Discovering How Mitochondria Communicate
The Moment We Discovered that Mitochondria Align Their Cristae With Each Other
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“Mitochondria are constantly communicating and cooperating not only with each other but also with other cellular compartments. The disruption of this crosstalk can lead to deep cellular damage and the onset of diseases.” — Silvia Barabino, Silvia Lombardi, & Mara Zilocchi
Exploring Mitochondrial Interactions
As a postdoctoral fellow, I wondered almost daily about how mitochondria talk to each other.
We knew mitochondria could share information by releasing small molecules like ATP and ions. In 2009, my colleagues Gyorgy Hajnoczky, Orian Shirihai, and others described the mitochondrial “kiss-and-run” where two mitochondria came close together, exchanged fluorescent proteins, and quickly moved away from each other.
More stable interactions called fusions involve two mitochondria completely merging to share their contents. Mitochondria may also undergo fission. During fission, a longer, filamentous mitochondrion splits into two or more globular ones. Orian discovered the fusion-fission cycle of mitochondria, which highlights how mitochondria are social organelles with a life cycle.
Questions about mitochondrial “social life” often followed me into the dark dungeon of the electron microscopy core at the University of Pennsylvania, where I spent countless hours.
I learned the art of electron microscopy during my PhD while visiting the legendary clinician-scientist Doug Turnbull in Newcastle Upon Tyne in the UK. He gave me carte blanche to use some of his research funds for imaging mitochondria. I hardly knew what I was doing, but I had an idea to test. I wanted to know what happens to mitochondria when we exercise. My hypothesis was that they would fuse and elongate, mimicking what two earlier studies had shown in cultured human cells when depleted of nutrients.
It turned out that my hypothesis was wrong. At least partially. During exercise, mitochondria didn’t simply fuse, they started to “kiss”. My imaging showed that there was an increase of dense inter-mitochondrial contacts sites, which we later named intermitochondrial junctions (IMJs).
Exercise enhanced mitochondrial networking.
Imaging Mutant Mitochondria
Back in Philadelphia, as a new postdoc trying to make use of my electron microscopy expertise, I started working with Meagan McManus. She was studying mouse models of mitochondrial diseases to understand how defective mitochondria contribute to heart disease and neurological disorders.
I first met Meagan when I toured her lab during my postdoc interview with Doug Wallace. She was wearing running shorts and in the middle of an experiment at her bench. I wasn’t sure if she had just come back from a run or was racing off to finish an important step before heading off.
By summer 2013, I was a postdoc in Doug’s lab. Meagan wanted to understand what happens to mitochondria in the hearts of mice with particular genetic mutations seen in mitochondrial disease. Impaired energy transformation in these animals led to the mouse equivalent of heart failure (dilated cardiomyopathy) and an early death., This was similar to what happens in humans with poorly functioning mitochondria and hearts. Our question was simple but profound: what did these struggling heart mitochondria actually look like?
To answer this question, I descended once again to the dark dungeon of the electron microscopy core laboratory.
That day at the microscope, I snapped about 200 high-resolution images of Meagan’s mouse hearts. These showed striking patterns of abnormal mitochondria. The core director Dewight Williams, was helping me apply a cool new method to create “tomograms,” which were three-dimensional reconstructions of cellular architecture. I planned to deploy quantitative methods to measure mitochondrial sizes, shapes, and the intermitochondrial junctions I’d seen earlier.
I was packing up to leave when Dewight called out, “We have some tomograms. You want to take a look?”
Visible Evidence of Mitochondrial Communication
What we saw was astonishing. Dewight played the reconstructed movie showing densely-packed mitochondria stacked against one another. Some mitochondria had neatly parallel cristae membranes—the hallmark of healthy mitochondria. Others had tangled and highly irregular “reticular” cristae—the unmistakable sign of poor mitochondrial health. This confirmed what I had observed all day.
But then, something unexpected appeared in 3D that I had not yet seen in the flat images. It was striking. In the unhealthy mitochondria, right at the inter-mitochondrial junctions, all the cristae were ordered. Within the junction between mitochondria, the inner membranes were perfectly aligned and more abundant than anywhere else in the organelle. It was as if one mitochondrion was organizing the cristae of its neighbor, informing where and how many should form.
“Oh this is cool! Meagan has to see this!” I rushed back to the lab, buzzing with energy.
Back at the lab, my heart was warmed to find Meagan, this time in yoga attire. No, she hadn’t just gotten back from yoga class, and no, she was not just about to head out on a run. By now, I knew this was just her natural state.
“Meagan, look at this! Something exciting is happening between the mitochondria in your mutant mouse hearts,” I said while plugging the USB flash drive into my computer. (It was 2013, and flash drives were still our lifeline.) As the movie started, I narrated what I had seen a few minutes earlier with Dewight: “Where the mitochondria touch, there are more cristae. And the mutant mitochondria surrounded by healthy ones actually have normal-looking cristae! Mitochondria influence each other!”
