I'm gone camping. Late last week I preprogrammed my blog to repost all these entries on microscopy. Enjoy!
I read far too many papers where the author claims that their favorite protein "localizes to the leading edge in migrating cells". Then they show a pretty picture like this one:
The problem is that the cell thickens right at the leading edge. So if your protein is freely floating around, there will ALWAYS be more of it (in absolute terms) at the leading edge then in the nearby lamella. To underline this point, the image above is of fluorescent dextran microinjected into the cytoplasm of a fibroblast. Dextran is a molecule that is certainly not a marker of the leading edge. Here's another example for you, it's a FLAG tagged mitochondrial protein. Notice that some of the tag is not in the mitos - the tag probably got cleaved off before the protein was inserted. Where does this tag seem to end up? You guessed it.
So how do you show that your favorite protein is at the leading edge?
Controls folks, they're called controls.
So how to proceed?
You need to label a free floating protein to measure the thickness of the cell (or to control for the thickness of the cell). Take a picture of this free floating protein (we'll call it BS protein or BSP) and of your favorite protein (YFP) in the same cell. Then calculate the ratio of YFP/BSP in the lamella and in the leading edge. If the ratio increases as you move from the lamella to the leading edge, then you can safely conclude that your protein is enriched in the front of the cell. If not? Well then your protein is certainly not enriched at the leading edge, it's probably just free floating in the cell.
For more on the mysterious leading edge and cell motility, see this entry.
Gah!!! Yes, yes, a thousand times yes. It's particularly bad when someone publishes a fluorescent probe that localizes to membranes and then makes the claim that *clearly* the whatever-it-is is localized to the leading edge. You're looking at two fat layers of membrane squashed into the same focal plane, people!
Not that this makes it easy when you are actually trying to find real l.e. localization, but thank you for pointing this out again.
A little bit of microscopy knowledge is indeed a dangerous thing.