Go go gadget extendo filopodia

I’m back from an intense semester of learning and teaching Developmental Biology.  One theme that emerged from my studies was that the development of organisms is centered around gene expression and cell to cell signaling.  Often times, one cell will differentiate into its mature form, and then release a signaling protein that tells neighboring cells what to develop into.  For instance, the nervous system is induced by signals released from the embryonic backbone.  There are a number of common signals that are used over and over throughout development, like BMP, Wnt and Shh.

A recent paper by Sanders et al., published in Nature, looked at how distant cells can signal to each other via the Shh pathway.  Unfortunately for Developmental Biology teachers everywhere, Shh stands for Sonic Hedgehog.  Oftentimes, strange or humorous gene names like this can be blamed on the fruit fly researchers who first discovered the gene, but in this case everyone is to blame.  This gene was originally discovered by researchers studying fruit fly embryonic development; they named the gene hedgehog because the mutant embryos had lots of tiny bristles all over, kind of like a hedgehog.  The mammalian researchers took it to the next ridiculous level, by naming the mammalian version of this gene Sonic Hedgehog.  The Shh protein is a secreted signal that binds receptors on other cells, which activate gene expression in the receiving cell.  Shh signaling is important for specifying many different cell fates, such as the different neurons in the spinal cord, the cells that become the vertebrae, as well as the formation of the digits of the hand.

Although Shh is secreted from the cell, it has chemical modifications that make it stick to the plasma membrane that surrounds the cell that released Shh.  How then can Shh induce the development of cells that are located at a distance?  Well, the answer is by stretching out long cellular extensions with Shh localized at the tip.

Shh Filopodia

Sanders et al. did live imaging of cells in the developing limb of the chicken using fluorescent proteins.  They did some genetic trickery so only a few cells were labeled in red and others in green.  This way they could detect individual cells in a sea of unlabeled cells and examine their structure in real time.  They observed individual cells extending long protrusions, called filopodia, from the cell bodies.  These filopodia could stretch long distances (150 micrometers, like 3-5 cell widths) and were dynamic-- retracting and growing over time. 

How to think about filopodia?  Imagine a stretchy balloon with a stick inside of it.  If you could push that stick into the wall of the balloon, the balloon would protrude from that one spot as the stick pushes it out.  That is like a filopodia, where the balloon wall is the plasma membrane and the stick is a protein called Actin.  Actin forms long chains that can grow, pushing out the membrane in front of it. 

The thin, string-like extensions from this cell are filopodia and are filled with Actin.  Image from proteopedia.org

The authors then labeled the Shh protein with another fluorescent marker and saw that it localized to the tips of filopodia.  Not only that, but the filopodia expressing Shh were more stable and did not retract as often.  In order for Shh to act as a signaling molecule, it has to bind a receptor on another cell.  Using a different color, the authors observed two co-receptors for Shh localized to filopodia from other cells.  They even saw filopodia from two different cells make contact with each other, where one cell expressed Shh and the other expressed the receptors.

This is amazing!  Instead of releasing a signal out of the cell with the hope that it goes to the right place and isn’t degraded, the cells literally grow to the right place with the signal on their membranes.  This is like hand delivering a note to your coworker, rather than making the note into a paper airplane and throwing it in the direction of their desk.

This is how I imagine this working.  Two cells that are located at a distance, reach out extensions and meet somewhere in the middle. The Shh signal would bind the receptor, causing changes to the pink cell.

A study like this could not have been done before recent innovations in live imaging and molecular biology to introduce the fluorescent proteins into the cells.  The filopodia are not preserved during the more traditional, static method of fixing cells with formaldehyde and then staining them.  Who knows what other tricks live cells use during embryonic development.  I suspect this is only the beginning.

Swapping eggs

This week’s paper describes a new technique that could be used to manipulate human oocytes (i.e. eggs) to prevent a group of diseases called mitochondrial diseases.  The paper was presented by Tachibana et al. in Nature along with a similar paper by Paull et al.  For the sake of brevity, I will only discuss the findings from the first paper.

