When cells don’t need chemical signals to find a wound after injury

Evolution over millions of years has given us the ability to function in our environment with little conscious thought—our heart pumps blood throughout our body, our digestive system pulls nutrients out of food, and when we get a cut, our body magically heals itself. But what if these things didn’t work? What if we had to tell our heart to contract like we tell our biceps when lifting a heavy box? What if digesting food required conscious thought or wounds didn’t heal? We probably wouldn’t survive very long. Luckily, our “built-in” systems keep us alive and thriving.

One of these built-in systems is wound healing. After getting a cut we might worry about how much it hurts or how long it will last, but we don’t have to consciously focus on healing. In order to function automatically, the cells that work to heal the wound need a way to know (1) that the wound occurred, (2) where the wound is, and (3) the size of the wound. And they need to know quickly. Cells gather this information using sensors that look for specific damage signals called DAMPs (damage associated molecular patterns). DAMPs form chemical gradients that alert surrounding cells that damage has occurred and the location and severity of the wound. (You can read more about DAMPs in my previous post HERE.) But although it has been well established that chemical gradients created by DAMPS are used to alert cells of an injury, scientists in the Philip Niethammer* group asked whether a mechanical sensor that indicates tissue damage would also be possible.

chatty-cells

After an injury, cells need to communicate with each other in order to find and heal the wound.

Unlike chemical sensors, mechanical sensors rely on physical sensations such as motion, temperature, and pressure. One potential biological mechanical sensor is cellular swelling.¥ Cellular swelling can result from tissue stress such as wounding and has been associated with recruiting leukocytes (white blood cells that are essential to protect the body from infection, especially at the site of a wound) to the injury site. The current theory to explain how cellular swelling recruits leukocytes involves the swollen cells rupturing to release DAMPs into the extracellular space (like a water balloon that gets too full and breaks). However, recent studies have demonstrated that cellular swelling can still initiate a wound healing response without cell rupture. These studies bring to light the potential role of mechanical sensors such as cell swelling in wound healing.

The first thing that the Niethammer lab found was that cellular swelling increased calcium (Ca2+) levels in cells surrounding the wound. The increase in Ca2+ caused cPLA2, a type of lipid found commonly in wound response, to move from the nucleoplasm (part of the inside of the nucleus) to the inner nuclear membrane (part of the outer ring, or “skin”, of the nucleus). Using this knowledge, the Niethammer group hypothesized that cellular swelling was sufficient to cause cPLA2 movement within the nucleus.

cellswellingfig

Cellular swelling causes cPLA2 movement from the inner nuclear membrane to the outer nuclear membrane. This process attracts leukocytes to the wound.

To test this hypothesis, the Niethammer group developed a novel method to make synthetic nuclei. Using giant vesicles made from dried lipids, they were able to mimic the “onion-like” inner and outer layers of the nucleus. After adding cPLA2 to the giant vesicles in the presence of Ca2+, they changed the environment of the vesicles to induce swelling. As they hypothesized, when the vesicles began to swell, cPLA2 moved to the outer layer of the vesicle. This means that cPLA2 movement only requires cell swelling and calcium! By working with only the bare minimum, they were able to strongly support their claim that mechanical sensing is possible in wound response which recruits leukocytes to the wound.

These experiments from the Niethammer lab are significant for a few reasons:

  • They have strong evidence to support a mechanism for wound sensing that differs from the current theory for how leukocytes are recruited to a wound. Mechanical sensing for wound response provides an alternative mechanism that may explain anomalies in cellular signaling. Furthermore, different areas of the body may require different sensing mechanisms. When thinking of wounds, you probably first think of wounds that happen on the outer layer of your skin, or your skin epithelial cells. However, there are epithelial cells throughout your body lining your organs and blood vessels to separate and protect them. Cellular swelling as a sensor is applicable to wounds such as those that occur in the digestive tract where the cells are in a hypotonic environment.¥
  • They developed a novel method to mimic nuclear layers that can be used to test how different extracellular environments can affect a cell. The giant vesicles are especially interesting because they reduce some of the complications that come from working with entire cells or organisms. Although it is important to take this complexity into account, a simplified approach allows researchers to isolate specific factors.
  • They show how simple biology can be. One of the beautiful things about biology is that, although constantly evolving to survive in ever-changing environments, it often takes the path of least resistance. That is, sometimes the simplest answer is the correct answer. Although other tissues may require more complicated sensing, biology takes advantage of instances when simple mechanical sensing is sufficient to relay an appropriate response.

