New & Noteworthy

Of Medieval Market Townes and Wasp Guts

February 3, 2016

As market towns like this one were a place where isolated medieval Europeans could find partners to take back home, so to are a wasp’s gut for yeast. Image from Wikimedia Commons.

Back before trains, planes and automobiles, people didn’t get around as much. And for the people of medieval Europe, this could be a real problem genetically.

At this time there were a lot of small, isolated villages scattered across Europe. If people in these villages stayed put, inbreeding might have gotten as bad as the poor Spanish Hapsburgs. Their last king, Charles II, was infertile, riddled with genetic diseases and his royal line died out with him.

One reason (among many) that this didn’t happen to people all over Europe was market towns. These were centrally located places where villagers came to sell goods. And where they also found partners to bring home to freshen up the gene pool.

Turns out that out in the wild, our friend yeast is in an even worse predicament than medieval Europeans. Because they are all clones of each other, they exist in isolated colonies with almost no genetic diversity.

Yeast are also way less mobile than people. They do have spores but these don’t tend to travel very far without help.

And yet, looking at yeast DNA shows that yeast definitely get around. There are all sorts of signs of various DNA mixing over time. So where are all these yeast hooking up?

A new study by Stefanini and coworkers in PNAS suggests that yeasts’ market towns are in the guts of wasps. It is there that various yeasts can meet and mate before heading back to their “villages.”

This makes sense in a lot of ways. First off, as we described in an earlier blog, there is good evidence that yeast winter in wasp guts.

So there are definitely a variety of yeast hanging around for months, waiting for warmer weather. The gut is also the kind of harsh place where spore dissolution, the first step in yeast mating, can happen.

When the authors looked at the yeast isolates from a wasp’s gut they saw a lot more outbreeding compared to other sources. This suggests that a lot of mating is indeed going on there.

The next step was to directly test how much mating can actually happen in a wasp gut. Stefanini and coworkers tested this by having the wasps eat five different yeast strains and then analyzing the isolates genetically over time. They compared the results from this experiment to the amount of mating that happens in wine must and under ideal lab conditions.

What they found was a whole lot of mating going on.

After two months, around 1/3 of the yeast in the wasp’s gut were outcrossed. This is OK but pretty comparable to what is found in wine must.

It was a different story after four months. Now 90% of the yeast were outcrossed. This is an even better result than scientists typically get in the lab. Clearly the wasp gut is a great place for a yeast to find a partner.

The authors also found that the S. paradoxus strain had to mate to survive in the gut. The only time they found this strain in yeast isolates was in hybrids with S. cerevisiae.

The next steps will be to see if this kind of mating actually has a big effect on yeast diversity in the wild. And of course what, if anything, the wasp gets out of hosting these cavorting yeast.

A market town was great for both the town and the visitors. People met up, sold goods, found partners and the towns prospered from all of this traffic. I can’t wait to find out if the wasp/yeast situation is so mutually beneficial as well.

Jerry Lee Lewis has a whole lot of shakin’ going on, just like a wasp’s gut has a whole lot of matin’ going on.

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Unfrying An Egg

January 20, 2016

Unlike the proteins in this egg, most aggregated yeast proteins get back to their normal shape after a heat shock. Image from Wikimedia Commons.

Eggs start out as slimy and awful, but can end up warm, firm and wonderful. All it takes is some heat to denature the egg proteins and voilà, a tasty breakfast.

Not that anyone would want to do it, but of course it is impossible to do the reverse. You can’t take a fried egg and turn it back into a raw one. The denaturation is pretty much permanent.

When a cell is hit with high temperatures, its proteins start to denature as well. And scientists thought that most of the denaturation of many of these proteins was as irreversible as the eggs. The thought was that many or most of these denatured proteins were “eaten” through proteolytic degradation. Although cellular chaperones are capable of disaggregating and refolding some heat-denatured proteins, it wasn’t known which aggregated proteins met which fate in a living cell.

