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Using microscopy to understand wound healing

17 February 2012

Researchers funded by the Biotechnology and Biological Sciences Research Council (BBSRC) are using advanced imaging techniques to uncover the early stages of wound healing in the hope of improving our understanding of how the human body heals itself.

When you cut yourself your body is able to heal the wound, often without scarring, because neighbouring cells move in to fill the gap. But how do those cells know when and where a wound has formed?

With funding from BBSRC, scientists at the University of Birmingham are using high powered microscopes to investigate this fundamental cellular puzzle.

Occludin (red) in a Green Fluorescent Protein expressing cell (Green) on the wound edge. Image: Dr Josh Rappoport, University of Birmingham

Occludin (red) in a Green Fluorescent Protein expressing cell (Green) on the wound edge. Image: Dr Josh Rappoport, University of Birmingham

Cell migration is the mechanism by which cells move to new locations. It is important in a number of clinical processes, yet no one really knows how it happens, particularly when groups of cells move together such as during wound healing. Sometimes cells don't move when we would like them to (as in ulcers) while other cells migrate when we don't want them to (like when cancers spread through the body). The University of Birmingham scientists have been investigating how a process called endocytosis could play a key role in the cell migration involved in repairing damage to organs such as the skin, lungs and kidney.

Endocytosis is the mechanism by which cells take in substances from their surroundings and previous research has shown that cells do not move properly when endocytosis is inhibited.

Using microscopes in the Birmingham Advanced Light Microscopy (BALM) facility, the researchers showed that migrating cells used endocytosis to take in proteins from their surface. The key, though, was to find which of these proteins was the most important.

Dr Josh Rappoport who led the study explains: "Normally, the epithelial cells which line surfaces in our bodies are bound tightly together to form a protective barrier like a line of people holding hands. When this line gets broken, like in a wound, the rest of the cells move forward to fill in the gap. We've known for a while that when these cells migrate they have to remove certain molecules from their surface, kind of like a ship raising its anchor, but we didn't know which molecules or why."

The team identified two candidate protein molecules which they suspected played a key role in migration. These proteins both help to moor a cell in place and act as sensors by which cells detects changes in their environment. Based on what researchers had seen in other types of cells, the team initially expected that the cells on the edge of a wound would use endocytosis to draw in the molecules that make structures called focal adhesions, which allow a cell to cling to its surroundings. However, when they used a powerful form of microscopy called total internal reflection fluorescence, or TIRF, microscopy to study migrating cells in the lab, they were surprised to find that endocytosis was not involved in rearranging the focal adhesions during wound healing. Instead, the team uncovered a different story.

Inhibition of endocytosis prevents a wound from healing in a cell culture. Image: Dr Josh Rappoport, University of Birmingham

Inhibition of endocytosis prevents a wound from healing in a cell culture. Image: Dr Josh Rappoport, University of Birmingham

Using another imaging technique called confocal microscopy the team found that a different protein, called occludin, was being taken in via endocytosis.

In a healthy tissue occluding holds epithelial cells together to form a single flat layer. When the researchers created a layer of epithelial cells in culture and then made a wound the cells on the edge pulled occludin in via endocytosis and then moved forward to fill the gap.

Interestingly though, when the researchers added additional occludin to the spaces between cells the wound was not filled as quickly.

"It seems simple, but how does a cell know when and where the wound has formed?" adds Dr Rappoport. "Cells generally grow until there is no space left, but when cell layers are wounded a big gap opens up for cells to fill. The can be compared to being in a crowded lift, or the tube. When the doors open and people exit, others move forward to fill in the space that has opened up.

"This process was slowed down by the addition of extra occludin but we don't know why exactly. It could be that presence of occludin left over at the wound edge gets in the way of movement or that by internalising the occludin at the wound edge the cell knows which way to move."

The research, published in the journal Biology of the Cell,has uncovered a key component in early events following wound formation. The next step is to understand how these components work together to create a fuller picture of the healing process.

Dr Rappoport and his team have produced a video explaining the science behind their work.

Contact

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