Transcription imaged in real time in single eukaryotic cells

Dr. Robert Singer and colleagues at Albert Einstein have observed transcription in single eukaryotic cells -- that's right single cells.

I will divide how into two groups for the initiated and the uninitiated so you can both see how cool this is.

For the uninitiated: In order for a cell to make proteins, it must first make a copy of its DNA to send to the cellular machinery that makes proteins. This is called an RNA molecule. While scientists have developed a bunch of ways to look at RNAs, all those methods up until this point were done on the collection of RNAs from many cells rather than a single cell.

This group developed a new way to image RNAs in a single cell that works sort of like this. They genetically engineered the cell such that when it produces the RNA they want to see it has an extra bit added to the front. This extra bit is a sequence from a bacteriophage -- a virus that infects bacteria. If you are bacteriophage, one of the big problems in the course of your day is how to make more bacteriophages. Bacteriophages are very tiny and in order to get all of their genetic material into a small space they have developed proteins called coating proteins that bind to the material -- in this case RNA not DNA -- and squish it into a package. Which brings us back to how they see RNA. In addition to changing the RNA they want to see, the group also made these cells express a protein -- also from the bacteriophage -- that binds to the part that they added on the front of the RNA. This is a coating protein. To this protein they tethered a protein that glows called GFP -- a protein that scientists stole from a fluorescent jellyfish. (What is the tally up to now? In this system we have a normal cell plus a bacterial coat protein plus a protein from a jellyfish. Mad science, I tell you, mad!!!)

When the RNA is created the fusion protein -- the bacteriophage coat protein plus the GFP -- binds to it. You can see the GFP using a special microscope as a point of light. You can even measure how much RNA there is and where it goes, all in a single cell. More information can be found in the press release here.

For the initiated: I looked it up and similar systems were used to observe transcription and even the motion in the cell of RNAs in E. Coli, but this is the first to do it in a eukaryotic cell. They create transgenic ameoba that overexpress a GFP fusion construct linked to a bacteriophage coating protein, MS2. They also modify the RNA they want to image -- dscA -- to contain multiple MS2 RNA binding domain loops. What results is a punctate distribution of fluorescence on top of a more diffuse background. They estimate that they are observing on the order of 10 copies of RNA.

When they image the cells over time they find some interesting results. 1) They expression in a group of cells is heterogeneous. At no time are all the cells expressing; rather they tend to express in clusters. 2) Expression occurs in pulses of variable but generally short duration (about 5 min).

A description of the technique used in E. Coli can be found here. Here is the paper itself. Sadly subscription are probably required for both.

Here is a video of a group of cells with the construct. If you look closely you can see puncta of expression.