Australasian Science Magazine

An electron microscope image of mitochondria undergoing herniation. On the bottom mitochondria (red) there are two membranes (lines) surrounding it on the left side, while there is only one membrane surrounding the mtDNA (green) on the right. Credit: Dr Benjamin Padman/Dr Kate McArthur

By Benjamin Kile, Kate McArthur & Tahnee Saunders

Mitochondrial DNA has been implicated in diseases such as arthritis, but how it escapes from inside the mitochondria and triggers these disorders has remained a mystery. Now Australian scientists have captured video evidence of mtDNA escaping for the first time.

Humans have two genomes. One is located in the nucleus. It is three billion DNA base pairs long, tightly wrapped up into chromosomes, and carries 99.94% of our genes that encode more than 90,000 proteins.

The second genome is tiny. Comprising just 16,000 base pairs that code for only 13 proteins, it is tucked away inside the mitochondria, a remarkable cellular organelle that generates the energy that powers the body’s biological processes. Each mitochondrion possesses several copies of this tiny circular genome of “mtDNA”, which has properties more like bacterial DNA than our own thanks to its evolutionary past.

Somewhere around 1.5 billion years ago, one cell engulfed a bacterium and an amazing thing happened: the two began working together. The cell likely provided protection for the bacteria, while the bacteria provided energy for them both. Over the course of evolution, as the two became one, the majority of the genes encoded by the bacteria’s DNA were transferred to the nucleus of the cell, but a select few remained, and these few genes make up the mitochondrial genome as we know it today.

DNA Triggers an Immune Response

Our immune system has two arms: the “innate” and the “adaptive”. The former is the body’s “first responders”, an army of white blood cells equipped with sensors that recognise pieces of microbial invaders. These “pathogen-associated molecular patterns” (PAMPs) can be anything that is unique to the pathogen and not a part of the human body. These include components of the bacterial cell wall and the DNA inside bacteria or viruses.

In addition to PAMPs, the innate immune system can also be triggered by components of our own cells should they become damaged or somehow find their way into the wrong place. For example, DNA belongs in the nucleus and the mitochondria; if it gets outside either of these two locations, the innate immune system recognises it as a danger signal, or “damage-associated molecular pattern”(DAMP), and goes off.

By its very nature, mtDNA is therefore a potent inflammatory stimulus if it escapes the confines of the mitochondria. Putting aside the fact that it looks like bacterial DNA, the cell’s defensive systems become activated simply because DNA has been detected in the wrong place. The very same cellular sensors that are normally on patrol to detect DNA from viruses will also potently recognise mtDNA, and will then trigger the exact same immune reactions that occur during viral infections. Inflammatory immune reactions to our cells’ own components (like mtDNA) are often to our own detriment, with potentially devastating consequences like autoimmune disease.

To date, more than 60 studies have detected mtDNA outside the mitochondria, in the cytoplasm of cells or in the circulation of patients. Elevated mtDNA levels in the blood have been associated with a wide range of diseases including sepsis, liver failure, HIV infection, Dengue virus infection, cardiac arrest, stroke, cancer, rheumatoid arthritis and lupus. In many of these settings, mtDNA-triggered immune signalling has been implicated in the progression of disease, and therefore understanding how and why mtDNA escapes from the mitochondria will be important to understand the fundamentals of these diseases.

Suicidal Cells

Our research into mitochondria and mtDNA began not with the immune system but a desire to understand cell suicide. The human body rids itself of billions of cells every day – the aged, the expended and the damaged – and one method it deploys is to have cells kill themselves. This process, known as “programmed cell death” or “apoptosis”, is regulated by molecules within the cell itself and occurs in a “silent” manner. That is, the cells that die by apoptosis are cleared away so quickly that they do not trigger an immune response from the cells around them.

This contrasts with other types of cell death. For example, physical rupture caused by trauma causes a cell’s contents to leak into the extracellular space and signal to neighbouring cells to drive an inflammatory response (e.g. pain, redness, swelling).

As the cell’s main energy supply, mitochondria are the prime target during apoptosis. Like a military strike, the apoptosis machinery destroys this key piece of cellular infrastructure to inflict a mortal wound.

This is achieved by two proteins called BAK and BAX, which punch holes in the outer layer of the mitochondria. These holes allow the release of a mitochondrial component into the cell’s cytoplasm, which triggers a family of killer enzymes known as “caspases”. In an event somewhat akin to handing an excited 5-year-old a pair of scissors, the caspases cleave thousands of substrates within the cell, rapidly driving its demolition from the inside.

However, unlike the 5-year-old, there is method to their madness. Specific caspase cleavage events ensure that not only are all major cellular processes switched off (such as gene transcription and protein translation) but the cell is packaged up into bite-sized pieces that are labelled for clean up, and their contents are contained so they don’t trip any alarms that would otherwise activate an immune response.

