Wound Healing & Jellyfish

When our bodies are wounded, we want them to heal as quickly as possible. Not only are injuries painful, but they can also be very dangerous. We see this when we look specifically at our skin.

Our skin is made up of sheets of cells called epithelial cells. Epithelial cells of the skin are attached to each other very tightly, creating a barrier that protects us from infection. Any damage to the skin allows bacteria access to the inside of our bodies, potentially causing disease. There are also sheets of epithelial cells on the surface of our eyes, and lining our intestines and lungs. Indeed, it is epithelial cells that allow us to have different organs with distinct functions. For example, they prevent the contents of the gut from leaking out into the body. When epithelial cell sheets are damaged, they need to be repaired as quickly as possible to prevent infections and allow organs to function as they should. 

When epithelial cell sheets are damaged, many other cells in the body rush simultaneously to limit the negative impact of the wound. In humans, blood clots to quickly close the wound. Special cells called immune cells rapidly migrate to the wound site. The job of these cells is to recognize and destroy any invading bacteria. Another type of cell called a fibroblast migrates to the wound. Fibroblasts secrete special proteins that create a temporary patch to the hole in the epithelial sheet. The epithelial cells on either side of the wound then spread out and "walk" forward to remove the temporary patch and permanently close the hole in a process known as re-epithelialization. Cell “walking” is called migration, and it only happens in very specific circumstances such as wound repair. All these events are triggered by a single injury, but they have to be carefully controlled and coordinated so that wounds heal correctly. We still don't have a complete understanding of how this all works, and our research team wanted to learn more.

It is extremely difficult to study wound healing in humans or other complex animals such as mice because so many healing processes are happening at the same time. It is also difficult to watch cells moving around in living animals. Luckily, many of the wound healing events in complex animals are similar to those in much simpler animals across the tree of life, from fish to insects to small worms. Our work has shown that even jellyfish heal epithelial wounds. In fact, the re-epithelialization process in one small jellyfish, named Clytia hemisphaerica (Clytia), looks very similar to that process in ourselves! This is fantastic for scientific research because jellyfish don't have blood, migrating fibroblasts, or immune cells. That means we can focus entirely on how the re-epithelialization process works without being confused by the other pathways controlling the fibroblasts and immune cells. Another great feature of Clytia is that they are completely transparent, so we can observe cells moving in real time in live animals. We can make wounds in the epithelium and watch them heal through video recordings. The wounds heal in only about an hour! One final advantage is that epithelial cells in Clytia are sitting on the “jelly” of the jellyfish, which makes up most of the animal. We can insert a small needle into the jelly and introduce dyes and other chemicals to see the effect that they have on the epithelial cells in living animals. This would be just about impossible in live mice or humans.

In our recently published work, we focused on a big question in re-epithelialization – how do epithelial cells know that there is a wound and start to transform from cells that sit quietly in one place to cells that spread and migrate? To understand this, you'll need to know a little bit about cell migration. All cells have an internal skeleton made of proteins that determine the cell's shape. One of these proteins is called actin. When cells are triggered to migrate, the actin in the cell skeleton accumulates at one side of the cell, so that the cell now has a front and a back. The actin in the front makes a bulge called a “lamellipodium” - which literally means “flat foot” - in the front of the cell. If the back of the cell lets go while the lamellipodium in front bulges forward, the whole cell moves forward. If actin cannot move to the front of the cell to form a lamellipodium, cells don’t migrate. All of this raises the interesting question of how wounding triggers actin to move to the front of cells to form a lamellipodium. 

This question is particularly intriguing because actin moves to the front of cells that are right at the margin of the wound (marginal cells), and also cells that are some distance away from the wound (submarginal cells). This is potentially great for wound healing, because if marginal cells and submarginal cells can make lamellipodia they can all migrate together to close wounds. However, we wondered how the submarginal cells know that there is a wound nearby. More specifically, how does wounding an epithelial sheet make actin accumulate at the fronts of the marginal and submarginal epithelial cells? 

To answer these questions, we looked for a small molecule that might act as a signal, spreading from the wound to the surrounding cells and letting them know that they should respond. One damage signaling molecule that has been studied in other systems is adenosine triphosphate (ATP). ATP is commonly known as the energy of a cell. ATP levels are very high inside cells, where they are needed to power many important processes. In contrast, ATP levels outside cells are very low. One of the few times that ATP levels OUTSIDE of cells (extracellular) go up is when cells are broken and ATP spills out. This has led to a very clever innovation – there are examples of both plants and animals that recognize extracellular ATP (eATP) as a signal that there is a wound! We know very little about how eATP signaling works to help wounds heal, so we decided to see if we could learn more by studying eATP in wound healing in Clytia.

In our first set of experiments, we injected ATP in an area OUTSIDE of the epithelial cells in a Clytia jellyfish and then made a wound to see if the eATP helps epithelial wounds heal. We found that wounds indeed closed faster with more eATP. We also injected a form of ATP that can't be used as an energy source. This worked as well as regular ATP, showing that ATP is acting as a signal rather than a source of energy to enhance healing. We then did the opposite experiment and injected apyrase, a protein that destroys ATP, outside of the epithelial cells before wounding. We found that wounds closed more slowly after apyrase injection. Together, these experiments confirm that the ATP released from cells during wounding in Clytia helps wounds to heal.

To better understand what the eATP is doing, we looked at the role of actin in healing epithelial wounds in Clytia. In unwounded epithelium, actin was seen diffusely throughout the cell. After wounding, as predicted, actin accumulated at the fronts of wound margin cells, and also at the edges of submarginal cells a bit away from the edge of the wound. To ask whether eATP is involved in the re-localization of actin, we injected apyrase before wounding. We found that actin still moved to the fronts of the marginal cells, but that accumulation of actin at the edges of the submarginal cells was greatly reduced! This suggests that eATP may be a signal, moving from the wound site to tell submarginal cells that an injury has occurred nearby and that they may be called upon to migrate.

We next asked how eATP OUTSIDE of the cell could cause a change in actin distribution INSIDE the cell. Many processes inside cells, including actin re-localization, are triggered by increases in calcium. Calcium levels inside cells are usually very low, while outside cells calcium levels are high. Could eATP allow calcium to enter cells in jellyfish? To ask this question, we used a yellow fluorescent dye called YO-Pro-1 that has many of the same characteristics as calcium and therefore can act similarly to calcium. When we injected YO-Pro-1 outside of epithelial cells, it did not enter cells. However, if we then injected ATP, the insides of the epithelial cells started to light up with yellow fluorescence. This tells us that eATP opens a channel in epithelial cells that could let in calcium, and gives us some clues as to how eATP may be affecting actin localization and cell migration.

We still have many experiments that we would like to do. We want to see if we can image calcium entering epithelial cells after wounding or addition of ATP, and we want to ask what happens if we prevent calcium entry. We also want to understand the channel that opens up in response to eATP. We predict that these experiments will lead to a better understanding of epithelial wound healing, both in the simple jellyfish Clytia and across the animal kingdom. This work could even one day impact the way we treat wounds in humans!

Written By: Jocelyn Malamy & Elizabeth Lee

Academic Editor: Neuroscientist

Non-Academic Editor: Operations Manager

Original Paper

• Title: Epithelial wound healing in Clytia hemisphaerica provides insights into extracellular ATP signaling mechanisms and P2XR evolution

• Journal: Scientific Reports

• Date Published: 1 November 2023

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