“Oh!” she gasped. “That’s crazy!”
The First Observations of Cristae Alignment
We watched the looping video several times. Then, she quickly pointed out, her voice jumping an octave: “And the cristae line up! The cristae line up between mitochondria!” She traced the alignment with her extended finger, following the junction where the two organelles met.
How had I missed this?
For years, I had poured over thousands of stunning electron micrographs—decades of work by the best mitochondrial microscopists. I never noticed or heard anyone else describe the cristae of one mitochondrion aligning with the cristae of another. Not even in the paper I recently unearthed, published in 1983—a year before I was born—by Russian scientists Lora Bakeeva and Vladimir Skulachev, describing “intermitochondrial contacts.” These contacts were intermitochondrial junctions.
Cristae are membranes, much like the outer membrane or the “skin” of a cell. However, cristae are unique in that they contain densely packed proteins that are required for energy production. This is where the magic of mitochondrial transformation and “dematerialization” occurs. The cristae are where electrons flow to finally reunite with oxygen. The reason we breathe is to bring oxygen to the cristae of our mitochondria.
I sat there, eyes fixed on the looping video. There they were, in full beauty. Mitochondrial cristae like nothing I, Meagan, or anyone else had ever seen or noticed before. They danced together in a way that transcended individual mitochondria. Instead of laying there as stacked cookie sheets, flat and parallel—like textbooks often portrayed them—they twirled and bent out of shape.
Looking at a single organelle, the movement made no sense. In fact, it literally escaped the senses.
Previous scientists and microscopists had looked for patterns inside each mitochondrion. But what we saw were patterns between mitochondria.
To see them, Meagan had to zoom out, shifting her gaze from an individual mitochondrion to examine a group of mitochondria together.
The truth was that the cristae of one mitochondrion “cared” about the status of the cristae of a neighboring one. So much so, that the membranes of one organelle bent out of shape to line up with the cristae of its neighbors. This meant moving away from their most thermodynamically favorable state, the flat cookie sheets.
What?! We watched the video again, and again, and again. We looked at each other, both gasping in amazement. Excited about what this meant and moved by the energy of the moment, we hugged.
Confirming and Extending Our Findings
“The cristae line up, one for one, between mitochondria,” I repeated slowly, trying to wrap my mind around this profoundly unlikely biological reality. This went against what we were taught.
We played another of Dewight’s 3D movies. And then a third one. We kept seeing the same thing—transmitochondrial cristae alignment. There was no denying it. It was real.
This is going to need quantification and many more experiments, I thought, anticipating the road ahead to share this discovery with the world in a scientific publication.
As I rode my bike home that day, many questions ran through my mind. How can we quantify this? Is this only true in the heart? Does it happen only in mice, or in other species as well?
To start answering these pressing questions, over the next few days I assembled some of the most striking pictures and videos we had. I also wrote to Clara Franzini-Armstrong, one of the most respected muscle electron microscopists. She had her own microscope, and her office was an extensive museum with collections of electron microscopy images. Clara had imaged nearly everything in the world with muscles.
At my request, Clara agreed to meet with me. I was anxious. What if this is just an artifact of my fixation and technique? What if it only happens in mice or mammals? I guess that would be OK, but based on the evolutionary history and conservation of mitochondria, my gut told me this was more universal.
We began our exploration by opening the album containing images of the flight muscle in arthropods, like the housefly and dragonfly. Boom! There it was, intermitochondrial junctions! Pushing the envelope further, we looked at a mollusk muscle in a scallop. There it was again: intermitochondrial junctions! In every album we opened, we saw them. The ability of mitochondria to form junctions is phylogenetically conserved, meaning it is present across different species throughout evolution.
“It’s real. I had never noticed these before,” admitted Clara with humility. She quickly reflected, “This will be a good paper. What else are you going to show?”
The Path to Publication
To start, I had to determine if this could be induced experimentally. My colleague Gyorgy and his team had recently developed a new way to pull mitochondria together with a special “linker” system. When I met with Gyorgy to show him the cristae alignment and the images of various creatures with intermitochondrial junctions, he agreed to run some experiments. We would link mitochondria, wait a bit, and then fix the cell’s mitochondria in place to look at their cristae.
The hypothesis was that bringing two energized mitochondria together would cause either new cristae to form, or reorient and align pre-existing cristae. Finally, after extensive experimentation and careful observation, we reached a pivotal moment.
Our findings revealed that the intermitochondrial junctions were not only inducible, but also had a profound impact on the membranous architecture of mitochondria.
