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  • Alex Cloherty

Use it up, wear it out, make it do, or do without

Updated: Aug 24, 2020

Autophagy is my favourite cellular pathway. I'm a bit of a hippie at heart, so I try to live by a phrase that my Mum often used as I was growing up: "Use it up, wear it out, make it do, or do without". It makes me happy to know that my cells are doing the same.

As I've written about before, autophagy is the recycling pathway of the cell. And, it is always in motion. Autophagy starts with the formation of a fatty envelope that can enclose not only worn out proteins from your own cell, but also dangerous microbial invaders that have entered the cell and need to be dealt with. These fatty envelopes are called autophagosomes, and they don't stop at enclosing the worn out cellular proteins or microbial invaders. After enveloping their cargo, autophagosomes acidify, which basically digests everything inside, like a stomach digesting food. In line with this stomach simile, autophagy doesn't only happen in response to detection of worn out proteins or microbial invaders, but also in response to starvation. Think of people in wartime. At first, people will only eat foods that are 'normal' in their culture - say, potatoes, onions, broccoli. But, if they are truly starving, people will turn to foods that are culturally abnormal. For instance, there are stories of the Dutch eating tulip bulbs out of desperation during the "Hunger Winter" of 1944-1945. If your cells are starving, they do the same. They will make extra autophagosomes in which to digest any non-essential materials that aren't normally eaten, but can possibly serve as cell-food.

A lot of my research relies on measuring autophagy - I want to see how I can manipulate this pathway to help our human cells mount a better response to infectious diseases. But, measuring autophagy is already in and of itself a difficult process. That is because autophagy is just that: a process. As with all parts of life, autophagy ebbs and flows naturally; there is an intrinsic flux at play. Your cells are constantly manufacturing new proteins, fats, and nucleic acids to maintain themselves, and those same biomolecules are constantly being broken down. It is beautifully transient.

Because the very proteins that carry out autophagy - by helping to form autophagosomes, or targeting specific cargo to those autophagosomes to ensure that the right stuff is digested - are also broken down in the process, we have to ask the question: what exactly should we measure here? The higher levels of these autophagy-related proteins at the beginning of the autophagic cycle? Or the degradation of these proteins at the end of it? And what would that measurement mean? Can we even properly measure those things, since autophagy is going on simultaneously at many different locations within every single cell? And to make matters even more complicated, there can be different proteins involved in carrying out autophagy depending on whether it's a response to invading pathogens, starvation, or other stresses to the cell.

For a long time, the best advice for measuring autophagy has simply been to measure it in several different ways concomitantly. That is the only way that you can be sure you are truly looking at an up- or down-regulation of autophagy, since each of the possible ways to look at this flowing process have downfalls. But recently, scientists have added a new tool to our kit for measuring autophagy - by elegantly using a trick from the adaptive immune system.

For a long time already, scientists have known how to grow antibodies that will stick to a very specific protein, or piece of a protein, that they want to analyze. To protect us against dangerous microbes, antibodies have to be very sticky, and very particular about what they stick to. The structure of an antibody is basically two grabby hands on a stick, and their job as a population of antibodies in your immune system is singular: grab any proteins that do not belong in your body. When antibodies successfully grab on to microbes, there are a few possible options - none of which is usually good for the microbe. If many antibodies grab on to the same virus or bacterium, it might be sort of swarmed out of commission. Imagine if you had a gazillion tiny lego pieces stuck to every inch of your body. You'd be in no condition to be very productive. I imagine that that's basically what a bacterium might feel like if it were entirely coated with antibodies. Alternatively, if only a few antibodies grab onto that single microbe, they can still incapacitate it, for example by acting as 'eat me' signs (I like to imagine this Alice In Wonderland-style) to immune cells that will then gobble up the 'eat me'-tagged microbe.

In the lab, we can use the very specific stickiness of antibodies to particular protein topologies to create highly precise probes for proteins of interest. Oftentimes, these probes will consist of an antibody raised to stick only to a specific part of the study protein, conjugated to a fluorescent protein, or fluorophore. We can then incubate this antibody-fluorophore conjugate with whatever biological sample we are interested in, wash the sample, and then voila! Wherever the sample fluoresces, the antibody, and therefore our protein of interest, lies. This even lets us measure changes in the amount of protein present. For instance, say you want to know if there is more of the protein you're studying present after a drug treatment. You can just measure how much fluorescence there is in your sample with the drug, versus without the drug.

Scientists from the University of Ottawa and Universite Laval, in Canada, recently used this trick to develop a new method for measuring autophagy. Because autophagy is such a central process for the cell, it is carefully and tightly regulated to maintain a balance between the building up, and breaking down of cell contents. The regulation of autophagy more or less depends on key autophagy regulating proteins interacting to switch each other on or off, depending on how fast or how slow autophagy needs to be flowing.

Although the topological changes to a protein are pretty subtle when it is turned off, versus turned on, antibodies are so remarkably precise that this subtle change can mean the difference between an antibody sticking, or not sticking to a protein. And this remarkable precision is exactly what those scientists in Canada used to develop a new antibody probe for measuring autophagy. They were able to create an antibody that only sticks to the 'on' version of a key protein regulator of autophagy. It is conjugated to a fluorophore, meaning that when autophagy is turned up, for example by starving cells, you can see higher levels of fluorescence in samples treated with their antibody. When the cells are fed and autophagy is turned down, you see less fluorescence. You can see their beautiful pictures of this here.

Harnessing the power of antibodies in this manner is actually pretty commonplace in biology labs, but I especially love when it is used to study processes involved in the immune system. I still find it wonderful that we can use the power of the immune system, to study just that: the immune system. Don't you agree?

Until next time,

~ Alex

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