Green Fluorescent Protein - A Jennifer Lippincott-Schwartz Videocast

In the past 15 years, the two biggest technical advances that have helped us Cell Biologists are RNAi and green fluorescent protein, aka GFP.

You see before the advent of GFP, researchers could only analyze the distribution of proteins in a living cell by first fixing and thus killing the sample. No information could be collected from a protein within it's natural envioronment, that of active cytoplasm. We could figure out where proteins were at a certain point in time, but not how they behaved over time. You see we were missing the temporal dimension. Sure there were exceptions, Yuli Wang had microinjected rhrodamine conjugated actin into tissue culture cells and Tim Mitchison did the same with tubulin, but outside the cytoskeletal field little was know about the dynamics of proteins in living cells.

Then came green fluorescent protein from the jellyfish Aequorea victoria. Douglas Prasher first isolated and sequenced the gene but lost his funding before he could do any work on the protein (and then left academic science!) Martin Chalfie heard about GFP (at a talk?) got the DNA from Prasher as his lab was closing. Then the Chalfie lab published the first paper where GFP was expressed in a single cell type in C. elegans.

Then Jennifer Lippincott-Schwartz got a hand on GFP.

There are so many cool imaging techniques that were developed in her lab, it is truly breathtaking. She developed a photo-activatable GFP, the FRAP (Fluorescence recovery after photobleaching), and FLIP (Fluorescence loss in photobleaching) techniques that allow the microscopoist to measure the mobility of molecules inside the cell. Recent she helped develop photoactivated localization microscopy. Here's the abstract from a paper that appeared last year in Science:

We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to approximately 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method--termed photoactivated localization microscopy--to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.

Along the way her studies have revealed much of how organelles are made, destroyed. In fact last year's breakthrough studies on the transient nature of the Golgi validated much of her early work.

Then a couple of days ago we stumbled onto this conference she gave at the NIH (where she works). So if you are interested in microscopy, the development of GFP or organellar biology, I strongly recommend that you follow this link and watch her seminar.

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Jennifer Lippincott-Schwartz gave a talk here a couple of months ago, and I thought it was absolutely terrific. The resolution of imaging was almost at the level of EM, and . I was particularly interested to see if I could use PALM (which was partly developed in her lab) for some of my own work. One big bottleneck right now though is the depth of penetration of light. Right now they need to use sections, and cannot yet use fixed wholecells....which throws a spanner into my grand plans. But Herman Hess (now at Janelia farms, who developed PALM) thinks over time it can only get better (he gave a talk here a few weeks ago also).

I guess you might be most interested in the FPP assay, amongst others, that can tell topology and membrane orientation of proteins.

Terrific stuff.

IIRC, isn't it W.W.Webb at Cornell who developed FRAP a long time ago?

PK,

I looked it up and your are right. I guess Jennifer was the first to apply FRET to GFP.

Hey Alex,
I dont think Lippincot - Schwartz developed FLIP for the first time either and I also have doubts about the distinction of being the first to demonstrate FRET with GFP. (I just cant seem to put my finger on who it was but I should find out)
As far as PALM goes, most of the credit for the technique itself goes to Eric Betzig and Hess. Of course they also now have a nice little story of how they started off! I think Schwartz's system made FRET very convenient in biological systems.
Sunil pointed out sectioning being the problem in his comment but I think the bigger concern is the time of imaging being about 12 hours.

Sunil,

I've yet to read the FPP papers - I should check them out (I think she has a couple of them out).

Kaushik,

Sorry, I meant to write FRAP in that last comment. I do agree with you on the PALM, I guess I should have explicitly said that she played a minor role.