Dr. Levin and Mr. Hyde

"A flatworm, a cancer cell, and a lump of intelligent frog skin walk into a bar. The bartender asks, what's the deal? The flatworm chirps: Why, we're all model organisms in the works of the scientist Dr. Michael Levin!"

(the bartender then promptly starts to dissociate and have a panic attack)

Try answering the following questions to the best of your ability.

Take your time, they're meant to be tricky! Close your eyes if you need to visualize :)

• What is a "self"? Is it a whole person? A brain? The brain cells that make up a brain?!
• Do you think individual cells have goals they try to achieve, akin to humans pursuing their own goals?
• Are humans and animals the only creatures that can "remember"?

Traditional Bio101 thought would give you some generalization like this:

Cells are the building blocks of organs, organs make up organisms, organisms form populations.

But what if I told you that's not necessarily true? What if cells didn't have to make up tissues and organs, organs didn't have to make up organisms, etc etc? What if the above looked something more like this:

More specifically, the idea that cells, organs, and organisms can be sovereign, individual "selves". This is kind of a revolutionary idea, and it is a central theme to the research of accoladed Tufts scientist Michael Levin.

The man himself!!

Before we get into the Nitty Gritty about what his lab has been researching, let me take some time to explain some basic background information to make things later on easier to understand. If you already know this stuff by heart, feel free to skip.

What is electricity?

You must think that I think you're an idiot. But trust me, I didn't entirely understand what it was until I formally looked it up myself!

When an atom has more electrons than protons, it possesses a negative charge and is called a negative ion. Likewise, an atom with a surplus of protons over electrons is called a positive ion. Due to Coulomb's law, positive charge and negative charge are attracted to one another, while same charges (negative-negative or positive-positive) repel. 

When the outer shell of an electrons of an atom (aka the valance electrons) get pushed out of their respective atoms for what ever reason, they become free electrons that seek to find positive charge (think of them as kind of having a codependency issue :p). A free electron moving onto an unwitting atom bumps out another valence electron, which goes on to do the same thing as the previous electron, creating a cascading effect. This cascade is no other but the electric current! Voila, electricity!

What is an ion channel?

Ion channels are a special class of proteins that regulate communication between cells. More specifically, they control the flow of ions from cell to cell by segregating the charges of cell membranes (membrane potentials).

Ions coming in, ions going out of a cell membrane

They're really important because they perform biological functions that don't depend entirely on genetics. They play vital roles in very quick biological events, like the transmitting of cell signals, release of neurotransmitters, and contraction of muscle fibers. If you look up "ion channels" on Google, you will get mobbed with all of the facts about their vital role in neurons and muscle cells.

In addition to ion channels, there are other ion related subunits found in cells that are vital to cell signaling and communication.

• Ion pumps - proteins that use chemical energy to pump ions against their concentration gradients (e.g. pushing positive ions towards a crowded space of other positive ions).
• Ion translocators - proteins local to each cell that stabilize the resting potential/ground voltage of that respective cell
• Gap junctions - will talk more about this in depth further down :)

Bioelectricity⚡🧬 

Electrical interactions that happen in biological systems, powered by ion channels, pumps, and translocators are well summarized into a phenomenon we call ~~bioelectricity~~.

Bioelectricity is responsible for many cell behaviors and functions. In addition to the several vital biological processes listed in the previous sections, bioelectricity is a fundamental impetus for morphogenesis, growth, and development of all multicellular life. It is an ancient property of living things, tracing its inception back to bacterial biofilms. 

Okay, let me elaborate on that big word I just italicized but didn't explain. 

Morphogenesis and metamorphosis

You ever wonder how you came from a single egg cell and grew into a well developed being by the time you were born? How did that egg cell that made you develop into the target morphology of a human being, and not a chicken, spider, or decepticon?

The cause for this is not as black-and-white as your genome coding for how your anatomy is assembled. After all, DNA codes for proteins, not arms and legs. Cells and emergent systems from cells (tissues, organs) are more plastic than the DNA that dictates their inner workings. An embryo that's cut in half can still develop into two perfectly fine organisms (this happens all the time, because that's how identical twins happen!). 

It's also been found that organisms undergoing metamorphoses share this same incredible plasticity– take tadpoles turning into frogs for example. The same genes that code for the weird, fishlike baby frogs we call tadpoles also code for the frogs they become. 