Mitochondria

So what are mitochondria?  Mitochondria are little compartments in the cell that make cellular energy.  They convert the energy stored in food into an energy source that the cell can use to drive chemical reactions.  In other words, they are absolutely essential for our survival.  The oxygen that we breathe in goes to the mitochondria to aid in this energy conversion, and we all know how vital oxygen is. 

There are two other interesting facts about mitochondria that relate to our story:

1) All the mitochondria in our body are duplicates of the mitochondria that were in our mother’s egg.  In other words, embryonic mitochondria are not made from our genomic DNA (gDNA) or from sperm contributions.

2) Mitochondria have their own DNA , which directs the synthesis of proteins that are necessary for their function.  This DNA is known as mitochondrial DNA (mtDNA) and it is only inherited from the mother, since all mitochondria originate from the egg.

If there are mutations in the mtDNA, then this can lead to problems with the synthesis of cellular energy, which can lead to human diseases known as mitochondrial diseases.  There are different types of mutations, which can affect people in different ways and with differing severities.  In this paper, the authors propose a way to prevent mitochondrial diseases from being inherited from generation to generation.  Let’s see how that works.

Nuclear transplantation

Let’s say you have a female patient with a mitochondrial disease, who wants to have a healthy child.  She is guaranteed to pass this disease on to her child via the mitochondria in her oocytes.  However, most of what makes the child “hers” is what lies in the mother’s genomic DNA, not in the mitochondrial DNA.  What if you could take the mother’s genomic DNA (plus the DNA from the father) and stick it into a healthy “enucleated” oocyte from a donor who has good, functioning mitochondria?  All the genomic DNA will have to be cleared out of the donated oocyte first, creating an enucleated egg.  The embryo that results from this nuclear transplantation will have genomic DNA from its mother and father, but its mitochondria will originate from the donor oocyte.  This would circumvent the mutated mtDNA that is in the real mother’s oocyte.

Tachibana et al. obtained human oocytes from volunteers and transfered the genomic DNA from one into another.  They then injected these oocytes with sperm DNA (like during real fertilization) and observed what happened.  Some oocytes failed to be fertilized and others died soon after, but a handful of oocytes survived into the blastula stage of development.  You can’t really grow a human embryo in a dish beyond the blastula stage and they are not allowed (yet) to implant these into women, so we don’t know what would happen to a child born from this procedure. 

They did carry out the above scenario with monkeys.  They transplanted the genomic DNA from one oocyte into another and implanted the blastula into another female monkey who carried the embryo to term.  The monkey youths are 3 years old now and doing just fine.  Their maternal genomic DNA is from one mother and their mitochondria are from a different oocyte donor. 

Isn’t this amazing?  I seriously doubt this procedure will be approved for human use anytime soon, because it’s too much like cloning, which basically follows the same procedure of putting genomic DNA into an enucleated egg.  It's a cool idea, though. 

Isolation and drug addiction

We all know that adverse, early life experiences can affect normal development and the ability to lead a happy and healthy adult life.  A number of recent studies have shown that rodents which are mistreated as pups have long lasting changes to their gene expression (i.e. epigenetics).  They are more anxious and have a harder time forming new memories.  A paper this week in Neuron builds upon these results, by studying the effects of social isolation on the “reward pathway” in the brain.

Reward pathway

What is a reward pathway?  Deep in the brain is a region known as the ventral tegmental area (VTA), which makes connections to the nucleus accumbens and prefrontal cortex.  When we do something that is naturally good, like eating or sex, the neurons in the VTA release dopamine onto the nucleus accumbens and we interpret that as “feeling good”.  This is our reward for doing something that will help us survive and procreate. 

The Reward Pathway in a brain cross section (from brainfacts.org)

Many drugs of abuse like cocaine, amphetamines and alcohol increase the amount of dopamine signaling in this pathway; this is one reason why drugs produce a “high”.  When this pathway gets overstimulated by increased drug use, the brain will try to compensate by making the pathway less efficient.  This is why drug users feel depressed when not on drugs and why higher and higher concentrations of drugs are necessary to produce the same high feeling.  This is a neurological explanation for drug addiction.  Drug abusers also start to make connections in their lives, and in their brains, between environments (a certain room, certain people, etc) and the feeling of reward.  Getting sober is so difficult because the brain has to unlearn these connections and the reward system has to recover back to its normal level of activity.