A new study out in Cell by Wallace and colleagues shows that at least in yeast, most eggs get unfried. After a heat shock, aggregated proteins in the cell return to their unaggregated form and get back to work.

Now those earlier scientists weren’t crazy or anything. The proteins they looked at did indeed clump up and get broken down by the cell after a heat shock. But these were proteins introduced to the cell.

In the current study, Wallace and colleagues looked at normal yeast proteins being made at their normal levels. And now what happens after a brief heat shock is an entirely different story.

The first experiment they did looked at which endogenous yeast proteins aggregated after they were shifted from their normal 30 to 46 degrees Celsius for 2, 4, or 8 minutes. The researchers detected aggregation using ultracentrifugation—those proteins that shifted from the supernatant to the pellet after a spin in the centrifuge were said to have aggregated.

Using stable isotope labeling and liquid chromatography coupled to tandem mass spectroscopy (LC-MS/MS), they were able to detect 982 yeast proteins easily. Of these, 177 went from the supernatant to the pellet after the temperature shift. (And 4 did the reverse and went from the pellet to the supernatant!)

After doing some important work investigating these aggregated proteins, the researchers next set out to see what happened to them when the cells are returned to 30 degrees Celsius. Are they chewed up and recycled, or nursed back to health and returned to the wild?

To figure this out they did an experiment where proteins are labeled at two different times using two different labels. The researchers first grew the yeast cells at 30 degrees Celsius in the presence of arginine and lysine with a “light” label. This labels all of the proteins in the cell that have an arginine and/or lysine.

Then the cells are washed and a new media is added that contains “heavy” labeled arginine and lysine. The cells are shifted to 42 degrees Celsius for 10 minutes and then allowed to recover for 0, 20, or 60 minutes.

After 60 minutes of recovery, the ratio of light to heavy aggregated proteins looked the same as proteins that hadn’t aggregated. In other words, aggregation did not cause proteins to turn over more quickly.

It looks as if aggregated proteins are untangled and allowed to go about their business. So after a heat shock the cell doesn’t throw its hands in the air and simply start things over.

Other experiments done by Wallace and coworkers in this study, that we do not have the space to tackle here, suggest that the cell has an orderly process for dealing with heat stress. After a heat shock, certain proteins aggregate with chaperones in specific areas of the cell. Once the temperature returns to normal, these stress granules disassemble and the aggregated proteins are released intact.

None of this will help us unfry an egg — a denatured egg protein is obviously significantly different than an aggregated protein protected by chaperones in a stress granule. But this study does help us better understand how our cells work. And that’s a good thing.

Unlike Mr. Bill’s dog, most aggregated yeast proteins can return from a heat shock.

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Join SGD at The Allied Genetics Conference

January 12, 2016

TAGC 2016 imageSGD will be attending The Allied Genetics Conference (TAGC) in Orlando, Florida, July 13–17, 2016! For the first time ever, the meetings of the yeast, C. elegans, ciliate, Drosophila, mouse, and zebrafish model organism communities will be united under one roof, along with a new meeting on population, evolutionary, and quantitative genetics.

Submit your abstracts now! Abstract submission will be open until March 23, 2016. If you want GREAT science and access to the leaders of the field, then TAGC is the place for you. SGD will be there, will you?

Clearing Customs in the Nucleus

January 6, 2016

Getting through the nuclear pore is like going through customs at the airport. And now we can see the mRNA make this journey in real time. Image from Wikimedia Commons.

Going through customs at the airport is a necessary evil. Once off the plane, you need to stand in line, scan for an open station, have various forms looked over and possibly stamped before you can pass through the airport doors and get into a new country.

And of course if there is anything wrong, you can be sent back to get your papers in order. A pain but it does help protect people.

Things work pretty similarly in the nucleus. The mRNA disembarks off the DNA, gathers up a set of proteins, and heads for the nuclear pore. There its proteins are checked and if everything is in order, it is allowed to proceed to the cytoplasm. And if there are problems, it is denied entry.

