Lighting up life, part II: Molecular tracers

Molecular biologists love proteins. We come up with all kinds of ways to look for them and detect their interactions, because that's how we understand what the parts of a cell do and how they do it. One way we can see them is through immunochemistry, where we put antibodies (from a mammal immune system) that are sensitive to the protein we want to see into a sample and then make the antibodies visible with a chemical. Most stains are not specific to exact proteins, and antibodies are, so this is a great, precise way to see where your molecule is in a tissue or cell sample.

Of course, a single stain isn't as good as two or three different colors showing where multiple molecules are, because then you can tell whether those proteins show up in the same places as each other: the same kind of cell, the same organelle inside the cell, maybe even so close you can't tell them apart -- which could mean that they need each other somehow. To get multiple colors, molecular biologists have used different fluorescent molecules, like fluorescein (greenish) and rhodamine (red). Currently there are a bewildering variety of fluorescent dyes available1. They come in any color, they're bright, and, as the products of a high-demand, high-innovation system, they generally work great.

Fluorescein can also be injected intravenously to visualize blood vessels in the human retina. It doesn't bind to anything specific, just makes your blood glow under UV and your spit look funny. Image by L. Bacud, from Wikimedia Commons.

So why do we care about some fluorescent protein, then? For one thing, if you start glomming antibodies onto a protein in a live organism, that protein may be attacked by its immune system. That's the point of antibodies: they attach themselves to a nasty bacterium or virus, and they alert the cells that come break it down. The antibodies can interfere with your protein's function by physically getting in its way, too. (Ever been attacked by three or four toddlers?) You don't want this to happen in a living system. It's bad for your organism, and your results will be all wrong. So when I said "samples" up there, I meant that your samples had to be dead. But in life proteins move. We can guess what they're doing based on snapshots, but a lot of the time it's hard to be sure. Plus, it can take a lot of processing to prepare as many samples as you need.

Ideally, then, we'd want fluorescent tags on our proteins so we could see them moving in live cells, doing their thing without interference2. This is possible with dyes, and there are some pretty good ways to do it3, but even those have major limitations on the techniques possible, the types of cells, and how long it takes to do such exacting experiments.

This is a statue of GFP. Really. "Steel Jellyfish", 2006. Photo by the artist, Julian Voss-Andreae, via Wikimedia Commons.

It turns out, though, that GFP is special, because it's made entirely of amino acids, coded for by genes rather than made in a bubbling beaker. Since it's made of the same things all proteins are made of anyway, molecular biologists can construct gene sequences that include the regular gene with GFP attached to it, and that protein can be made in live cells 4. Light 'em up and watch 'em dance!

When we learned to put GFP wherever we wanted it, it was a revolution. The Nobel Prize in Chemistry for 2008 was awarded to Osamu Shimomura5 for isolating GFP from Aequorea victoria as published in 1962, Martin Chalfie for using it in the nematode worm Caenorhabditis elegans to highlight individual cells in 19947, and Roger Y. Tsien for deciphering why GFP shines and altering it to get new colors like yellow and cyan in the 90s 8.

In conclusion, enjoy these images of ridiculous glowing animals, made possible by jellyfish and molecular biology.

Next time: more fluorescent proteins and what they're good for. -- KMP

[1] Alexa Fluor is just one popular brand.
[2] Well, minimal interference. Any addition occupies space, and I'll talk about tetrameric RFP next time.
[3] Griffin BA, Adams SR, Tsien RY. 1998. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281:269-272.
[4] Doesn't that get in the way just like antibodies? Sometimes. But most proteins are a lot bigger than GFP, so it works easily most of the time, and there are a few workarounds too.
[5] While working at UW Friday Harbor Labs, home of the pictured sculpture of GFP, where you can talk to people who worked with Shimomura to isolate the protein[6].
[6] It was isolated from "squeezates". Squeezates, people. What was I saying about creative naming in science? Sometimes our Wikipedia-like earnestness is more beautiful than any poem. (Shimomura O, Johnson FH, Saiga Y. 1962. Extraction, purification and properties of aequorin, a bioluminescent protein from luminous hydromedusan, Aequorea. J Cell Comp Physiol 59:223-239.)
[7] Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. 1994. Green fluorescent protein as a marker for gene expression. Science 263(5148):802-805.
[8]Heim R, Prasher DC, Tsien RY. 1994. Wavelength mutations and post-translational autooxidation of green fluorescent protein. Proc Natl Acad Sci USA 91:12501-12504.


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