Plasticity

Before we talk about the paper, I need to introduce one more concept.  Neurons become activated when channels in their membranes open and positive ions rush in.  They can then pass on this signal to another neuron by releasing neurotransmitters (like dopamine) onto the next neuron.  The activity in the neuron and the amount of transmitter it releases into the synapse can change over time, based on that neuron’s previous experiences.  This is known as synaptic plasticity.  There are short-term changes, like facilitation, and longer-term changes (we’re talking hours and days here).  One of the more famous types of long-term plasticity is called long term potentiation (LTP) and is thought to underlie learning and memory.  When drug users start to become addicted, these types of long term changes to neuronal activity are occurring throughout the reward pathway.

Social isolation and VTA neurons

The experiment begins when young male rats were either housed together in groups of 3 or alone.  The researchers then recorded neuronal activity of VTA neurons under various conditions.  They found that rats that were isolated for more than 3 weeks, specifically during the equivalent of the rats’ early adolescence, can more easily induce LTP in the VTA neurons.  In other words, rats that had no social interactions during a critical period had more sensitive VTA neurons.  That is to say, their reward pathway is primed to be overstimulated, just like during repeated drug use.

What are the behavioral manifestations of having a sensitive reward pathway?

The next set of experiments they did is called conditioned place preference.  The rats were placed in a cage that had two different compartments, with different wall colors and floor textures.  The rats were then injected with amphetamine in one of those particular compartments, so they learned to associate the drug high with that environment.  The rats were then given a choice between the two compartments and inevitably they went to the room that was associated with the drug.  The researchers found that isolated rats had a greater preference for the drug room and developed the preference sooner than the control rats.  Social isolation as an adolescent causes an increased rate of learning an association between drugs and environment.  This could make these rats more vulnerable to drug addiction. 

What about unlearning the drug association?

After the drug testing, the rats were exposed repeatedly to the drug room, but this time they didn’t receive any drugs.  This is called extinction of a memory and it is measured by the rats losing their preference for the former drug compartment.   Socially isolated animals had a significantly slower rate of unlearning the preference.  Their memory was more resistant to extinction.  If their VTA neurons are overly sensitive, then it may be harder to rewrite that connection in the brain between environment and reward.

In the context of drug addiction, these findings are big.  An adverse early adolescence can prime the brain to develop addiction more easily and make it harder to sober up.  If the VTA neurons start firing every time you go through an environment associated with drugs, you’re going to want to take a hit again.  The authors bring up an interesting point that social isolation generally causes a depression of neuronal activity in places like the hippocampus (the site of learning), so maybe the increased activity in the VTA is the way for the brain to maintain some sort of homeostasis – some areas increase, some decrease, but overall the brain may have normal amounts of activity.  This is an interesting way of looking at this problem.  I suspect that social isolation offers little in the way of rewards, so the reward pathway is trying to compensate by getting more sensitive.  It will be interesting to see if there is also a connection with changes in gene expression.  The authors explain how the VTA neurons get overactive, from a cellular point of view, but what actually initiates those changes?  And how can social interactions feed into the biology of the cell? 

Burning carbs in the Andes

I am finally back from a very busy semester.  I taught physiology classes at Mills College and UC Berkeley this semester, so I have been interested in new topics in human physiology.  This week’s paper by Schippers et al. came out recently in Current Biology and describes adaptations that mice must make in order to live at high altitude.  They compared the metabolism of mice that live at 4000m above sea level in the Andes where the oxygen content of air is about 13%, to mice at sea level, which contains 21% oxygen.  We all know from experience that we need oxygen to survive and it’s harder to exercise at high altitudes, but why do our bodies actually need oxygen?

