Wednesday, 16 December 2015

Fluorescent endosomes in a live cell

Lately I’ve been visiting conventions with a talk about fluorescent proteins. It’s a technique that allows to visualize different processes in living cells. For the most time in the history of biology we had to make do with fixed cells, which are obviously quite dead.

To do that, first we have to perform a transfection. This is a technique of transient genetic modification of the cells we want to look at.

If it sounds like a beginning of a sci-fi thriller - actually this is a very popular method. For a molecular biologist, as common as using a hammer for other professions. It is also much safer, because while you can hit your finger with a hammer, a transfected cell on a plate remains a cell on a plate and does nothing in particular.

We do not actually change the cell’s genetic material (most of the time that is). We make her take up plasmids. A plasmid is a circular DNA particle, used by bacteria as a handy upgrade to her genome. It usually codes antibiotic resistance, and since bacteria can exchange plasmids between each other, this is how antibiotic resistance spreads in nature.

Well, we can prepare a plasmid so the mammalian (or other) cultured cell can use it. And make her take it up, for example with lipofectamin. Mammalian cells usually lose the plasmid in a few days, or die from it, but we don’t care. We want to visualize them in a short time from the transfection (there are methods to make stable modifications to the cells, but I don’t want to dwelve into that right now).

Then, we can:
1. Make a cell produce a protein it usually doesn’t produce. Either because it can’t or because we made her lose that ability, or it’s a mutated protein. And investigate what happens.
2. Make a cell produce a protein it usually produces, but much more, to collect it later and investigate it in a large batch.
3. Make a cell produce that protein, fused with a fluorescent protein, to watch what happens live.

Fluorescent proteins were a discovery awarded with the Nobel Prize in 2008. While newspapers wrote about it, it is hard to imagine what we do with it, except for creating shiny mice (for the record, they do not fluoresce in the dark. They emit light when excited with another light, but with a chromatic shift - it has a different color than the original one).

What do I do with that, for example?

I watch endosomes.
I’m not sure if endosomes are organelles you learn about at school, so briefly: they are vesicles responsible for transport and/or degradation of other proteins. There’s a lot of kinds of them in the cell.
Faulty endosomes were found in neurodegenerative diseases such as Alzheimer’s, Parkinson’s or ALS. In other diseases too, but I am obviously biased as a neurobiologist.

These are endosomes. Live. Small balls flying across the rat fibroblast, cultured on a glass bottom plate, in the tasty medium.

In green, we see vesicles with EGFP-Rab5.
RGFP is green fluorescent protein, enhanced version. Rab5 is a small protein that appears on early endosomes.

In red, we see vesicles with Tag-RFP-EEA1.
Tag-RFP is a new red fluorescent protein. EEA1 is another protein found in early endosomes; it binds to Rab5. You can see that they go together.

Colors in this movie are digital; they reflect the real ones only because I made it so. The original movie is always black and white, recorded in two channels. I could make them violet and yellow and that wouldn’t matter, but this is less confusing.
I kind of like like them. Do you?

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