Thursday, January 6, 2011

Moving Targets within a Cell

Anyone who has had an introductory molecular biology class knows that the genetic information for almost all living things (this idea gets trickier if you count viruses as living things) is stored in the form of DNA, or deoxyribonucleic acid. DNA is an elegantly simple, stable molecule that is a candidate for being the longest naturally occurring molecule on the planet (in your human cells, a single molecule of DNA would be two meters long if stretched from end to end – and that’s just from one cell!). From your introductory biology class you should also remember that, while DNA stores the genetic information, this information has to be turned into protein (the stuff that actually IS you) via an intermediate called RNA, or ribonucleic acid.

That one little “deoxy” difference between DNA and RNA makes RNA a very dynamic (and frequently unstable) molecule. In chemical terms, “deoxy” is referring to the lack of an Oxygen atom (or hydroxyl group) in DNA that is present on RNA:

Oxygen, if you don’t already know from principles of how a fire burns, is a very reactive species in the chemical world, so it makes sense that the presence of an extra oxygen atom in the RNA molecule makes it more unstable than DNA. However, the dynamic nature of RNA makes it really handy for all kinds of other chemical reactions and cellular processes – another way to say “unstable” is to just say “reactive”; and in your cells, millions of chemical reactions take place all the time, so a reactive RNA is really useful. In fact, it turns out that your cells can regulate on a finer scale whether a gene is turned on or off at the RNA level than at the DNA level.

Think about it: if you want to turn a gene on (genes are made of DNA), you need to get all the molecules situated just right over the gene so that it can make copies in its intermediate form of a particular kind of RNA called messenger RNA, or mRNA. Those mRNA copies can then go out of the nucleus into the rest of the cell and be turned into protein. Of course, you only have two copies of a gene in your cell, a pitifully small number. How can you make billions of copies of a protein from only two copies of DNA? Well, your cell makes thousands of mRNA copies of the DNA gene. Those thousands of copies of mRNA go out into the cell and are used by other molecules to make protein – usually, one mRNA copy is read over and over again to make lots of protein, and that’s how you get millions of copies of a single protein in a short amount of time.

When a cell is done making and using this protein, it can go down to the DNA to “shut off” the gene by removing all those molecules that are making mRNA copies, which will in turn keep any new protein from being made. That is, as long as the mRNA copies that are already present simply go away once the DNA gene stops making them.

But what exactly happens to an mRNA copy after the DNA gene is turned off? Does it just degrade and disappear? Can it linger around? Can it be “turned on” and “turned off” like a DNA gene can? These are all questions that people who work on RNA biology have been asking for quite some time, and the answers can be pretty amazing and pretty complex.

While other classic research has shown that mRNA copies can hang around and be turned “on” or “off” regardless of what’s happening to the DNA gene, a study published in 2007 showed some pretty cool results demonstrating that RNA copies can be trafficked around inside a cell by riding along one of the biggest structural proteins inside a cell: microtubules.

Michael Blower at Harvard University, in collaboration with Karsten Weis and Rebecca Heald at UC Berkeley showed unambiguously that mRNA copies can bind to microtubules and play an important role in localizing their protein products to a specific site within the cell. As you can imagine, trying to get enough molecules to do experiments can be hard sometimes, especially if you have to do all your experiments from a single cell. However, scientists have clever ways to amplify material, and they have a habit of studying animal models that have REALLY big cells to make this easier. This is where females from Xenopus laevis, or frog, come into the picture. Xenopus females lay incredibly large eggs (an order of magnitude bigger than human eggs) and in very large numbers (400-500 at a time!). Blower and colleagues used these massive cells to extract and purify allll the microtubules (MTs) from the rest of the cell – what they found is that there are mRNA copies stuck to the MTs!

This graph is showing you all of the mRNA copies in the Xenopus eggs and whether they are bound to MTs or not. The X-axis is showing you “enrichment on MTs” and the higher the number, the more it’s enriched. The Y-axis is showing how many mRNA copies are enriched at that amount. For example, there are 140-160 mRNA copies that are approximately -1.4 Log2 enriched, and only 5-10 mRNA copies that are about 1.3 Log2 enriched. Notice that a lot of the transcripts are not enriched; in fact, they’re in the negative numbers indicating that they aren’t found on MTs but elsewhere in the cell (makes sense). However, a select few mRNA copies are actually enriched. Fortunately, the experiment that gave them this pretty graph also gave them the identities of each and every one of those mRNA copies.