*Enyedi, B.; Jelcic, M.; Neithammer, P. The Cell Nucleus Serves as a Mechanotransducer of Tissue Damage-Induced Inflammation. Cell. 165, 1160-1170, (2016).

¥ Cells swell when the inside of a cell is exposed to a hypotonic environment, or environment that has more water than the inside of the cell. Biology needs balance, and in order to balance out the amount of water inside and outside of the cell, water begins to enter the cell—therefore causing swelling.

Illuminating biology with biological glowers

Utilizing Biological Phenoma to study Biology: Bioluminescence

Quick: Is the glowing water in this picture photoshopped or real?

bioluminescent waves

Phil Hart [Copyright Phil Hart under a Creative Commons attribution-noncommercial-share alike 2.5 license]

The correct answer is real! Yes, there is such a thing as naturally occurring glowing water. And it is made possible by a phenomenon called bioluminescence.

Bioluminescence is a chemical reaction that happens in a living organism to produce a beautiful glow. Last month a few of my friends were swimming at La Jolla Shores and noticed that the water around them lit up with every movement of their bodies. What made the water glow? Phytoplankton emitting light when agitated—every movement from waves, currents, fish, or, in this case, swimmers, sent colorful flashes throughout the water. Wanting to witness it for myself, we went out again to experience the “glowing waves”. Unfortunately, we were too late in the season to experience the full beauty of bioluminescent plankton lighting up the waves, but by rapidly swishing our hands underwater, we were still rewarded with small sparks of light. Although my encounter with bioluminescence fell short that evening, I do get to see it in action every day in the lab.

Today I introduce how scientists utilize bioluminescent properties to study biological phenomena at the molecular, cellular, and organism level. Throughout history humans have benefited from the natural glow produced by bioluminescent organisms such as the use of dried fish skins as a source of light in coal mines instead of lamps. Usage of these organisms has led researchers to ask the question: what makes these organisms glow? Seeking to understand bioluminescence at a molecular level has led researchers to produce fluorescent “tags”. These “tags” can be attached to an unlimited number of molecules and proteins to answer fundamental biological questions.

Bioluminescence is a chemical reaction that takes place in a living organism to produce light. Although the most well-known organism to produce this glow might be the firefly, the majority of bioluminescent creatures are found in the ocean. Different organisms may vary in how they produce their unique glow, but the basic mechanism involves the enzyme* luciferase reacting with the light-emitting compound luciferin to produce light.

The beauty of naturally-glowing living organisms led researchers to investigate the underlying chemistry of this unique phenomenon. One researcher, Osamu Shimomura, began a lifelong relationship with bioluminescence when he found the structureǂ of luciferin in 1955. What followed was a quest to discover how different organisms produced their own unique glow. While investigating the bioluminescent jellyfish Aequorea victoria, Shimomura discovered green fluorescent protein (GFP). This discovery, and the consequent development of GFP, revolutionized biology. Scientists could now attach fluorescent proteins, such as GFP, to a molecule of their choice and literally watch biology in action by watching the fluorescently tagged molecules. In 2008, Shimomura, along with Martin Chalfie and Roger Tsien, received the Nobel Prize in Chemistry for their discovery and development of GFP.

Since the discovery of GFP, fluorescent proteins have been developed in a wide variety of colors for an extensive number of molecules and proteins. Although both fluorescent proteins and bioluminescence produce light, they work very differently. Rather than using a chemical reaction, fluorescent proteins produce certain light colors by using energy from other specific wavelengths of light. Fluorescent proteins create their unique glow when they are “excited” by a wavelength of light specific to the protein.¥ For example, GFP

GFPexcitationandemissionemits green light at ~510 nm after shining blue light at ~490 nm on the protein. By manipulating the protein, these light patterns can be optimized to produce different colors!

