The TRAP technique gets back to basics

When it comes to studying the gene expression of cells, or cellular states, there is constant compromise. The act of isolating cells and putting them into suspension is stressful, and unlike anything the cell has experienced. This can drastically change cellular processes and transcription. Even the technique we talked about before, Dropseq, which can do rapid single cell genomic studies, has this unavoidable caveat because the neurons must be removed from the brain. A neurons health depends highly on its environment, more so than other types of cells. Disrupting a neuron by taking it out of the brain, removing it's communication with fellow neurons, and introducing stresses, can quickly change genetic expression. To avoid these pitfalls, scientists have tried to develop systems that shorten the collection time of cellular materials. This keeps cellular exposure to stressful events at a minimum. The technique we'll talk about today, in a unique way, marks relevant genetic information using cellular machinery, and makes it easy and quick to collect.

To develop this technique, named TRAP (short for Translating Ribosome Affinity Purification), the scientists relied on their understanding of translation. After DNA is transcribed into mRNA inside the nucleus of the cell, it is shipped off into the cytoplasm or towards the endoplasmic reticulum. The mRNA is then recognized by ribosomes. The ribosome is the molecular machine responsible for translating mRNA into proteins. It is comprised of two distinct subunits, each made up of proteins and ribosomal RNA (rRNA). The researchers' goal was to separate and identify the mRNA from a specific subset of cells in the hopes that the genes the cells were expressing would expose new cellular traits. In order to do this, they wanted to separate the ribosomes, and the attached mRNA they were translating, from the cells they were interested in. They took advantage of their ribosomal knowledge to engineer one of the proteins that make up the ribosome to express EGFP at its N-terminus, as a type of tag. To express this specific EGFP-tagged ribosome only in the neurons they wanted to study, they used a promoter for a marker gene. This is a gene specifically expressed in the neurons they are studying. They would then genetically express that promoter in front of the gene they inserted for the EGFP-tagged ribosomal protein. In this way, the only cells that express the EGFP-tagged ribosomal protein would be the ones that expressed their marker of interest. The EGFP tagged protein would then incorporate itself into the normal ribosomes found in the cells. This is shown in the figure above, where only mRNA attached to the EGFP tagged ribosomes (green) would come from the cells they want to study.

The next step would be to isolate the EGFP-tagged ribosomes, and the mRNA its holding. In order to do this, the researchers first froze translation using a chemical name cyclohexamide. This stops all of the ribosomes in their place on the mRNA they are translating. From here, they collect an entire region of the brain that contains their neurons of interest, including many others they aren't interested in. They then homogenize the cells - blending them, more or less, making a cellular soup full of all the different cellular machinery, and most importantly, ribosomes from all of the cells. After this, the cellular soup is incubated with small magnetic beads designed to bind to EGFP. This will cause the EGFP-tagged ribosomes to stick to the beads. The pictures to the right show an electron micrograph of a bead incubated with EGFP ribosomes (right) and without EGFP ribosomes (left). You can see, when the ribosomes have EGFP, they are attached to the bead, and even have mRNA strands coming out of them. By incubating with these special beads, and using a magnet to collect them, they can select and isolate only the translating mRNAs from the neurons that express the EGFP-ribosomes; the neurons of interest. This data will be very accurate too, because the cells were stressed for only a few minutes prior to being homogenized.

From here, they went on to sequence and analyze the collected mRNA. This would paint them a picture of the genes that are highly enriched in their neurons of interest. In this report, the researchers compared the gene expression profiles of two closely related but distinct populations of neurons; dopamine receptor 1 (D1) expressing medium spiny neurons and dopamine receptor 2 (D2) expressing medium spiny neurons. These cells are part of a control center in the brain called the striatum that coordinates movements for the body. The diagram to the left shows a gene expression array comparing both populations of neurons. This plots the expression of every gene for both populations of neurons. If both populations show the same level of expression for a given gene, you'd expect that gene to fall on the line going down the center of the graph. This line means that it is expressed equally in the D1 and D2 cells. However, if it is highly expressed in one group of cells, and not another, like Drd2 or Penk1 in the D2 neurons, it will be far away from the line because there is nearly two times as much expression of those genes in the D2 population compared to the D1 population.

By comparing the genetic expression of these two closely related populations of neurons, they start to get incredibly accurate molecular characterizations of these neuronal species. They can distinguish important differences that define subsets of cells. By having information like this, we can make more accurate inferences as to what cells do, discover cells that are vulnerable to disease, and find further subclasses of neurons within that population. As an example, the TRAP technique has been used recently to study the aging process in neurons, and led researchers to find a potential modulator of Huntington's Disease. It's great to see that high impact data can come from new methods based on fundamental science. Sometimes, new biological techniques depend too much on complicated technologies. I think the biggest lesson from this method is that to make great strides in science one must take time to learn the foundational principles. Having a sound understanding about basic biological processes allows you to take advantage of something the cell does, and turn it to your advantage.

References:

Heiman. M,. et al. A Translational Profiling Approach for the Molecular Characterization of CNS Cell Types. Cell 135, 738-748 (2008.)

Heiman, M., Kulicke, R., Fenster, R.J., Greengard, P., Heintz, N. Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP). Nature Protocols 9, 1282-1291 (2014).

Image Credits:

All the images are modified from the Heiman et al. Cell paper cited above.