Experiment one — A rose by another name
Where lab weed is defined and fluorescent marker proteins explained, at least a little.
Sunday 8/17 First experiment or “a rose by another name”
Early Thursday afternoon, I did my first piece of lab work. Darren introduced me to the Leica SP-5 confocal fluorescence microscope and I took a few movies of a growing root. No philosopher could term this an experiment, I was not varying x to see if it made any difference to y. Is any piece of lab work “an experiment”? This is a semantic issue that I will leave to the wordwrights. I was mucking about in the lab, call it what you will. What I was doing had a two-fold purpose. First, getting to know the confocal computer control and the microscope it is built around. Second, making movies to serve, with any luck, as sufficient raw material for my image-analysis collaborators, although more than likely I shall have to take a few more movies.
The movies I made were of the root of a special line of lab weed. “Lab weed”? Yes, lab weed is the plant, and here comes a rant. Lab weed is what I call the popular model plant species, Arabidopsis thaliana. After all, the species rampages among the greenhouses and growth chambers of universities and research institutes around the world. Plants with a long history of use by humans have vernacular names, like pea or rice, that are in the common language as well being in fact common; but A. thaliana is an ephemeral plant, too small and unremarkable to have been much spoken about before its discovery ofthe biological laboratory niche. It is true that collectors have recorded “Thale cress” and “mouse-eared cress” in use by British gardeners but these names, though perhaps still extant, are hardly recognizable.
Another choice would be to call the little weed “arabidopsis” after its genus. That’s reasonable, many plants from azaleas to zinnias are named after their genera. But oddly, popular opinion among scientists is that this should be written as “Arabidopsis” or “Arabidopsis”. The first one is obviously wrong because Arabidopsis means the genus, by long established rules of taxonomic nomenclature. One of the smarter things that Linneas did to ensure his system became the law was to decree that all scientific names of organisms were to be Latin, thus ensuring that neither the French, the Germans, nor god forbid the Swedes, would be in a privileged position vis a vis plant and animal names. An equally hoary tradition among printers of English is that foreign words are set in italic type. Thus, genus and species names are in italics because they are Latin. Conversely, non-scientific names (like pea or rice) are in the vernacular language (in this case English) and thus are not set in italic. What about “Arabidopsis”? What is the meaning of the capital letter A? None. It is not a proper name (compare “Queen Anne’s lace”), and most importantly, we do not wish to refer to the genus, Arabidopsis, which contains about ten species, and that capital letter bespeaks generic (i.e., genus level) rank.
I don’t know why scientists, and I might add journal editors, have gotten this so bolloxed up. Maybe everyone has more important things to do than to care about how things are named? Maybe plant scientists are insecure and they feel they will get more attention from a Latiny thing? But there it is, and so lab weed.
End of rant, back to the movies. I know, I know, everyone wanted another fable but all I gave them was a rant. Sorrrrrreeee!
As I said, there is a special line of lab weed that I imaged. This line expresses a protein that is resident in the cell’s plasma membrane (the membrane that surrounds the cell) and that has an attached fluorescent protein called, prosaically, green fluorescent protein. Another thing that scientists do is to abbreviate almost anything and so hardly anyone ever writes “green fluorescent protein”, and instead it is GFP. This acronym is getting to be almost as famous as DNA. The discoverers of the protein were awarded the Nobel prize a few years ago.
The opinion of the Nobel committee and the widespread popularity of GFP arises from a few key features. Frist, the protein is intrinsically fluorescent. This means that when the cell makes synthesizes GFP, the protein molecule is automatically ready to see. There were other proteins in use but to become actually fluorescent the cell or organism making them had to be bathed in some effector molecule (this is always tedious and often toxic to the organism). Second GFP is small, compact, and inert. This means that when the GFP is used to tag another protein, the GFP rarely interferes with that protein’s regular business. Incidentally the fact that just about any protein in the cell tolerates having a GFP on its back without a flinch seems to be saying something interesting about how we should think about the inner workings of cells, but I am not sure what it is.
OK, so in the line of lab weed that I have, GFP has been added to a protein that spends most of its time in the plasma membrane. I don’t even know what that protein is. This is a little embarrassing but at the end of the day (or any time of the day) it doesn’t really matter, at least not for what I am doing. What does matter is that the line synthesizing the tagged version suffers no ill effects from the extra protein. This is true as far as everything that has been observed to date in this line, and it is a workhorse at CPIB. Thus, ill-effects if they occur at all, are at least subtle. For me, the key deal is that the GFP-tagged protein in this line is in the plasma membrane, and that there is lots of the protein, and in every cell of the root. Why? Because when I image the root of that line through a fluorescence microscope, I can see every cell as though I had drawn around it with a pen. FIGURE.
And so we come to the actual work (there’s a good word for what I did in the lab, “work”). I put the seedling in a suitable dish, with a coverslip for a bottom surface, and placed some water and a small square of agar on top of the root tip. The agar block is a nice gentle way to hold the root steady and right up against the coverslip surface, a proximity that is ideal for imaging. I imaged with a 20x lens, which allows me to have a reasonably large field of view, but still high enough magnification to see cells clearly (FIGURE). Fortunately the confocal is a powerful instrument with a sensibly streamlined interface so I needed only a little bit of instruction to learn how to get images, which I think will be just fine for my purpose. I imaged the root as it grew, with various time intervals between images. That is, the thing I varied was how long between frames in the movies. So hey, maybe it was an experiment! I used 1 min, 30 sec, and 15 sec, and took ten frames worth, for two different roots. I am not sure if ten frames will be enough for the image analysis team, but I think so. I also don’t know which frame interval will suit them best. That is the reason for the variation.
The plan now is to hand off these movies to my colleagues and see if they can work out how to track each of the cross walls as it moves. But before I can do that, I have to get the movies out of the proprietary format of the Leica confocal and put them into nice simple TIF files. This also requires me to be able to log onto the shared drive where the movies are stored. These things are supposed to be simple but will probably be a bit of cowbird the first time around. That’s on the agenda for this week. Stay tuned!