Sure, you can spend inordinate amounts of time micromanaging an animal's genes to change its phenotype, but clearly it seems like there's a way– and a way there is– to modify behaviors at a higher level. Let's investigate!

Planaria, a model organism

This is a planarian flatworm. 

Wow, so derpy!

Planaria are really remarkable animals. They have incredible regenerative abilities, which make them effectively immortal. To reproduce, planaria impregnate each other, then proceed to tear themselves in half, with each half regrowing into a new worm. 

Because of their intensely regenerative properties, you can cut a planarian in half– nay, up into hundreds of pieces, and each piece will form back into a whole worm! 

A very rough idea of how planaria regenerate tissues and limbs follows these steps:

1. Signals (either nervous, metabolic, or immune) from the site of injury begin to build up in the body
2. Pluripotent stem cell reserves (making up about 30% of a planaria's body!) flock to the site of injury
3. Stem cells proliferate into a blastema, a bud of tissue which differentiates into the structures needed 
4. The missing structures fully form into their target morphology, as in the structures that were prior injured. Cellular proliferation, differentiation then stops. Regeneration is complete!!

The process of regeneration in planaria raise some Very Important questions– how do the stem cells know where to go when receiving those first signals? When they get there, how do the stem cells form into a blastema? When does the blastema stop growing and not turn into cancer?

A lot of the responsibility of answering these questions rely on the idea of the gap junction. 

Gap junctions

Gap junction proteins are voltage gated channels that connect adjacent cells to each other. They allow cells to transfer small molecule signals from one to another at a rapid rate. 

Looks... suspiciously familiar, doesn't it?

Gap junctions are an ancient ancestor of synapses. Neurons take advantage of these mini information highways to form our central nervous system! And these gap junctions have been at it for way longer too. 

(fun fact: Levin's lab uses SSRIs and other drugs meant for your brain to mess with planaria, since those neurotransmitters go through gap junctions, causing them to sometimes open or close) 

Gap junctions are what make individual cells cooperate together. In embryogenesis/morphogenesis, they coordinate stem cells to work together and form structures. 

They are vital to form and structure. At a high level, they are what change the anarchy of sovereign cells, all acting in their own self interest and competing against one another for resources, into that hierarchy I showed you at the very beginning of this post. 

Levin's lab demonstrates this in a myriad of ways. Through closing off gap junctions in a cut up planarian missing its tail for a few days, instead of regenerating a tail the worm made... another head?

With the power of bioelectric voltage gradients, by manipulating ion channels and gap junctions, we can now change the target morphologies– the "homeostatic setpoints"– of living organisms. We can do this in a permanent way, too. If you kept the gap junctions of this two headed planarian off some more and cut them in half again, you would have two pairs of two headed worms. And just as simply, you could turn them back on, amputate them once more, and have one headed worms again.

Gap junctions allow for some primitive form of "memory"– more advanced than computers and cruder than neural memory. They allow for cells to share information (metadata) with one another. This could cause signaling cascades of many sorts! If one cell has a calcium ion spike, the ones surrounding it will too. “Memory” from one cell can infiltrate into another :o

Cancer

Cancer has a strong relationship to bioelectricity. A goal of the Levin lab is to unveil this link and make it relevant to the field of oncology. In their words, 

"One view of cancer (distinct from the current paradigm of intrinsically "cancerous" cells resulting from specific DNA modifications) is as a problem of organization within a "society of cells". Cancer is, in some sense, a disease of geometry - a failure of cells to attend to the signals that normally organize their behavior towards the patterning needs of the host"

How cool is that! Cancer can be interpreted as a disease of communication and bioelectric signaling rather than a disease of mutation and genetics. Of course there will always be a big genetic aspect to some cancers– breast cancer with the BRCA gene and myeloma affecting some ethnic backgrounds more than others– but for a large part, this could be caused by malfunctions in bioelectric signaling.

A funny way I like to look at it is that all the cells in your body form a Voltron by communicating with each other.

The bioelectricity they don't want you to know about! :p

When one of the characters, let's say the guy who controls the arm of Voltron, can't listen to the rest of the characters and coordinate with them (maybe his comms are shut off, sort of like when gap junctions are shut off wink wink), causing him to flail around crazily, messing up the whole Voltron.