bioluminesenceTsien

Source: Tsien Lab

Researchers can use these proteins to image biological events in real-time and real-space. This process is called fluorescent microscopy. Fluorescent microscopes work by first hitting a sample with an excitation light. A camera attached to the microscope then catches the light emitted from the sample. When specific molecules (proteins, compounds, parts of cells, etc) are tagged with different fluorescent proteins, researchers can watch, analyze, and quantify what is happening—whether this is of molecules by themselves, inside of a cell, or even inside of an organism. Advancing technology in fluorescent proteins, microscopes, and other emerging biological approaches are pushing the limits of how small we can go (can we look at very specific interactions inside of a cell?) and how many things we can watch at the same time (can I see 6 things inside of a cell at the same time rather than only 3?). These advances are enabling us to find answers to critical questions such as:

  • Why do some cells act differently than others, even when treated the same? This question has applications in cancer research, healing responses, and why certain drugs work for some people and not others.
  • Can we predict the movement patterns of cells in response to a specific stimulation? Take a look at my previous post on wound response to see an example of the importance of understanding cellular movement.
  • What are the differences between the actions of a single cell and the actions of a population of cells? Single cells do not always act the same, and yet, a population of cells has a very predictable response. Why?

Although still young, the use of fluorescent proteins in biology is an exciting and continually improving field. By applying fluorescent microscopy to quantitative biology methods, we can better understand biological events to answer some of the most complex biological questions, such as those listed above. Most importantly, the resulting images provide a snapshot into the hidden world of biology by providing a new level of detail into how we function as living organisms.


*An enzyme is a protein that accelerates chemical reactions. Here, it accelerates the chemical reaction that makes luciferin light up.

ǂ In order to understand how things work, scientists often need to first find the structure of their molecule or protein of interest. Structures can be found using a variety of methods.

€ A wavelength of light is a unit of measurement in a light spectrum. That is, each color of light that we see has a specific wavelength—this is the visible light region (390 nm –780 nm).

Light Spectra

¥ Each fluorescent protein has a specific excitation spectrum and emission spectrum. A spectrum is a pattern consisting of peaks and valleys at different wavelengths across the visible light spectrum. This spectrum is unique for every fluorescent protein. For example, the spectrum of GFP has an excitation peak at ~490 nm and an emission peak at ~510 nm. ­ The emission spectrum consists of a peak that emits the color for that wavelength – the green that you see in GFP. This happens only after a specific wavelength of light, or the peak found in the excitation spectrum, excites the fluorescent protein.

GFP spectrum

 

How do cells find a wound after injury?

How do cells know where they are? It’s an important question we often take for granted, especially in situations like wound healing where cells that are closer to the wound need to respond differently than cells that are farther away. If cells don’t respond correctly it can lead to chronic wounds (wounds that don’t heal), excessive scarring, and even cancer. Here I highlight a study recently published in Currently Biology that demonstrates how hemocytes*  find their way to a wound. The Paul Martin Lab at the University of Bristol and the Michael P.H. Stumpf** group at the Imperial College London worked together using a combination of in vivo (meaning, experiments done in a living organism) wound data and computational simulations to answer the question of how hemocytes know which direction to travel following an injury in Drosophila (fly)*** embryos.

HowDoCellsKnowWhereTheyArev3

One way that cells figure out where they are is by using information from the wounded cells themselves. Injured cells release DAMPs (damage associated molecular patterns) which tell the neighboring healthy cells how far they are from the wound. To illustrate how DAMPs work, imagine you are sitting in the stands of the Shamu show (or what used to be the Shamu show, at least) at SeaWorld and you want to figure out how close you are to the tank. It should be easy enough, but here’s the catch: you can’t open your eyes and you can’t move. One way you could figure out how close you are is by measuring how wet you get when Shamu splashes the audience. If you are close to the tank, you will get soaked. If you are farther from the tank, you will stay dry. DAMPs work in a similar way. Cells that are closer to the wound receive a lot of DAMPs, and cells that are further away receive less. However, scientists still aren’t sure what exactly these DAMPs are and how they travel across cells to activate them to initiate a healing response. Here, Martin and Stumpf determine how DAMPs lead hemocytes to an injury.

