and Pieribone VA. 2007. Dynamic Regulation of Fluorescent Proteins from a Single Species of Coral. Marine Biotechnology 9: 733-746. doi:10.1007/210126-007-9025-1.
• Labas YA, Gurskaya NG, Yanushevich YG, Fradkov AF, Lukyanov KA, Lukyanov SA, and Matz MV. 2002. Diversity and evolution of the green fluorescent protein family. PNAS 99(7): 4256-4261. doi:10.1073/pnas.062552299
• Matz MV, Marshall NJ, and Vorobyev M. 2005. Symposium-in-Print: Green Fluorescent Protein and Homologs; Are Corals Colorful? Photochemistry and Photobiology 82: 345-350.
• NCBI:Blast. http://blast.ncbi.nlm.nih.gov/Blast.cgi. • Palmer CV, Modi CK, and Mydlarz LD. 2009. Coral Fluorescent Proteins as Antioxidants. PLoS ONE 4(10): e7298. doi:10.1371/journal.pone.0007298. • Shimomura, O. 2005. The discovery of aequorin and green fluorescent protein. Journal of Microscope 217(1): 3-15. • Traylor-Knowles et al. 2011. PocilloporaBase: pocilloporabase.org. • Technau U and Steele RE. 2011. Evolutionary crossroads in developmental biology: Cnidaria. Development 138(8): 1447-1458. • Ugalde JA, Chang BSW, and Matz MV. 2012. Evolution of Coral Pigments Recreated. Science 305: 1433. • Zimmer M. 2009. GFP: from jellyfish to the Nobel prize and beyond. Chemical Society Reviews 38: 2823-2832.
Image Sources • [1] http://www.nwf.org/Global-Warming/Effects-on-Wildlife-and-Habitat/Coral-Reefs.aspx • [2] http://marinebio.org/oceans/coral-reefs.asp • [3] http://www.immunok.com/Nematostella vectensis • [4] http://thagreenplanet.yolasite.com/ • [5] http://www.reeffrontiers.com/photos_corals/showphoto.php?photo=285&title=echinopora-forskaliana&cat=522 • [6] http://www.coralbiome.com/store/502-869-large/discosoma-sp-red-superman-.jpg • [7] http://www.thomsenlab.org/people.php • [8] http://www.fishlore.com • [9] http://www.saltyunderground.com/article_info.php?articles_id=47 • [10] http://media.photobucket.com/image/recent/p96531/P3310253.jpg • [11] http://www.ne.jp/asahi/mc/minatomachi/pocillopora1a.htm • [12] http://gallery.divebums.com/v/Kevin-s-UnderWater-images/Invertebrates/album67/050327Woods_ID2_w.jpg.html • [13] http://www.wetwebmedia.com/pix%20of%20the%20day%20marine/potd%20sw%20arch%20260-279/swpotdarch266.htm • [14] http://www.flickriver.com/photos/boogieswithfish/434365820/ • [15] http://www.cetamar.com/producto.php?id=762 • [16] http://www.usca.edu/biogeo/zelmer/sansal/cnidaria/stony/branchpillar/ • [17] http://species.wikimedia.org/wiki/Scleractinia • [18] http://www.soutassaltwater.com/product.php?productid=16222 • [19] http://www.reefcentral.com/forums/showthread.php?t=1066476 • [20] http://news.sciencemag.org/sciencenow/2011/07/scienceshot-coral-genome-reveals.html • [21] http://www.nano-reef.com/forums/lofiversion/index.php/t87732-50.html • [22] http://reef.pantshead.com/?p=267 • [23] http://reefkeeping.com/issues/2004-05/totm/index.php • [24] http://www.bluewatervisions.com/bahamas.htm • [25] http://web.nhm.ku.edu/inverts/meg/index.htm • [26] http://microbewiki.kenyon.edu/index.php/Zooxanthellae • [27] http://www.biomedcentral.com/1471-2164/10/333 • [28] http://www.wetwebmedia.com/mushroomidfaqs.htm • [29] http://www.crm.gov.mp/programs/monitoring/help.asp • [30] http://spider.iwr.uni-heidelberg.de/~pimhof/research.html • [31] http://summerofresearch.blogspot.com/ • [32] http://discoverysedge.mayo.edu/alzheimers-research-tau-protein/