As it turns out, the caspase activator isn’t the only thing released when BAK and BAX damage the mitochondria. By studying cells in which the caspases had been disabled, we found evidence that mtDNA stimulates an anti-viral response during apoptosis. This suggests that the role of the caspases is to suppress the triggering of innate immune sensors by DAMPs when DNA is detected in the cytoplasm.

This also raises the question of how these sensors get access to mtDNA in the first place. How does mtDNA escape the mitochondria during apoptosis?

The Great Escape

mtDNA is kept within the innermost compartment of the mitochondria, behind two protective membranes. Until now it was thought that BAK and BAX puncture only the outer membrane during apoptosis, but there had to be something more to the escape plan. Using a brand-new form of microscopy called Lattice Light-Sheet Microscopy (LLSM), we were able to observe for the first time what happens inside an apoptotic cell in real time. The results provided a remarkable surprise.

After BAK and BAX punch tiny holes all over the mitochondria, these holes start to drift together to form even larger holes. These “macropores” allowed the inner membrane of the mitochondria to balloon out through the opening and into the cytoplasm, taking the mtDNA with it. Once outside its protective outer layer, the inner mitochondrial membrane started to break down, releasing mtDNA into the cytoplasm.

We called this process “mitochondrial herniation” because, like a hernia, it involved the bulging of an inner compartment through an abnormal opening in the wall that usually contains it. This is the first time a mechanism for mtDNA escape has been described in any setting.

Is herniation the mechanism by which mtDNA escapes during autoinflammatory diseases such as arthritis or lupus? Is it the same mechanism that occurs during infections like HIV or Dengue fever? And, if so, can we manipulate herniation to prevent mtDNA’s escape and potentially halt the progression of disease?

Cells See the Light – And Survive It

Light microscopy has always been a powerful tool for studying biology, but until recently it has been limited in its application because cells are inherently sensitive to light. The majority of cells in our body never see light and are not designed to tolerate it. Documenting a live process inside cells has been a problem because most of the time cells die from phototoxicity. Scientists have been constantly forced to compromise, trading off spatial resolution, time, clarity of image or cell viability against one another.

Advances in microscopy over the past decade have dramatically expanded our ability to observe living cells at high resolution without killing them. Lattice Light-Sheet Microscopy differs from conventional microscopy in that it rapidly scans thin sheets of light (as opposed to laser beams) at angles perpendicular (rather than parallel) to the angle at which they are eventually collected. A sheet of light takes less time to light-up an entire cell than a laser beam does, is gentler than a focused laser beam in terms of energy, and the perpendicular arrangement for collecting the light circumvents out-of-focus light that normally contributes to noise (or blur) in the image.

The result is an extremely gentle method with the resolution required to see inside living cells in real time for hours on end without toxicity to the cells. This is a perfect solution for imaging mitochondria during apoptosis.

Excitingly, these advances in light microscopy have been matched with similar technological improvements in so-called “super resolution” microscopy. When combined, the two approaches are extraordinarily powerful. As we all know (even before the advent of the “selfie”), a single snapshot of a living thing can be profoundly misleading, no matter how high its resolution. When that high-resolution information can also be placed in context (i.e. in time), our understanding of the true situation deepens. Together these techniques provide us with the means to observe and dissect dynamic cellular processes like never before.

After we had videoed the process of mitochondrial herniation from start-to-finish with live-cell LLSM, we were able to pinpoint the exact time when the inner membrane ballooned out into the cytoplasm, and by using electron microscopy, we could get a snapshot that was 200 times magnified again, showing the exact regions of the inner membrane that broke down and allowed mtDNA to escape. Without LLSM we would never have known when to look, and even had we chanced upon the right moment, we could not have recognised what we were seeing.

A Whole New World

Even as you are reading this article, mtDNA is escaping from your mitochondria in the billions of cells that are dying by apoptosis in your body. Luckily, the caspases are ensuring that your dying cells are cleaned up before the mtDNA can be seen by your immune defence mechanisms, and you are none-the-wiser.

What remains to be seen is how mtDNA is escaping during autoimmune diseases and viral infections. Is it through a similar mechanism, or something entirely different? In the same way that we discovered mitochondrial herniation, we can apply live-cell and high-resolution microscopy techniques to further investigate mtDNA escape during diseases, leading to better understanding and hopefully a cure one day.

If mitochondrial herniation has been occurring on such a wide scale within us every day, and has only now just been detected, imagine the biology that we have not yet observed. Recent advances in live cell imaging are allowing us to watch biology within cells, tissues and even whole organisms in real time like never before. Just as the Hubble Telescope allows astronomers to see exciting new worlds out there, we biologists now have microscopes that will let us explore similar new worlds. Ours are just on a much smaller scale.