Laborious quantifications over the following weeks provided the necessary data to confirm the robustness of this discovery, and the resulting manuscript was submitted to Nature Communications. The handling editor who received the submission said, “If there was a mechanism, this would be a Nature paper.”
They wanted to know what protein caused this phenomenon. What is the native molecular unit holding and aligning the cristae in place at intermitochondrial junctions? This is the same question I repeatedly received from colleagues when I presented this finding at conferences and seminars. “How are you going to find out the mechanism?”
The truth is, I didn’t see what finding the mechanism would bring us.
A Difficult Choice
With the exciting discovery of intermitochondrial junctions, we had two choices.
We could invest a lot of time performing molecular screenings to possibly discover a protein, or sets of proteins, that disrupted the cristae alignment when the proteins were removed. To confirm the relevance of the protein(s), we’d have to test if when expressed at higher levels (overexpressed), they would enhance cristae alignment. Next, we would have a molecular “target” that a pharmaceutical company could develop into a drug. With that, we would look for disease models where cristae alignment is disrupted, as in Meagan’s mice, to see if the new drug candidates improve cristae alignment.
If successful, we would look for human diseases in which to test our lead drug candidate. Then, we’d develop randomized controlled trials and run a multi-billion dollar program to get that drug on the market. Lastly, we’d lobby to get it approved, market it to as many people as possible, and hope that the side effects aren’t too severe.
This first path looked appealing and meaningful if we could accept the following: 1) That the cristae-alignment is the problem to cure, rather than an expression of a deeper energetic disturbance; 2) The need to ignore that the healing process is an integrated process, and that blocking/altering one part can upset other parts of the system and interfere with the healing process; And 3) Being lured by the potentially lucrative path towards drug development. Like the positive pole of a battery attracts electrons, the prospects captivate many brilliant scientists. The allure of a swift mechanical solution has frequently steered scientific endeavors in this direction.
I opted for the second choice. From this discovery we would derive a specific feature of mitochondria to a broader biological principle.
After hundreds of hours in the dark electron microscopy dungeon, numerous collaborations, and a published paper, we learned an important lesson: mitochondria exchange information.
Are there other ways in which they communicate? Might this uncover a broader, internal universe of information and energy exchange?
The Next Frontier
A couple years later, again sitting at the electron microscope, now with Amy Vincent, a student in Newcastle, we examined the muscle of a patient with a rare mitochondrial disease. This patient had a similar mutation to Meagan’s mice.
What we found that afternoon opened another door of inquiry. In front of our eyes, made visible by the beam of electrons scattered by the structures of mitochondria, were mitochondrial nanotunnels.
We were observing, for the first time in humans, evidence that mitochondria send out thin tubular structures, akin to the feelers their bacterial ancestors extend to explore their surroundings. In individuals with mitochondrial disease, the mitochondria extend more of these nanotunnels, as if “reaching out for help.”
It appears as though the sick mitochondria that cannot flow energy as easily are attempting to establish a supportive network, working together to sustain the essence of life.
This observation leads us back to our primary objective: to discover how energy and information flows between mitochondria to make life possible.
Discovering transmitochondrial cristae alignment was a step in this direction. It showed us a layer of communication that could be central to how mitochondria flow and transform energy within our cells.
Now we just have to understand how mitochondrial energy flow and transformation fuels dozens of activities that sustain our everyday lives. That’s a story of energy resistance, which we’ll talk about here in the next few weeks.
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Communication among mitochondria reflects the social organization of our bodies and our energetic nature.
This was my favorite paper ever written on mitochodnria because it helped me understand the link between melanin and cristae alignment. For me, this solved the "C-value paradox" and the "Regulatory Conundrum" of human evolution.
The reason humans look, think, and operate so differently from chimps, despite sharing 98.8% of the same protein-coding genes, is not because we have different "parts." It is because we have a fundamentally different timing and energy distribution system. Cristae geometry is a fossil for how we did it using an ancient system. Melanin is a chiral chaotic polymer that absorbs all frequencies of light. It is capable of directly communicating with the IMJ sans the genetic metabolic machinery on the IMM. The three divers who saved the EU from Cherobyl on May 4, 1986 are the experiment buried in this paper. All three survived and never got radiation sickness. They did get radiation tans on their legs because of what they had to walk through to save Europe. Their survival meant that radiosynthesis has to have a way to lead to cristae alignment. The "radioactive tan" was the physical manifestation of the body rapidly deploying melanin to create a chiral, spin-selective shield. This allowed electrons to continue "tunneling" through the IMJ even as the delicate genetic machinery was being bombarded. Because this mechanism is driven by physics (CISS and semiconduction) rather than biology (transcription/translation), it is nearly instantaneous and incredibly "hardy." It is the same reason we found out in the last decade that the Chernobyl fungi grow faster in the fire, because they aren't fighting the radiation; they are "plugging into" it.