Likewise, in cancer, we see cells that once cooperated with their surrounding cells lose communication with them. Levin's lab found that skin cells which had their gap junctions shut off ended up developing into metastatic melanoma.

Figure B here shows the abnormal, tumor like lesions caused by malignant melanocyte cells. This was caused by the misexpression of a potassium channel gene called KCNQ1.

When a cell becomes cancerous, it develops its own goal states and starts behaving like an individual organism, reverting back to an “ancient” unicellular past. It treats the rest of the body around it as a farm of resources to grow and flourish off of. Cancer grows well in hypoxic conditions, not much unlike our own ancient unicellular ancestors.

Bioelectricity has interactions with cancer genetics too, by the way. In a study done in 2015, it was found that the ion powered movement of butyratein through cells activates histone deacetylase, which regulates tumorigenesis (Chernet et al, 2015).

Xenobots

Xenobots may be Michael Levin's most famous work. You may have seen this in the news cycle a couple of times:

Here's one in Turkish!

It even got onto Stephen Colbert and CNN.

(the two guys who uploaded these videos, Sam Kreigman and Josh Bongard from UVM were also crucial to the development of these things. they deserve big credit for this too)

So what are xenobots? In a broad sense, they are the first unique living organisms not designed by natural selection but by algorithm. Levin, Kriegman, Blackiston, and Bongard prodded together skin and heart muscle cells of Xenopus laevis (a model organism of frog) embryos to from into the shapes shown below.

More specifically, the skin cells they used are called epithelial cells, which were used to create structural integrity and protection for the xenobots. The heart muscle cells were used for motility, because tissues that could contract (like muscles do in animals) allow for movement. Using Kriegman's computer evolved xenobots as a template, the skin and muscle tissue is able to be modeled into shapes ideal for different tasks

Above, a digitally evolved xenobot (in silico) moving in virtual space. Below, an in vivo xenobot modeled after its digital counterpart moving around physical space.

Because xenobots are designed algorithmically in a virtual environment (where the conditions of fitness can be changed on a whim), they can possess different qualities which can solve different tasks. Some xenobots evolved leglike appendages which allow them to "walk" around, others grew cilia (hairlike protrusions) that give them the ability to "swim" around. Most awesomely, some xenobots were even evolved to be able to mop up loose particles around the petri dish it lived in.

Xenobots have been evolved digitally into all sorts of shapes! Try spotting the Among Us xenobot

Another incredible capability that xenobots possess is their capacity for regeneration. Xenopus embryonic cells (which xenobots are made out of) are highly regenerative, meaning if you cut a xenobot half it will reform. Take a look!

After the forceps mess up the structure of the xenobot, it heals itself back to ship-shape.

Very recently, Levin, Kriegman, Blackiston, and Bongard showed that xenobots can kinematically reproduce. New xenobots have been designed to be able to push together Xenopus cells into shapes and structures that resemble xenobots themselves.

So, when are these things going to take over the world?

I'm glad you asked. Right now, we have every right to feel concerned about gain of function research. There is significant evidence that the SARS-COV2 virus was created in a Wuhan lab, and look where that got us. When I first heard that xenobots could replicate, I even made a joke on my Twitter that they could destroy the world by making paperclips.

Mazel tov to Sam and team!!! As long as we don't let them make paperclips I think we'll be OK https://t.co/fXDBPuP4L2

— Alex Kesin (@alexkesin) November 30, 2021

But the creation of xenobots is not dangerous. Fortunately, they:

1) can't do many bad things right now. I don't even know if they can survive out of petri dishes, and I'm sure that cytotoxic compounds can kill them just fine

2) have a conveniently programmed time to die. Because there is no way for them to currently consume food, the individual Xenopus cells survive off the yolk that they are "born" with. This means it takes about a week before the xenobots cells starve and die. 

3) The "reproduction" of xenobots is not quite like mitosis, where heredity is involved. As of now, the xenobots need fresh, lonely frog cells floating about to be able to push around into new xenobots. Its not like they can "bud" off new xenobots. 

And as Levin had noted, frogs shed their skin cells into lakes all day... yet we don't see xenobot swarms destroying power grids or turning into some crazy hivemind monster.

While I'm definitely more convinced than not whether they're safe, the public seems to be wary of their existence. Especially people in YouTube comment sections (whose wisdom is obviously some of the most impartial, empirical, and rigorous).

