Long Live the Proteins!

Proteins are the workhorses of the cell. They catalyze important reactions, provide support and scaffolding, transport packages through the cell, signal between cells, and do many other vital functions. Proteins are made by following instructions from genes. Protein expression is very dynamic, with old or damaged proteins being replaced by new proteins on a daily or even hourly basis. This process is known as “protein turnover”.

Protein turnover is important for two reasons:

1) If the protein is involved in a signaling pathway, you don’t want it sitting around being active if its purpose is to respond to a signal. For instance, if a hormone activates expression of Gene X that makes Protein X, you wouldn’t want Protein X hanging around all the time. It should only be expressed in the presence of the hormone signal. Therefore, these types of proteins have markers on them that tell the cell to degrade the protein after a certain length of time.

2) Proteins get damaged from normal wear and tear in the cell. Damaged proteins may start acting inappropriately, so they need to be destroyed before they start running amuck. Damaged or unfolded proteins may also start binding with each other to form big aggregates that clog up the cell. Something like this is what happens in Alzheimer’s and Parkinson’s Disease.

Therefore, as molecular biology students, we learn that protein turnover is an important housecleaning procedure in the cell. Imagine my surprise, then, when I read a paper this week about extremely long-lived proteins (that’s the official scientific term and it’s abbreviated ELLP). A few ELLPs were already known, such as a protein that insulates neurons, and proteins that control how tightly DNA is packed into the nucleus. The major finding of this paper by Savas et al. was of a new group of long-lived proteins that make up the nuclear pores. They did one experiment, made one figure and blew my mind.

Pulse chase experiment

In order to look for ELLPs, the authors did a “pulse-chase” experiment. The idea behind this type of experiment is to introduce some sort of a marker that can be traced for a long time. The “pulse” is the marker that is introduced for a limited time and the “chase” is waiting to see what happens to that marker. The authors want to mark proteins that are made at the beginning of life and then see how long those original proteins last in the body.

Proteins are made of amino acids which have nitrogen in them. Therefore, in order to label proteins, the authors used an isotope of nitrogen (15N), which is heavier than normal nitrogen (14N). By using a technique called mass spectrometry, they can tell the difference between proteins made with normal nitrogen and those made with the 15N isotope. They fed newborn rats with a diet that was rich in 15N, which then became incorporated into their newly synthesized proteins. The young mice were switched off the 15N food after 6 weeks, so later in life, if a protein has 15N in it, it must have been made during the first 6 weeks of life.

They examined liver tissue from these rats 6 months later and found that only a few proteins had 15N in them. This is consistent with previous findings which show that the proteins in the liver rapidly turnover. Using another rat, they examined tissue from the brain after 12 months and found many 15N proteins. In other words, the proteins in the liver are made new all the time, whereas in the brain, some proteins that are made during the first weeks of life remain there a year later. Keep in mind that the lifespan of a lab rat is about two years, so 12 months is half of its lifetime. It’s unbelievable to me that a protein would last at least half of the lifespan of an animal. It should be noted here that they only used one rat for each time point, so their sample size is essentially one.

The authors found some well known long-lived proteins, which shows that their experimental approach was valid. They also, surprisingly, found a set of long-lived proteins that are part of the nuclear pore complex (NPC).

Nuclear Pore Complex

DNA is kept in a membrane enclosed compartment called the nucleus. The nucleus has doorways, or pores, through which proteins and other molecules can pass in and out. Small molecules can easily go through this doorway, but bigger molecules, like proteins, need an escort to get through. The escort only binds to molecules that are supposed to go through the pore. The nuclear pore complex is this door between the nucleus and the rest of the cell (called the cytoplasm). The NPC checks that everything big that is trying to get through has an appropriate escort. The NPC is analogous to a doorman at a club who will only let in people who are on the guest list. There are some things that need to stay in the nucleus (like DNA and the machinery to express it) and some things that have no business being there, so it’s important that the NPC regulates this transport process.

Electron micrographs of NPCs from cytoplasm side (left) and nuclear side (right).

From http://learn.genetics.utah.edu/content/begin/cells/membranes/

One NPC is made up of 100s of proteins all bound together to create a water filled pore through the nuclear membrane. Only some of these NPC proteins were found to have the 15N isotope 12 months later, so some subunits of the NPC are rapidly turned over, like most other proteins. However, these findings imply that although individual components of the NPC may be replaced, the entire NPC as a whole is built to last for a very long time.

Conclusions

These findings are important, because long-lived proteins may relate to cellular aging. One can imagine that as cells age, their long-lived proteins become damaged, including those in the NPCs. In fact, this same lab has previously shown that NPCs in the brain cells of aging rats deteriorate and become increasingly more leaky. The NPCs may not work in the same way anymore and may allow toxic cytoplasmic proteins into the nucleus without the appropriate escorts. These proteins from the cytoplasm could cause damage to the DNA inside the nucleus, which would lead to more dire consequences for the cell, and may lead eventually to cell death. It is still unknown if there is any advantage to ELLPs and why they aren't replaced like all other proteins.

Objects in mirror may be farther than they appear

This week’s paper describes a newly discovered visual method used by jumping spiders to determine the depth of objects. Jumping spiders need to have good depth perception so they know how far to jump to attack their prey, which include fruit flies. Organisms use different methods to determine depth, such as binocular vision in humans and motion parallax in birds and other insects. For one of the first times, the authors describe how defocused images in the eye can be used to measure absolute distance of objects. The article by Nagata et al. appeared in a recent issue of Science.