So they did the next logical thing: if one experiment suggests that certain mRNA copies are stuck on MTs, then this should be visible in the cell. They decided to check it out using a microscope by labeling an mRNA and MTs to see if they look stuck on each other (the scientific word for “stuck on each other” is “overlap”). Don’t you worry about how they labeled it – that’ll be an entry for another blog post some time.

The red color indicates MTs and the green color indicates potential mRNA copies that should overlap (blue represents the DNA, but just ignore that for now). Notice that the left two panels show green mRNA copies that actually do overlap in the same region as the red MTs – this is exciting and confirms their finding. On the right you see a negative control – or an mRNA copy (called net1) that should NOT bind MTs, and indeed you don’t see any green staining, do you?

This is really cool, but the people who gave these researchers money to do research are … well, they’re taxpayers! And taxpayer money (funneled through the National Institutes of Health, or NIH) means that there’s usually a pretty strong interest in human experiments, not frog experiments. So the next question was, “If this happens in frogs, can it happen in humans?” And that’s exactly what they checked – so they took some human cells that grow in culture (no human eggs or anything like that, just human body cells), purified allllll the MTs away from the other stuff, and did the same initial experiment:

Notice again that a few mRNA copies are enriched on MTs, but most are not. This is a pretty cool idea: the same thing that can happen in frog eggs is going on in human cells too! But why would mRNA copies want to be on MTs? If mRNA copies are located on MTs, does that mean protein is made on the MTs as well? The process of reading mRNAs and making protein from them is called translation. So the scientists in this paper performed an experiment to test whether translation (the process of making protein from mRNA copies) is occurring on MTs. A clever system was adopted from previous work devising a method of labeling sites of active translation with a derivative of the antibiotic puromycin. Puromycin kills cells by getting lodged into the molecular machinery that turns mRNA copies into protein. So scientists made a version of puromycin that is tagged with a fluorescent molecule to see under a microscope. By injecting small amounts of this glowing puromycin into cells, it lodged itself into molecules called ribosomes, and labeled active sites of translation (such small amounts were injected that cell death was not a concern):

It worked! And it appears that translation is occurring on MTs. Notice the green puromycin in the left panel, which labels active translation, overlaps with the red color labeling the microtubules. You'll also note that it seems to be concentrated green at the tips of the red staining. That area is called the spindle pole, and the right panel shows a different green marker, ribosomes. Ribosomes are the molecular machinery that promote translation of mRNA copies into protein. So translation machinery is located on the MTs and at the spindle poles, and we know that active translation is occurring on MTs because the puromycin stains it too!

This is a pretty clever trick to answer their question, and they also proceeded to show that the same phenomenon occurs on MTs in meiosis as well as mitosis (remember that meiosis is cell division of the sex cells in your body, but mitosis is cell division of the rest of the cells in your body - the focus is on cell division because that's when lots of MTs organize to form really clear structures that you can look at). The last question they attempted to address was, “Is the process of translation necessary to move the mRNA copies to the MTs?” This is more important of a question to answer than you realize, but it is difficult to know what is responsible for moving mRNAs around a cell.

One hypothesis is that the translation machinery moves mRNA copies to MTs, so that’s one of the easier questions to answer. This time, the experiment required a bigger dose of purmocyin to stop the process of translation altogether. If translation is stopped altogether, then the mRNA copies may or may not be located on the MTs. The results are below:

The microtubules are in red and the mRNAs are in green. The top row is normal cells and the bottom row shows cells treated with the translation inhibitor puromycin – notice that the green mRNA copies still localize to the MTs even when translation is inhibited with puromycin in the bottom row.

These experiments, among others, make this a great paper. However, there are many questions that are left unanswered. Why are some mRNA copies localized to MTs but not others? What molecules and processes are responsible for bringing the mRNA copies to the MTs? If the mRNA copies are not allowed to localize to the MTs, will something bad happen? You can easily see how the experiments for the next possible paper are shaping up.

If you found these results interesting, you might also like this incredible video made at Harvard, animating the life of a cell. In it, you’ll see one depiction of how scientists currently think mRNA copies and other cargos are moved along microtubules (to some pretty awesome music no less).

Stay tuned for more science next week!

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