Fruit Fly Muscles and Your Friends' Health

I wrote this article to participate in the AWSAR 2019 competition. Turns out previous years' winners can't apply. So here it is for everyone.

Consider the value your muscles bring to your life? Imagine for second, whatever you did since this morning. Now picture doing it without using any muscles. Unless you were sleeping and completely still, not a twitch or blink of the eye, you depended on your muscles. Arguably, working muscles are as important to quality of life as a working brain. Some people lack this luxury. Due to genetic mutations running in some families, members lose their muscles at different stages of life. This condition is called muscular dystrophy. A close friend of mine was training to be a fast bowler in the Ranji trophy, in his late teens. A decade since the onset of muscular dystrophy, picking a cup of tea, a laptop out of his bag or himself out a chair are formidable tasks. He would have made an excellent fast bowler. Maybe you know someone who has or will suffer from muscle disease. One in 3,500 of us suffer from muscular dystrophy. We do not know enough about all the genes necessary for keeping muscles innately healthy. If we did, maybe we could treat my friend, and yours.

Before attempting to understand muscle diseases, let us understand muscles. Skeletal muscles, the kind used to move limbs, are made of long thin tubes that can contract, a little like elastic wires. Their ability to contract comes from long strings of molecular springs inside each cell. These “strings” are attached to cell-surface making sure the whole cell acts as one contractile unit. Many such cells bundle together like wires form a cable. Several such “cables” form what we think of as muscles. A common muscle disease is Duchenne Muscular Dystrophy where the molecule attaching the “strings” to the cell surface is defective. 

We can identify some mutations in people that can lead to muscular dystrophy. In several patients, we don’t know which mutations cause the disease. We hope if we know the mutations, we might find out which bodily functions are affected, and then treat the disease. Knowing surely if a mutation or mutations cause muscle diseases takes rigorous testing.

How would you decide if a genetic mutation causes a disease? At a basic level, if many patients have the same mutation, but it is absent in healthy people, that mutation may partly or wholly cause the disease. To be certain, you’d have make the same mutation in other people and show that those people now suffer from the same disease. The next step would be to remove this mutation and show the patients improve. As you can imagine, causing disease and discomfort to fellow human beings is unethical, specially with the absence of any guarantees. Even if we could do this easily, which we cannot, it would be plainly wrong.

To avoid human suffering in search of treatments, over the past century we have identified animals where this is possible. Some muscles in mice and fruit flies, share the same basic design as human skeletal muscles. In fruit flies, the ones that drone around old bananas, we have developed the ability to make genetic changes almost at will. They are also quicker and cheaper to work with and less ethically problematic than mice. The basic principles and molecules that affect body formation and by extension are affected in diseases like cancer, were first identified in fruit flies. In many cases, human proteins can substitute fruit fly proteins with few adverse consequences for a fruit fly. That means, that flies and humans share some of the same cellular machinery.

In fact, certain mutations in human patients have similar effects on fruit fly muscles. Mutant flies stop flying and their flight muscles become delicate and weak. This presents an opportunity! If we find out what aspects of muscle form and function goes wrong with mutant fruit fly muscles, it is likely that similar changes happen in human patients. Further, if we could treat diseased mutant fly muscles to health, we might be able to treat human muscle dystrophy patients on the same principles.

Fruit fly muscles however present a special technical challenge. They are too large for complete regular microscopic inspection and can get damaged during dissection. It is hard to observe them fully as is inside an animal without deforming them. Deforming muscles while trying to observe them can lead to completely inaccurate analyses. Sometimes, technology determines what questions a lab can address. We needed a way to see and measure muscles inside a fruit fly without cutting them out. So how does one “see” muscles inside a fruit fly without cutting them?

We can see objects because they reflect light. For instance, a flower reflects sunlight and becomes visible to us. But, you can’t see what is inside the trunk of a tree, because the bark reflects light before it can reach under the surface. You probably don’t see bones inside your hand (please consult a doctor if you do). That’s because bark, your skin and flesh are opaque to light. But a sheet of good quality glass, can be completely transparent to light, making it invisible from certain angles. 

But bones inside us are visualized. You’ve probably seen pictures of them. Airport security uses a similar trick to check what’s inside bags without opening them. Those pictures are generated with X rays. X rays are light waves with very high energy. They can pass through objects that are opaque to visible light. X rays get absorbed by nuclei in atoms that are heavy. If many such heavy atoms are packed together tightly into a nugget under X-rays, we can detect on the other side of the nugget, is its X-ray shadow. Calcium is tightly packed in bones so you can clearly see their shadow in an X ray picture but not skin and muscles. To take 3D pictures, many shadow images of the same object are taken from around it. A computer collects these shadow pictures to calculate what the object looks like in three dimensions. This process is called Computer-assisted Tomography, more popularly called CT Scanning. High resolution CT scanning or MicroCT scanning allows us to look at the intricate details inside small objects, whether they are stones, skulls of lizards or complete insects.

To see muscles as they are inside fruit flies, we faced a problem with employing MicroCT scanning. Insect muscles are also transparent to X-rays. To “see” muscles, we’d need a heavy element to sit with them, the way calcium stays firmly in bones. Iodine is one such heavy element that sits in muscles. Researchers have tried many compounds of heavy elements to visualize the insides of animals through MicroCT scanning. A problem with previous methods was that they dissolved these compounds, or elements in alcohol and placed animal tissue in this solution. Animal tissues do take up these stains and their MicroCT scans reveal muscles. Evidently though, soft tissue and muscles in some sense dry (dehydrate) in alcohol. This drastically changes the shape and size of muscles. While they can be seen inside an insect, you see they are deformed. You wouldn’t know whether this was diseased muscle or not.

We realized that for accurate measurement, these muscles would have to remain in a water based solution. These  MicroCT scans of fruit fly muscles looked completely accurate. This allowed us to accurately measure their shapes and volumes. To know what diseased muscles look like, we now had to define what ‘normal’ means in this context.
Fruitfly CT Scan

This method allowed us to ask how muscles in flies change throughout their life. We found that one subset of muscles is three times larger in females as compared to males. Specific muscles grow in adult females, but all but one muscle grow in males. As a fly ages, the separation between contractile cables inside muscles increases and might be the reason they cannot fly as well anymore. Strikingly, left and right muscles do not have to be equal in volume. Imagine, your left chest muscles being 25% larger than the right! In flies this is normal. In fact, through our studies (Chaturvedi et al Royal Society Open Biology 2019) we have defined what normal means for this muscle group in fruit flies. We showed that muscles degrade inside flies that have a mutation in the gene sply which causes similar muscle diseases in humans. 
Sply Mutant Fly muscles
We also showed that this method can be used on mosquitoes and honeybees with some tweaking. With a microscope we discovered microbes. With these unprecedented observations came novel fundamental questions. Our data raise a few of them.
Honeybee CT scan
Mosquito CT scan

All together, we can now accurately measure the effects of mutations on muscles. Combined with powerful computing and automation, the detection of muscle defects can be scaled up. Possible treatments for muscle diseases can be tested on fruit flies much more quickly, accurately and cheaply. We hope that with help from this method, scientists world over will be able to more accurately understand how our muscles work, treat muscle diseases and explore the fascinating realm of insect flight.