A couple of new studies out in the Journal of Cell Biology use imaging microscopy to give us a close up view of the bustling airport that is the nucleus of a yeast cell. It is utterly fascinating.

Both studies showed that mRNAs often hang out at the nuclear envelope, pausing at a nuclear pore and then sometimes moving to a new one. And that factors both in the nuclear pore and bound to the mRNA affect this scanning of the nuclear envelope.

The basic strategy with both studies is to fluorescently label specific mRNAs in a live yeast cell and follow its journey from the nucleus to the cytoplasm. To do this, they also needed to fluorescently label the nuclear pores, the custom stations in the nuclear envelope.

They labeled the mRNA using the bacteriophage PP7 RNA-labeling system. Basically, they load up the untranslated region (UTR) of a specific gene with sequences that form specific loops. Once transcribed, these loops are then bound by fluorescently labeled PP7 coat protein. Now they can track this labeled mRNA.

To more easily track mRNAs, they chose low expressing genes. That way they could follow a single mRNA more easily. They also needed to get rid of the yeast cell wall so they could see inside the cell better.

Overall they found that at least in yeast, the mRNA takes around 200 milliseconds to get exported to the cytoplasm. Very little of this time is spent in the nucleoplasm; the mRNA very quickly makes its way to a nuclear pore.

Once there things slow down. The mRNA stays at a nuclear pore or slides along the nuclear envelope to a different pore in a process the authors call scanning. Eventually the lucky successfully make it through the pore to the cytoplasm where they can seek out a ribosome for translation. Around 90% of the mRNAs they studied made it through.

They had a couple of different ideas about why the mRNA hangs around the nuclear envelope for so long. One is that the extended stay at the pore is to make sure everything is in order with the mRNA. It can’t pass through customs unless all of the right forms have been filled out properly.

Another possibility is that by scanning it is looking for a nuclear pore that is competent for exporting. It has to search for an available customs agent.

Now that the authors had established a system to look at mRNA export, they next set out to see which factors play important roles. As you might guess, mucking with parts of the nuclear pores or the proteins that bind the mRNA can throw a monkey wrench into the process.

In the first study, Smith and coworkers looked at what happens to the process when one of the key mRNA binding proteins, Mex67p is mutated. This protein is known to interact with the nuclear pore.

Losing the nuclear basket means mRNAs fall away from nuclear pores more easily. It is like getting to the head of the line and then having it close for lunch. Image from thornet on flickr.

It has also been proposed that Mex67p is important in making sure the trip through the pore is one way. Once the mRNA goes through, it releases Mex67p which makes the mRNA let go of the cytoplasmic side of the nuclear pore. The imaging studies here confirmed that Mex67p is indeed important for mRNA directionality.

Using a temperature sensitive mutant of Mex67p the researchers found that the mRNA they tracked stayed at the nuclear envelope about three times longer than in a wild type strain. The process was also much less efficient with only 32% making it to the cytoplasm instead of the 90% seen in the wild type strain. And of the 14 mRNAs which failed to make it through the pore, 7 headed back through the pore to the nucleus.

In the second study, Saroufim and coworkers concentrated on a part of the nuclear pore called the nuclear basket. This is the first part of the nuclear pore that the mRNP, the mRNA plus its proteins, encounters.

They found that deleting or mutating two key parts of the nuclear basket, MLP1 and MLP2, made the mRNA linger for a shorter time at the pore. The mRNA no longer scans the nuclear envelope.

But that didn’t mean the mRNA passed through to the cytoplasm more quickly. No, it just tended to fall back into the nucleoplasm and then have to reattach more often.

It is as if you had to deal with a customs agent who keeps sending you back into the airport. Or agents who keep putting up the “Out to Lunch” sign as soon as you get to the head of the line.

These two studies give researchers a way to study mRNA export in live cells in real time. As we piece together which proteins play what role, we will get a better handle on this important part of gene expression.

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

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