This is a very good article. I am truly glad that Dr. Campbell did such a good job. I think it is definitely ready for open access. I only have a few suggestions on how to improve it, at the author’s discretion, and a couple of minor corrections.
I feel uneasy about calling the article “Fluorescent proteins”, as well as using this name throughout the text, as if the GFP-like proteins were the only fluorescent proteins existing in nature. There are plenty of other fluorescent proteins emitting in the visible range, such as photosystem complex, rhodopsins, hemoglobin etc –all those containing an externally synthesized chromophore moiety. Not to mention that all proteins containing aromatic residues in their sequence are fluorescent in the UV range. So I would lean towards calling the piece “GFP-like fluorescent proteins”. But then, not all GFP-like proteins are fluorescent! Take pocilloporins, for example. Honestly, I cannot think of a nice catchy title for this group of proteins. All that comes to mind is the bland “GFP-like proteins” or “Fluorescent and colored proteins of GFP type”. So I will leave it for Robert to decide.
None of the organisms that were studied in our 1999 paper (Matz et al, 1999) were actually corals (class Anthozoa, order Scleractinia). Discosoma sp. is a mushroom anemone (order Corallimorpharia), Zoanthus sp is a zoanthid (order Zoanthidea), Anemonia majano is a sea anemone (order Actiniaria), and Clavularia is a “soft coral” (order Alcyonaria). They can be called “reef Anthozoa” or “coral allies”, but not “corals”.
The story of the discovery of Anthozoan fluorescent proteins
There is an interesting story about the discovery of Anthozoan FPs which may be worth mentioning, at least in part. Coral and sea anemone fluorescence has been known for decades, and the need for more fluorescent proteins besides GFP was widely recognized by the biotech community. So why did it take so long to make a connection? The reason was in the mindset: everyone (including us in Lukyanov’s lab in the beginning of the project) assumed that FPs, just like the GFP from A. victoria, must be found in bioluminescent animals, and they should be green. It took the intervention of Yulii A. Labas (who, to everybody’s greatest regret, passed away this year), an evolutionary biologist then at the Institute of Ecology and Evolution of the Russian Academy of Sciences in Moscow, to break out of the box. Being interested in evolution of bioluminescence for most of his career, Labas realized that GFPs, like all other components of bioluminescent systems, must not have evolved from scratch for the purpose of bioluminescence, but must have been recruited from other places and functions. So it made a perfect sense for him to look for GFPs in non-luminous, but fluorescent animals – such as corals and sea anemones. Now, Labas was arguably the closest living approximation of a “mad scientist” - old, hunched, grey-bearded, chain-smoking, completely fascinated with science, and shooting all sorts of ideas (ranging from improbable to completely crazy) at anybody (literally, anybody) who would listen. And I was a young and very snobby graduate student. So when one day Labas called me and said that a friend of his – a professional aquarium keeper Andrei Romanko – has some amazing fluorescent creatures in his tank in his tiny flat in the outskirts of Mosow, and I should come to check them out – my feelings were rather mixed. Then, imagine us staring into the tank, with Labas stabbing his nicotine-stained finger into the glass at the bright green tentacle tips of a sea anemone (that was Anemonia majano) shouting “See?? See??? GFP!!!” GFP my ass, thought I (I remember this exact thought with an exceptional clarity). It is just bloody too bright. But I took advantage of Andrei Romanko’s generosity and snipped off a few tentacles, along with a yellowish piece of Zoanthus (why am I doing this?.. GFP must be green for heaven’s sake! But I cannot disappoint the old looney), and a Clavularia polyp which was also unnaturally brightly fluorescent. Then, a couple of weeks later, Anemonia cDNA library yielded the first snippet of an obvious GFP-like sequence. The world became a whirlpool and everybody got so excited. Then Andrei Romanko called me from work, saying: “Look, I just got a live rock that I have to deliver to a customer tonight, with three tiny mushroom anemones on it, and they are red! If you come right now, we can steal one of them!” Needless to say, I was there as soon as Moscow’s subway would carry me. That’s how we got the sample that yielded DsRed. By the way, since I took only a part of the mushroom anemone, the rest happily regenerated and until this day lives in Romanko’s tank.
