Throw another adipocyte on the fire

Humans are able to live in so many different climates, in a wide range of temperatures and yet our inner core body temperature remains nearly constant.  This ability to thermoregulate has something to do, of course, with clothing and the ability to cool and heat our living spaces, but our bodies also offer many adaptations to regulate body temperature.  If it’s too hot, we sweat, releasing excess heat through evaporative cooling.  If it’s too cold, we shiver, producing heat in our working muscles.  The production of heat through physiological mechanisms is called thermogenesis and also includes a non-shivering version.  Today’s paper is about non-shivering thermogenesis, which is when our fat cells produce heat.

Non-shivering thermogenesis

To understand how non-shivering thermogenesis works, we need to take a step back and discuss cellular respiration.  The cells of our body store energy from food in the chemical bonds of a molecule called ATP.  During cellular respiration, a cell will convert glucose or fat into carbon dioxide, while slowly tapping into the energy in those food molecules in order to make ATP.  The final step of cellular respiration is that the energy from the electrons in glucose are passed from protein to protein, releasing energy that is used to pump protons into a membrane-bound cellular space.  You can think of these protons as a form of potential energy, like stuffing a closet full of balls.  When you open up the closet door, all the balls come tumbling out, releasing their potential energy in the process.  During cellular respiration, this potential energy is used by an enzyme to make ATP.  During non-shivering thermogenesis, though, the potential energy stored in all those protons stuffed into a small space is released by the cell as heat.  Thus, the energy from food is used to heat the body rather than being stored in ATP.

The main type of cell that does non-shivering thermogenesis is brown adipocytes, or fat cells.  Brown fat is very common in infants, but is also found in adult humans in the upper chest and neck.  The purpose of brown fat is to provide heat for the body.  Thus, non-shivering thermogenesis is activated by a drop in body temperature.  The cold temperature is sensed by the brain, which activates the sympathetic nervous system (the “fight or flight” response), which signals to the brown fat cells to express the genes necessary to bypass ATP production and release heat instead.  In a recent paper published in PNAS, Ye et al. describe how a different type of fat cell is able to skip all the nervous system steps and sense the cold directly (red arrow in diagram).  It is pretty cool that the fat cells are able to sense temperature, as if they were neurons, and can act autonomously to remedy the situation.  No need for a brain here!

Independent thermogenesis

Through a series of experiments, the authors demonstrate that a particular type of fat cell will express genes necessary for non-shivering thermogenesis when exposed to cold, independent of sympathetic nervous system activation.

In one experiment, they grew fat cells at different temperatures and measured gene expression using a technique called quantitative PCR (qPCR).  The idea behind this technique is that if a gene is highly expressed, there will be a lot of mRNA in the cell (remember the “central dogma” of molecular biology) and qPCR is a method for measuring the concentration of mRNA for a particular gene.  They focused their measurements on thermogenic genes that are known to be part of the non-shivering thermogenesis mechanism, such as Ucp1, which is the enzyme that actually allows the protons to fall back across the membrane, thereby releasing their energy as heat.  They found that these fat cells that were exposed to the cold expressed more Ucp1 mRNA, even in the absence of any nervous system.  These are just cells in a dish, so this must be an intrinsic property of fat cells.

It wasn’t just any fat cell that had this response.  In fact, brown adipocytes did not express more Ucp1 in the cold.  It was a different type of fat cell called a white adipocyte.  What is white fat?  The majority of fat in our body is white fat and its purpose is to store fat for energy (for cellular respiration) and to act as a thermal insulator, so we don’t lose as much heat through our skin.  There is one subtype of white fat that has been shown to do non-shivering thermogenesis and it was this type that could express thermogenic genes, like Ucp1, in the cold, independent of the nervous system.

Okay, so these white fat cells don’t need input from the nervous system, but do they still use the same intracellular pathway to turn on expression of these genes?  Normally, when a fat cell is activated by the sympathetic nervous system, it sets off a molecular cascade of events inside the cell, which involves activation of molecules in a pathway called the cAMP pathway (as shown in the diagram).  The authors inhibited this pathway in various ways and found that the cells could still respond to the cold as before, so this effect must use a different pathway.

There are still a number of open questions, such as: how do fat cells sense temperature?  Do they use the same types of receptors as temperature-sensitive neurons?  Why are some white fat cells independent, but brown fat cells need the nervous system to activate thermogenesis?  One thing that is clear, however, is that white fat cells are clearly important for temperature regulation as well as fat storage.  The authors suggest that tapping into thermogenesis might be a good way to help obese patients get rid of excess energy storage by releasing it as heat.  This pathway that is independent of the sympathetic nervous system could allow medications to target only the fat cells without involving the sympathetic nervous system which controls so many other functions in the body.

Something to think about as the cold Bay Area summer sets in.

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.

There's more than one way to get out of a nucleus

This week’s paper totally blew my mind.  It’s so amazing to me that we know so much about how cells work and yet this paper describes a totally new process that has never been observed before.  How’s that possible?  What else remains to be discovered?

