Lysosome Storage Diseases
What does that mean? Basically, your cells take up cholesterol from your diet. Similar to how when we eat and our food travels to our stomach, cholesterol from outside of the cell accumulates in the lysosome.
From there, the cholesterol goes to the Endoplasmic Reticulum to be made in to other things or be sent to different organelles. In lysosome storage diseases (LSD), cholesterol cannot get out of the lysosome. There are many different versions of these disorders, resulting in many different rare diseases.
Neimann Pick Type C (NPC)
In the Vaughan lab, where I have been spending my summer, we are looking at a lysosome storage disease called Neimann Pick Type C (NPC for short). NPC affects the perkinje cells in the brain, and causes dramatic brain damage. Damage to the brain causes several other symptoms that are pretty diverse for each patient. NPC affects about 1 in every 150,000 people.
By people I mean children.
Before the age of ten.
They die before they complete adolescence.
A long time ago there was a famous coach at Notre Dame named Ara Parseghian. His children lived in Tucson, AZ, which is where I grew up. They had four children, and three of them were diagnosed with NPC, and they all passed away.
Because there were no treatments available for these children, their mother set up a foundation named after their grandfather to further research on this rare disease. My lab for this summer is part of the small community of researchers trying to figure out what happens in NPC and how to treat it effectively.
NPC1
The first step was to identify the protein coded by the recessive gene in NPC. They found that most of the mutations affected a protein called NPC1.
To explain that picture up there, there are many parts (domains if you are a scientist) to this protein. The first part that matters are the many sequences that pass through membranes. The next part is a dileucine signal, meaning that the protein goes to the lysosome. Add those two things together and you get a lysosome membrane protein. The last part to pay attention to is the sterol sensing domain (SSD).
What do you get?
A protein in lysosomes that binds to sterols, cholesterol in particular.
If this protein is messed up, then moving cholesterol from the lysosome is not going to work very well is it?
What happens to NPC1?
This is the part that our lab is trying to figure out. Because this is such a young field of research, there is not a lot of information on the mechanisms or pathways. There are two different models that are currently being focused on.
Endoplasmic Reticulum Assisted Degradation
The first hypothesis relating to the effect of mutation on NPC1 was presented by Dan Ory in this paper. In this model, the mutations in NPC1 cause the protein to be folded differently. Most of the time, when a protein is not folded like it should be, then it is broken down before it can move on to other parts of the cell. Because most protein folding and modifications happen at the ER, that is where the mutant protein is recognized and set up to be degraded.
So if the protein that normally binds to cholesterol to be transported is degraded, then you don't have anyone to hold on to cholesterol. If cholesterol is not bound to anything, then it gets stuck in the lysosome. BOOM. Lysosome storage disease.
The key to this theory is that when NPC1 binds to cholesterol, the lysosome sends everything to the ER in little packages called vesicles. NPC1 is in those membranes and travels to the ER again that way.
The Vaughan Model
The lab that I am participating in this summer has a different theory as to why cholesterol is not leaving the lysosome. Before I explain, I will give some background.
There are these things called membrane tubules. Think of long highways that stuff can travel on through the cell. It kind of happens like this (but not exactly):
Before I got to ND, the Vaughan Lab had noticed that these little structures come off of the lysosome. Interestingly, these membrane tubules were not present in mutant cells. Their hypothesis was that cholesterol traveled through the cell via these membrane tubules, and NPC disease happened because the membrane tubules were not present in the mutant cells.
But wait? What does that have to do with NPC1?
That takes another protein.
STARD9
In an attempt to understand the mechanism better, the Vaughan lab looked for other proteins involved in making membrane tubules. What they found was a new motor protein called STARD9. Remember the video above? (if you didn't watch it, this is your chance) STARD9 would be the guy moving everything along essentially.
In healthy cells, they could see that NPC1 and STARD9 were in the same place.
This did not happen in the cells that represent NPC.
Many people say that this happens because of the degradation model that I described above. However, in our microscopy experiments, the Vaughan Lab could see the mutant protein in lysosomes. If they made it to another organelle, then it did not degrade.
Where I Come In
Now if you have been reading this blog post, you probably are wondering what this has to do with me. If you look at my tweets and my instagram posts, you would not get all of that theoretical stuff. I am pretty sure NPC is rarely mentioned...
These are the things that happen in the Vaughan lab...
My job is to help identify lysosomes and then see if I can find the mutant NPC1 protein inside the lysosomes. The parts I need are: glowing proteins, a Live Cell Imaging microscope, and good cells.
There is another protein that are in lysosomes called LAMP1 (lysosome associated membrane protein 1). Everyone uses it as a "tag" for lysosomes. When I look at cells expressing the genes for this protein along with a florescent tag, I can see it glowing that color under the microscope.
Our lab has a version that glows green. That's fine and all, but all of our mutant proteins glow green. If we wanted to say that our mutants were in the same spot as LAMP1, then we would need it to glow another color.
I am making a red version. Well...cherry.
This involves me cutting up a bunch of DNA and then running it down the gel to get the sequences I want so I can put it together in a new way. The sequences I want have a specific size, and if I cut it properly, then I can see it really well.
If you want to know...it did not work.
Where Oh Where is NPC1?
If you follow me on Twitter or Instagram, this part of the project is probably what you have seen the most. As I said earlier, if you have a tag on a protein, you can see it glowing under the microscope. So I put some of the DNA in my COS7 Cells, wait 48 hours, and then I use our fancy microscope to look for cells with glowing proteins.
I named the microscope Hal. This is Hal and I. We're really tight if you didn't know.
Now there are a few challenges to getting a good picture. When you put these weird plasmids in the cells, they freak out and crank out these proteins all the time, which makes them pretty sick. Also, if you do not properly take care of your cells, they are going to die. I had that experience happen twice. One time it was so bad that this happened...
Well actually I was just an idiot who couldn't keep her cells alive.
Why Does This Matter?
Obviously my contribution to the lab as an undergrad is minimal. However, by using images taken from the microscope, we can do some computations to determine if the mutated NPC1 protein is in the lysosome with LAMP1.
If the numbers are good, then it would be another piece of evidence supporting Dr. Vaughan's model.
And if we have a better understanding of what happens to the mutated protein, then we can study why the membrane tubules are no longer present in the mutant. This can help us understand how cholesterol normally travels through the cell. By finding locations and mechanisms, better treatments for NPC disease (and other related diseases) can be developed.
What Are You Up To Now?
No. That is NOT safe lab practice.
Ok. Lab dance parties are pretty fun, but that is not exactly what is going on (at least not when anyone is watching). Basically I have failed a million and two times, but I am working on improving. I also am learning a lot about what everyone else is doing and how everyone else's experiments work. The Vaughan lab is full of cool people.
Lab Buddy: Read "Other Undergrad who sits in the same back corner room."
Of course that does not necessarily mean that I have learned any extra social skills by being in a basement lab all summer. I am pretty sure I have started to become more of an awkward scientist....
But really, I am thoroughly enjoying my time in the lab. My cells are currently very healthy, and I am getting closer to making my own "glowing protein." Hopefully I will have some valuable results by my REU symposium...
So there. Now you know what I am doing.
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