Looks like I got carried away a bit… Obviously, most of the above is somewhat non-scientific and will not go well with the general structure of the article. Still, I would like to ask Dr Campbell to mention the key role of Yulii A. Labas in the discovery. Labas was one of the smartest people I ever knew, a great scientist who, due to the peculiarities of his personality, just could not fit well into the academic system. I am deeply grateful for his help and attest that without his “out-of-the-box” thinking and enthusiasm we would not have gone too far in the project.
It may be helpful, at the beginning of the article, to delineate the difference between fluorescence and luminescence. In my experience, four out of five PhDs confuse them.
There is no such taxon as Galaxeidae. My guess is that Karasawa et al cloned their protein from the coral Galaxea sp. (class Anthozoa, order Scleractinia, family Oculinidae).
The work of Schlichter and Fricke, on fluorescence of Leptoseris fragilis to enhance photosynthesis, is most likely is not related to GFPs since their pigment was extractable with chlorophorm.
While the visual material in the article is of exceptional quality, I think it would be good to add a photo or two of fluorescent corals. These are quite stunning. I will be glad to provide the images should Dr. Campbell deem such a thing valuable.
Speaking of which, we just published an extensive analysis of coral FPs: Alieva, N.O., Konzen, K. A., Field, S. F., Meleshkevitch, E. A., Hunt, M., Beltran-Ramirez,V., Miller, D. J., Wiedenmann, J., Salih, A. and Matz, M. V. Diversity and evolution of coral fluorescent proteins. PLoS ONE 2008, 3(7): e2680. May be worth mentioning.
Also, I think it would be really great to include a couple of videos shot with FPs as markers.
I feel that one important aspect of FPs application was overlooked: the photoswitchable FPs, such as PA-GFP by Lippincott-Schwartz et al, Chudakov’s kindling FPs, and Kaede line and Dronpa by Myawaki’s group. These enable a whole range of new applications in imaging cellular processes, plus applications in sub-optical resolution microscopy such as STORM and PALM.
Finally, I really like the note in the Conclusions on the lack of biology research on Aequorea. I would strongly suggest expanding it: in fact, the biology and ecology of animal fluorescence remains an area of wild speculations with very little research devoted to it, despite the huge impact the FPs are making on biotechnology, and truly striking visual appearance of some of these animals. The mechanisms of color determination in GFP-like proteins is another very under-studied area, which may be rather surprising. We still don’t know what makes a cyan or red fluorescent protein at the level of structure-function relationships. Biotechnology is content with the fact that through random mutagenesis and selection some color modification can be achieved, but clearly, the ability to design a marker of required color, and with biochemical properties precisely tailored for the needs of a particular application, would be a great step forward.
Mikhail V. Matz Assistant Professor University of Texas at Austin Integrative Biology Section 1 University station C0930 Austin, TX 78712 phone 512-992-8086 cell, 512-475-6424 lab fax 512-471-3878 web http://www.bio.utexas.edu/research/matz_lab
Author : Addressing reviewer comments
Regarding the use of Fluorescent Proteins as a title. I did not choose the title for the article but, like you, I have been unable to come up with a better description for this class of proteins. I have now added a section called "Notes on terminology" at the end of the article to address this issue.
Regarding the species in the 1999 paper. I have changed the text of the article such that the species are now referred to correctly.
Regarding the story of the discovery of Anthozoan FPs. What a wonderful anecdote! I have set aside the text of the story as a section and linked to it from the main article. I have added a sentence to acknowledge the critical role of Labas.
Regarding the difference between fluorescence and luminescence. I have added some definitions to the "Notes on terminology" section at the end of the article. I agree that many students get these things mixed up.
Regarding Galaxeidae. I have changed Galaxeidae to Galaxea sp.
Regarding the work of Schlichter and Fricke. I have removed this reference as well as the sentence about enhancing photosynthesis because I can not find a different reference that supports the hypothesis. The sentence has been rewritten to better emphasize our overall poor understanding of the biological roles of fluorescence (and the lack of research on the topic).
Regarding including an image of fluorescent coral. Dr. Matz has generously provided an image that is now incorporated into the article.
Regarding the PLoS ONE article. I have now cited this article (where appropriate) and added it to the list of recommended reading.
Regarding including a time lapse movie. I have now included a link to the Hamamatsu fluorescent protein digital video gallery.
Regarding photswitchable FPs. I have added a section on photoswitchable FPs.
Regarding lack of biological research on biological fluorescence. I have added another sentence about this in the section on Function.
Thank you for your valuable comments! Robert E. Campbell