Following an injury, hemocytes travel to the site of the wound to begin the healing process by following a gradient of DAMPs. However, the specific identity of these DAMPs and how they travel remains unknown. Since the technology to physically measure gradients formed by DAMPs is yet to be fully developed, the Martin and Stumpf groups developed a mathematical model to predict how the DAMP gradient forms based on how hemocytes traveled to a wound following an injury. They began by injuring flies and quantifying how hemocytes moved towards the wound. Quantifying hemocyte movement involved several parameters including how long it took the hemocyte to reach the wound, when the hemocyte began to move after the wound, and how many times the hemocyte changed directions while moving towards the wound.

The results of their experimental data combined with the computational model show 3 main points that differ from the current theory of DAMPs.

  1. The Martin and Stumpf labs found that DAMPs are released from the cells immediately surrounding the wound. This differs from prior predictions that DAMPs are released from the wounded cells themselves. The current theory of DAMP release is kind of like a water balloon that explodes when punctured. All of the water comes and gets anything in close proximity wet.
  2. The model revealed that there is only a single “wave” of DAMPS that lasts for only 30 minutes (as opposed to multiple releases over an extended period of time). This is significant when you think about how long a wound takes to heal-days and even weeks! That’s like taking a 2-week long road relying only on directions given to you in the first 30 minutes.
  3. DAMPs that recruit hemocytes to a wound travel by diffusion. That is, the healthy cells surrounding the wound do not use any sort of active mechanism to ensure that the DAMPs travel to the appropriate distance.

The Martin and Stumpf groups were also able to use their model to determine how hemocyte recruitment changes with multiple wounds, closely spaced wounds, and large wounds.

Multiple Wounds. For multiple wounds, they found that hemocytes become desensitized, or no longer respond, if the wound takes place less than 3 hours following the first wound. This makes sense when you think about how an organism needs to efficiently allocate its resources. If all of the hemocytes went to the new wound, then the old wound would not be able to continue healing. However, this might imply decreased healing for wounds occurring close to the first wound within a short time period. They also examined how hemocytes travel to wounds that are varying distances from each other.

Closely Spaced Wounds. Wounds that are very close together are essentially seen as “one wound” by hemocytes whereas wounds that are far apart are seen as two separate wounds. However, wounds at an intermediary distance displayed an interesting hemocyte behavior. Here, hemocytes between the wounds struggled to decide which wound to travel to, resulting in a decreased number of hemocytes at both wounds. It would be interesting to study whether this behavior causes wounds located at intermediary distances to heal less efficiently, but as of yet, no one has.

Large Wounds. The Martin and Stumpf groups studied the effects of very large wounds on hemocyte recruitment. Interestingly, they found that some large wounds healed appropriately while others did not. This is similar to clinical observations where chronic wounds cannot always be determined at the initial stage of wounding (meaning, you can’t tell if a wound is going to be chronic based simply by looking at it). This is the first time that a chronic wound model has been shown and will be of interest to future scientists to study what factors impact whether a wound will be chronic or not.   

So what does this mean in the broader world of science??

One of the most interesting thing that the Martin and Stumpf accomplished here was their ability to develop a mathematical model to predict the formation of the DAMP gradient. Mathematical models, although used heavily in other sciences such as physics and chemistry, have been slow to be adopted in biology. Biology can get messy, and therefore mathematical models can be difficult to implement. However, when technology to directly observe biological phenomena is still lacking, mathematical models can be a powerful tool. Although their model cannot tell you what the exact DAMPs released from the wound are (that will require more experiments), it provides a lot of clues to where researchers should be looking. This is a great example of the power of quantitative biology to solve biological problems.

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*Hemocytes is the term for blood cells in invertebrates. In this case the invertebrate is a fly. Hemocytes are a part of the immune system and are required in the wound healing process.

**Weavers et al., Systems Analysis of the Dynamic Inflammatory Response to Tissue Damage Reveals Spatiotemporal Properties of the Wound Attractant Gradient, Current Biology (2016), http://dx.doi.org/10.1016/j.cub.2016.06.012

***Why flies? The use of Drosphila embryos as a model is common in wound studies. They are transparent, making them easy to image under microscopes. Furthermore, flies are easy to genetically alter and, as far as immune response goes, work similarly to humans.