Three commenters in a row cowering in fear of the ferocious frog cells

Symmetry

An important aspect of living things that the Levin lab studies but I find is not highlighted enough is symmetry. Here is an excerpt where they explain generally their reason for interest:

"The vertebrate body plan is basically bilaterally-symmetrical; however, consistent and well-conserved asymmetries of the brain and visceral organs are superimposed upon the fundamental structure. Strikingly, it is now known that even single mammalian cells in culture maintain a consistent left-right axis. We are working to understand the mechanisms by which the embryo aligns the left-right axis with respect to the other two axes, and imposes this spatial information on macroscopic cell fields prior to the morphogenesis of the asymmetric organs. In contrast to popular models of asymmetry initiation by extracellular fluid flow during gastrulation, our lab studies much earlier, intracellular events that break symmetry and establish consistent asymmetry as a form of planar cell polarity, using physiological mechanisms to amplify cytoskeletal chirality across cell fields."

Huh?

At first glance, this reads confusingly. So let's break it down a bit.

"The vertebrate body plan is basically bilaterally-symmetrical; however, consistent and well-conserved asymmetries of the brain and visceral organs are superimposed upon the fundamental structure."

Interestingly, all vertebrate animals (basically any animal with lots of bones) are always left-right symmetrical. Have you ever seen a creature that look exactly like it does upside down? How about back and front? If you split a person in half (very sorry for the graphic image), the two halves are perfectly chiral (mirrored).

"We are working to understand the mechanisms by which the embryo aligns the left-right axis with respect to the other two axes, and imposes this spatial information on macroscopic cell fields prior to the morphogenesis of the asymmetric organs."

 Embryos too are like this– and makes sense, because our chirality needs to start at some point. By "macroscopic cell fields", I think what they're trying to say is that individual cells create more complicated structures that are symmetrical in their own right (emergent symmetry).

Remember gap junctions? Well, they seem to be an important reason for why this symmetry occurs. Again, in their own words:

"We identified a dependence of asymmetric gene expression on early communication between left and right sides in the chick and frog. For example, expression of left-sided markers depends on events occurring on the right side, during very early stages, suggesting that the two sides need to coordinate their decision with respect to the L-R identity of each. One mechanism for communicating between cells and tissues involves gap junctions: multimers of connexin proteins form channels between cells and pass small molecules, subject to complex regulation by various signals."

It makes perfect sense– for such a complicated aspect of morphogenesis, gap junctions play a crucial role in determining that at scale, the organism that it's forming into is symmetrical. There is no other way to coordinate the left and right sides in unison without gap junctions working well (which is possibly a reason why congenital deformities happen– they could be interpreted as disorders of symmetry).

Plasticity of the "self"

We have come full circle! Now that I've given you a tour of what Levin's lab has discovered, it's time we try to take a stab at those first couple questions I asked you. Levin has pretty solid answers for them in his recent preprint, Technological Approaches to Mind Everywhere.

1. What is a "self"? Is it a whole person? A brain? The brain cells that make up a brain?!

A self, as defined by Levin, has the following characteristics:

• the capability to pursue tasks/goals
• it is reinforced to generate positive behaviors for survival (its bad traits get it punished, its good traits have it rewarded)
• it can hold complex information/memory

Let's see if I can argue something you might not traditionally think is a "self" is actually a self. Take the mighty thermostat– it pursues the goal of keeping a room at a stable temperature (its homeostasis) (1). When a thermostat doesn't turn on at all, it will be replaced– when it works perfectly well, it stays (2). "Smart" thermostats can even be programmed to change their temperatures depending on the time of day! (3)

If that isn't a self, I don't know what is. (here is a Twitter thread I took part in talking about other homeostatic systems)

      2. Do you think individual cells have goals they try to achieve, akin to humans pursuing their own goals?

Well, as we have previously explained, cells do have their own individual goals. As I mentioned about cancer earlier– cancer cells shift their goal state away from the rest of the body's. While cells have their own goals, gap junctions and lines of cellular communication allow cells to cooperate with each other and make complicated structures at the benefit of each cell. 

Levin says, "even advanced animals are really collective intelligences." Cells are intelligent organisms themselves that form us– we are the superorganisms they compose.

An amazing example of this in action is how we've been able to coach neurons in a dish into being able to play a flight simulator together.