In this week’s issue of Cell, Speese et al. describe a new method for molecules to exit the nucleus.  The nucleus is where the genome is stored and where genes are expressed into RNA (see Fundamentals section for more details).  The nucleus is surrounded by two nearly impenetrable membranes, an inner and outer membrane.  All biology students are taught that there is only one way into and out of the nucleus: the nuclear pore complex (NPC).  It’s an elaborate and selective protein pathway that spans the inner and outer membranes of the nucleus (I’ve written about the nuclear pore complex before in the context of long-lived proteins).  If the molecule has the right kind of chaperone, then it can pass through the NPC to get into the cytoplasm. 

The lab that did this research studies a signaling pathway at the Drosophila neuromuscular junction (that’s what I study too!)  The neuromuscular junction is a synapse (remember synapses?) between a motor neuron and a muscle.  The pathway that they study is called the Wnt pathway and they’ve shown that it helps regulate the development of the neuromuscular junction.  The neuron releases a signal that binds a receptor on the muscle.  The active receptor, called Frizzled, is taken into the cell where it gets cut into two pieces.  One piece enters the nucleus and they have observed that it accumulates into big aggregates (we’ll call them “foci”).  But what happens next?  They suspected that the foci may leave the nucleus, but how could such a big glob of proteins get through the relatively narrow nuclear pore complex?  Ay, there’s the rub.

Nuclear Lamins

The first thing the authors investigated was whether nuclear lamins might be associated with the nuclear foci.  Nuclear lamins are the resident proteins in the nucleus that help spatially organize the chromosomes and provide scaffolding for the nucleus and nuclear pore complexes.  Lamins are important medically because there are a number of “laminopathies”, human diseases associated with mutated lamins.  One particular lamin, called LamC, does appear in the Frizzled aggregates in the muscle nucleus.  In fact, if you get rid of LamC, then the Frizzled receptor no longer forms foci in the nucleus.  The association with LamC, though, doesn’t explain how the Frizzled receptors function in the nucleus.

Perhaps Frizzled binds to DNA; that’s the major molecule in the nucleus after all.  Nope.  The authors found that there is no DNA associated with the Frizzled clumps.  Well, perhaps it binds mRNA then.  Remember that DNA is copied into small pieces of mRNA which then travel out of the nucleus and direct protein synthesis (see Fundamental #1).  Sure enough, they find that there are some mRNA molecules bound in the Frizzled/LamC aggregates.  It is not that uncommon to find proteins bound to mRNA in the nucleus and usually they all transport out of the nucleus through the pores.  mRNA can only be translated into protein in the cytoplasm, so they have to leave the nucleus to be functional.  The problem is that these Frizzled globs are way too big to go through the pores.  The aggregates must disassemble before leaving the nucleus, right?  Right?  Not necessarily.

Nuclear budding

The researchers did lots of imaging of the Frizzled foci and found something strange.  They sometimes would see an aggregate surrounded by nuclear membrane on both sides.  In other words, the aggregates appeared to be in the space in between the inner and outer membranes.  What?!  They also imaged the foci over time and could see them staying intact as they leave the nucleus.  In the end, what they believe is happening is that the particles get surrounded by the inner membrane and then pinch off into the intermembrane space.  At that point they are surrounded all the way around by a sphere of membrane.  That membrane fuses with the outer membrane, spitting out the particles into the cytoplasm.  See the figure below for a visual representation of this process.  The role of LamC in this process is probably to help promote rearrangement of the membrane scaffold to allow nuclear budding. 

It turns out that this same sort of process happens during infection by a herpes virus.  The viral genome goes into the host nucleus and directs formation of new viruses.  The complete virus is much too big to exit the nucleus through the pores, so it uses this method of nuclear budding to get out of the nucleus.  Everyone assumed that this process was specific for viruses, but now we see that it’s actually a normal cellular activity.  In fact, the virus probably hijacks the apparatus for normal nuclear budding to get itself out of the nucleus.  Viruses always know more than the scientists.

Synaptic translation

Now we’ve solved the problem of how the Frizzled/LamC/mRNA complexes can exit the nucleus, but what happens next?  How does this continue the Wnt signaling pathway?  The authors followed the path of one mRNA that binds Frizzled in the nucleus.  They found that the mRNA localizes near the synapse (in the muscle side).  It’s been shown before that local protein synthesis at the synapse can direct synapse development and plasticity.  When LamC is impaired, this mRNA no longer goes to the synapse, which really goes to show how important lamins are in cellular signaling.

Review time:

1) Signal from neuron to muscle activates the Frizzled receptor

2) A piece of the receptor gets cut off and enters the nucleus (via the NPCs)

3) The Frizzled piece binds up with LamC and particular mRNAs

4) LamC helps rearrange the nuclear membrane, so budding can occur

5) The large Frizzled particles exit by budding straight through the membranes

6) Frizzled and the mRNA travel to the synapse

7) The mRNA is translated into a particular protein that directs synapse development

This helps solve a problem about the laminopathies (diseases caused by mutation of a lamin).  Often these diseases cause muscular dystrophy.  Well, maybe what happens at the Drosophila neuromuscular junction could happen in humans too.  If a lamin is mutated, it may prevent membrane budding, so RNA-protein complexes cannot exit the nucleus.  If they get stuck in the nucleus, the mRNA cannot be translated at the synapse, so no new proteins will be made and the neuron-muscle synapse will be impaired.  That would explain defects in the muscle system caused by these diseases.