Oxygen in cellular respiration

Most all physiology can be explained by the following equation, which describes the process of cellular respiration:

Glucose + 6 Oxygen (O2) --> 6 Carbon dioxide (CO2) + 6 Water (H2O) + 34 ATP

Glucose is a simple carbohydrate (sugar) that we use as a direct energy source.  Glucose, which is 6 carbons long, gets broken down step by step in a series of chemical reactions.  At each step, a little bit of energy is released by the reaction and that is stored in carrier molecules.  These carriers then donate the energy in the form of electrons, which is then harnessed to make another molecule called ATP.  ATP is cellular energy.  The chemical bonds in ATP store high energy and can be used to drive other cellular reactions, like pumping ions, or the process that causes muscle contraction.  Without ATP we die. 

But what does oxygen have to do with this?  Well, the electrons that are donated by the carrier molecules must hop from protein to protein in what is known as the “electron transport chain”.  The final electron acceptor is oxygen (O2), which forms water with that extra electron.  That’s it.  That’s why we breathe, that’s why our heart pumps blood— our tissues need energy (ATP) to perform cellular tasks and in order to get energy from glucose, we need oxygen to accept the final electron.  Carbon dioxide is produced as a byproduct and is removed from the body during exhalation.

Glycolysis is anaerobic respiration and does not use oxygen. Note that oxygen is used as the last step of the electron transport chain to make the majority of the ATP. (www.phschool.com/science/biology_place/biocoach/cellresp)

I should also mention here that the more our tissues are active and working, the more ATP they need and the more O2 needs to get to the cells.  When we exercise our muscles are very active, so that’s why the heart rate and breathing rate increase; our body needs to intake more oxygen and distribute it faster to our muscles.

You can see that oxygen plays a critical role in our cells, so the mice at high altitudes are going to have a harder time getting their cellular energy.  How do they manage to run around when there is so little oxygen?

Energy sources

As I mentioned above, we can make ATP directly from glucose (a carbohydrate).  We can also make ATP by using fats as an energy source.  There are two differences between these two energy sources:

1) When we use fats as an energy source, it always requires oxygen.  Glucose, on the other hand, can make a limited amount of ATP without oxygen, which is called anaerobic respiration.  This is useful during short vigorous activity, but we cannot make enough ATP by anaerobic respiration for sustained exercise.

2) For a given amount of oxygen, more ATP is produced from carbohydrates, like glucose, than from fats.  However, the amount of ATP created from a single fat molecule is greater than from a glucose molecule.  In other words, if you have plenty of oxygen, you should be burning fats.  But once oxygen becomes limiting, either because you’re working so hard, or because you’re at a high altitude, then carbohydrates should be used.

Given this information, the authors hypothesized that the mice at high altitudes will burn more carbohydrates than mice at sea level.  They have a limited amount of oxygen in the air, so they need to use it in the most efficient way to produce the energy they need.

High altitude mice burn more carbohydrates, but fatigue sooner

The authors did all their tests in the same experimental conditions, with the same amount of oxygen in the air for both sets of mice.  Under normal oxygen conditions and when there was low oxygen content, the high altitude mice burned more carbohydrates than the other mice during moderate exercise.  At rest, they also burned more carbohydrates under low oxygen conditions.  The authors found that the activity of enzymes associated with breaking down carbohydrates were greater in the high altitude mice, specifically in the heart muscles.  The heart has to work harder at high altitude to get enough oxygen to the tissues, so it makes sense that these muscles, in particular, would be burning carbohydrates preferentially. 

The high altitude mice, therefore, have adapted to the low oxygen environment by having more active enzymes to break down carbohydrates rather than fats.  One problem with that, though, is that the carbohydrate storage is less extensive than fat storage.  The researchers found that high altitude mice fatigued more quickly than the sea level mice, which burned more fats.  They suggest that the fast fatigue is a result of using up all the carbohydrate stores.  These mice, though, don’t travel long distances and just need short bursts of speed to escape predators.

I really like how so many physiological processes can be explained through understanding cellular respiration.  It’s so logical that animals at high altitude need to use oxygen more efficiently, so they use carbohydrates more for energy.  It’s simple.