Introduction to retinas

By Sunshineconnelly at en.wikibooks

The lenses in our eyes focus incoming light onto the back of our eyes, in an area called the retina. The retina is made up of specialized cells called photoreceptors, also known as rods and cones in humans. These cells express proteins, also called photoreceptors, in their membranes that change their activity when light hits them. Photoreceptors respond to certain wavelengths of light. For instance, in human cone cells, there are receptors that are activated most efficiently by red light; there are others for green and for blue light. When the photoreceptors become activated by light, they send this signal on to other cells in the retina and eventually to the brain, which interprets this as a visual image.

The light that hits our retinas needs to be focused on the retina in order for us to perceive a sharp image. In people who are near or far-sighted, their eyeballs are the wrong shape, so the light that hits the retina is not precisely in focus. Imagine there’s a pinpoint of light in front of you, if that activates a small precise area on the retina, it will be perceived as a small pinpoint. If the light is not focused properly onto the retina, more photoreceptors will be activated and it will be perceived as a bigger, blurry smear of light.

A near-sighted eye will focus the image in front of the retina. Corrective lenses (glasses) can make it so the image will be correctly focused onto the retina. Image from glassian.org.

Depth Perception

There are a number of ways that animals perceive depth in a field of view, but I will just mention the two main ones.

1) Binocular vision: In animals with two eyes that have overlapping fields of view (like us), the brain can calculate depth by comparing the angles of the light hitting both eyes.

2) Motion parallax: If you move your eyes while looking at a visual scene, objects that are closer will appear to move more than objects that are further. This works in humans, though it’s not our main way of determining depth. For many organisms which have eyes on the side of their head, like birds and insects, this is how they perceive depth.

In this article about jumping spiders, binocular vision was ruled out, because when all of the spiders’ eyes were covered except for one, the spiders could still easily jump accurately onto their prey. Motion parallax was also ruled out because jumping spiders do not move their heads before they jump. The authors asked what then could be responsible for the excellent depth perception of the spiders.

Spidey vision

Jumping spiders have four pairs of eyes (eight total eyes). In the front of their head, they have two anterior lateral eyes (on the side) and two principal eyes. The principal eyes are larger and are used for depth perception. The retinas in the back of the principal eyes have 4 layers, L1-L4.

Nagata et al. demonstrate that retina layers L1 and L2 have photoreceptor proteins that are most sensitive to green, and considerably less sensitive to red light. Layers L3 and L4 have receptors that are sensitive to UV light. Among layers L1 and L2, the first layer is at such a position to allow for light to be fully focused onto it. Layer 2 is closer to the lens, so unfocused light will hit it on the way to L1. This fact gives the first clue as to how spiders can detect depth.

Imagine there’s an object A closer to the spider than object B. Because of the way that lenses bend light, the image of the closer object A will activate a larger area in layer L2 of the retina, whereas object B will activate less receptors in L1 (see figure below, adapted from Nagata et al., Figure 1D).

The number of activated receptors in L2 could send a signal to the brain about how far away an object is. The bigger the area of activation in L2, the closer the object. In other words, the images will all be focused in layer 1, so that will produce a visual representation of the real object, but the “defocus” signal in layer 2 of the retina will encode information about depth of field.

Testing the theory

Different colors of light have different wavelengths and therefore have different focal distances. In other words, different colors will focus through a lens at different distances. A pure red light and a pure green light will focus onto the same spot of the retina only if the red visual image is further away from the lens than the green (figure below adapted from Figure 3A).

Remember that the receptors in the L1 and L2 layers of the retina are most sensitive to green light. When the spider is in natural light, it probably responds best to the portion of light that is in the green spectrum. The brain has learned that a certain amount of defocus activation in L2 under green light illumination (which would naturally be present in sunlight), translates to a certain distance away from the spider. The authors tested this by putting a spider in a container with a few moving fruit flies. Then they changed the illumination to pure green light coming from LEDs. The spider was still able to judge distance accurately and could catch the fruit flies without a problem.

Next, the authors changed the illumination of the chamber to pure red light. Let’s predict what will happen in this case. Although the receptors in L1 and L2 prefer green light, red light at a high enough intensity will still activate the photoreceptor cells the same amount (the authors did controls to test this). The spiders are used to getting depth information from green light, though. When a certain number of photoreceptors in L2 are activated, the brain knows that this is a certain distance under green light. A object lit up with red light that is farther away would activate the same number of photoreceptors. However, the brain doesn’t know that this is red light now and doesn’t know about different focal distances of light. Therefore when x number of photoreceptors are activated, the brain will think the red object is a closer green object. One would predict that the spiders would jump inaccurately to shorter distances with red light.

The results of the experiment under red illumination prove that this model is correct. The spiders consistently jumped too short and often missed the fruit flies altogether.

Conclusion

This is one of the first examples of an organism using “defocus” to determine depth. The layout of the two green-sensitive retinas allows for the spiders to use one retina to create an accurate representation of the object and the other retina to determine depth. The authors suggest that this information from the spider could be used to inspire developments in artificial computer vision. I like the idea of technology learning from biology which developed through years of evolution.