Each neuron is unique! The dish, however, is plated with electrodes, which can reward or punish proper behavior to specific neurons for the emergent network as a whole. Thus the system as a whole learns how to properly fly the virtual airplane, even during adverse conditions.

      3. Are humans and animals the only creatures that can "remember"?

No. 

Memory is a complicated idea that should not necessarily be misconstrued with "consciousness". It's been found that our good old friend, the planarian, can remember how to do specific tasks after their head has been cut off! So my last question is a bit of a trick question– you don't even need to be a thinking "creature" to necessarily have memory. After all, the worms LOSE THEIR FREAKING BRAINS!! BUT STILL REMEMBER!!

Take another example– reindeer's antlers. Reindeer antlers seasonally grow and then fall off. The antlers don't need to be told where to grow– the tissue they stem off of "remembers" on cue when to start growing! Antlers injured at critical places too will remember the "wrong" way to grow next season. This is memory taking place outside of the brain... how cool!!

Okay, still not convinced about the plasticity of the self? I will give you two more bizarre examples that Levin mentions in the TAME paper.

Even though caterpillars go through extraordinary metamorphosis into butterflies– having their brains liquefied in the process– they still retain memory they learned as caterpillars when they become the entirely different creature of a butterfly. Butterflies avoid a scent associated electric shocks they learned about when they were caterpillars, and even might retain memory of what kind of leaves to lay their eggs on from when they were barely out of eggs themselves. Even though butterflies and caterpillars are fundamentally different animals: in shape, diet, and environmental niche. To a degree, the same goes for mealworms and flies; tadpoles and frogs; potato shaped babies and human shaped humans.

My favorite pet example Levin brings up though has to be how some twins who are conjoined at the head can share many complicated behaviors. The "oneness" of human beings as we know it is challenged by these two Canadian sisters, Krista and Tatiana:

Through their thalamus being connected, the sisters can:

• see what the other is seeing (they tested them by blindfolding one and asking her what the other sister was looking at)
• taste what the other sister is eating
• feel what the other sister is feeling (they tickled one sister and the other would get jumpy!)

While each sister is her own unique person, it is undeniable that cognition can transcend the self.

So what in store for us? Levin has a hunch that in the future, we will have "chimeric" selves– a living organism that traditionally would have its own distinct parts, but acts as one. An example would be a creature that is part vacuum cleaner, part human, and part tiger. This sounds disturbing at first, but in a way it's already happened. It has been evidenced that mitochondria, before being part of cells, was its own independent organism. Now, they are an organelle just like any other in cells– a part of a whole!

The promising future we have ahead

Levin's work as a thing to admire is already groundbreaking. But excitingly, a lot of what we've learned through his research will soon be applicable to benefit the world greatly. Here are several innovations that are being spun out of the lab:

• Levin's lab has found ways to manipulate voltage channels and bioelectrically surpress the cancer causing effects of oncogenes. They were able to shrink and eliminate tumors this way!
• Through causing deformities in tadpoles (dubbed Picasso tadpoles, since their facial features were put in all the wrong places), Levin was able to show that as the tadpole becomes a frog, an "electric face" is cast onto the facial tissue, reorganizing it into a proper frog face without any deformities.
• By manipulating ion channels in tadpole gut cells to emulate behaviors of an eye, the gut cells developed into... well... an eye. A fully functional one, in fact! This means we can develop new organs by manipulating bioelectric networks in our bodies– imagine if we could grow new eyes for the blind, or repair old ones.
• Xenobots have many amazing use cases– cleaning up ocean waste, scraping up cholesterol in arteries, and delivering drugs are a few of many possible ways they can perform tasks that current technologies have great difficulty performing precisely.
• Regrowing limbs!!! Levin's lab has been able to regrow Xenopus frog legs by coaxing them to regrow with bioelectricity manipulating hormones.
  
  
  
  Levin is actually working on a company called Morphoceuticals that has the goal of regrowing limbs in mice. You can check it out here!

          Michael Levin's ultimate goal for his lab and the synthetic biology community as a whole is to develop what he calls in his own words an "anatomical compiler". He wants it to be easy to CAD and 3D print out a creature of one's own design with the click of a few buttons. And to that, I say:

Acknowledgements: a big thank you to Dr. Levin and Ikey Croog for proofreading this and pointing out mistakes :)