Pieces from my Poster

Abstract: 

As the target of many mood effecting drugs such as antidepressants, cocaine, and 3,4 methylenedioxymethamphetamine (MDMA, ‘ecstasy’), the human serotonin transporter (hSERT) is of significant clinical importance and the subject of many pharmacological studies. Current understanding of how antagonists bind to hSERT is advancing rapidly due to recent crystal structures but remains unclear. Data suggest binding of antagonists occur at a binding site located at the center of the transporter. In fact, our lab as well as other labs have elucidated several residues at this site that are critical for high-affinity citalopram (CIT) binding1,2. However, there are studies suggesting the binding site is located more cytosolic and positioned above the outer gate3. Recently, to further characterize CIT binding, we performed quantitative structure activity relationship studies (QSAR) using CIT analogs and the hSERT mutants Y95F, I172M, S438T. Results from this study suggest that the I172M and S438T mutations may result in complete loss of CIT binding at central high-affinity binding site (HABS) causing CIT to bind at a secondary site. In contrast, CIT binding appears to be maintained at the HABS in the Y95F mutant. In order to test the presence of a secondary binding site, we generated cytosolically-located mutations in wild type, Y95F and I172M hSERT backgrounds and evaluated their impact on CIT binding using [3H]5-HT competition uptake assays. 

Introduction:

Previous research on hSERT has identified Y95 on transmembrane 1a (TM1a) and I172 on TM 3, as critical for high-affinity binding of many antidepressants. It was found that an amino acid mutation of Tyr to Phe  at residue 95, or Ile to Met at residue 172, resulted in a significant reduction of antidepressant potency1. Our studies on a library of CIT analogs revealed the compounds interacted with the I172M and S438T mutants in a manner distinct from WT and Y95F where many CIT analogs exhibited lower inhibitory potency (IC50) in WT and Y95F backgrounds. In contrast, the IC50 of compounds in the I172M/S438T backgrounds was largely unaffected and in some cases displayed increased potency. This led to the hypothesis that mutation at either I172M or S438T eliminate the ability of CIT to bind at the HABS and divert CIT binding to a secondary binding site elsewhere in hSERT. CIT in the WT or Y95F mutant is thought to bind in the HABS. This hypothesis explains the QSAR analysis that suggest alteration of pharmacophores on CIT differentially impact WT/Y95F versus I172M/S438T mutants.

hSERT is in the same transporter family as the bacterial Leucine transporter (LeuT) from Aquifex aeolicus. LeuT protein was stable enough to form crystals and obtain high resolution x-ray diffraction structure and some subsequent co-crystal structures revealed determined that tricyclic antidepressant inhibitors bind in an upper region of the transporter on the extracellular face of the transporter3. However, the majority of biochemical data suggests that SERT antagonists bind at a more central binding site. The possibility that two sites exist suggests that our hypothesis of a secondary binding site existing in the upper region of hSERT is reasonable.

Methods:

Side view of transporter

Selection of residues for mutation to identify LABS: An hSERT comparative model (LeuT) was visualized in MacPyMOL and candidate residues (magenta side chains) were chosen based on their proximity to the cavity above the outer gate residues R104 and E493 (orange and blue side chains). Amino acid substitutions were introduced to alter residue hydrophobicity, size, and charge. Mutagenic forward and reverse primers were designed for each substitution. Silent restriction sites were also built into the primers to identify whether or not the desired mutation was generated. 

Top view of transporter

Site-directed mutagenesis and construction of mutant plasmids:  Mutants were created using PCR mediated site-directed mutagenesis QuikChange II (Stratagene).  DNA purification and analysis:   Plasmids were amplified in bacteria and isolated using the Wizard+ SV Miniprep kit (Promega). DNA concentration and purity were determined spectrophotometrically. DNA was digested with the appropriate silent site enzymes and separated on 0.75% agarose gel. Bands were visualized under UV. Positive clones were verified by sequencing at NorthWoods DNA Inc.

Expression of DNA in HeLa cells: Mutant SERTs were transiently expressed in HeLa cells and analyzed for uptake of radiolabeled  serotonin. Paroxetine and non-transfected cells were used to determine specific uptake. Functional mutants were tested in a [3H]5-HT competition uptake assay to determine changes in CIT potency.

Results: All of my results and discussion is hard to explain without the graphs and poster, so you will just have to talk to me about it in person if you are interested :)

Catch up of July

I can't believe I am already on week 9. This blog will be extra long, because I have to fill you in on July 12-August 3.
Outside of the lab I have:

• Traveled to Fargo
• Ridden a ferris wheel inside the original Sheels sporting goods store.
• Been kidnapped, blind folded, and taken to the school planetarium to watch HP 7.1 on the big screen
• Watched HP 7.2
• Eaten at the locally famous "Red Pepper", Blue Moose, Little Bangkok, Rhombus Guys. As well as Happy Joe's and Applebees.
• Had a hall dance party with glow sticks, strobe light, and black light
• Built a fort
• Watched the sun rise from the top of the parking garage
• Played kickball
• Went swimming at a hotel pool
• Went star gazing
• Walked across the Mississippi river headwaters
• Bowling
• Sardines in Walmart
• Slack line
• 9 mile tandem bike ride at 11pm
• Baked a cake
• Cooked pancakes and bacon on my griddle

In lab:

• PCR: Creating the mutation on a template DNA plasmid
• Transformations: inserting the DNA into E. coli
• Culturing E. coli: Growing cells on petri dishes, picking colonies, and growing those colonies in flasks
• Minipreps out the wazoo: purifying DNA from E coli
• Restriction digests: cutting the DNA with special enzymes that recognize specific base pair sequences
• Gel electrophoresis: pushing the digested DNA through agarose to separate cut DNA pieces of different lengths (This is a check to make sure we have the right DNA) We can view the bands of DNA under a UV light.

• Sequencing the DNA: a long complicated processes that I don't really understand, but it gives me the base pair sequence of the DNA to make sure the correct mutation was created and no additional ones were made. Each wave in the chromatogram pictured below represents a different base pair.

• Culturing HeLa cells: Taking care of the immortal cell line of HeLa cells. Passing them into new flasks every 3 days to keep them from getting over crowded, and giving them new food to eat. 

• Plating HeLa cells: Counting my cells and putting 50,000 into each well of a 24 well plate.
• Transfecting my mutated DNA into my HeLa cells: Inserting the mutated DNA into the HeLa cells on the plates.
• Uptake of radioactive serotonin with a serial dilution of inhibitor: The DNA that was inserted into the HeLa cells sits over night and the cells will read the DNA and start creating serotonin transporters in their cell membrane. I then add inhibitor (citalopram-an antidepressant drug generically referred to as Lexapro) at various concentrations. Then I add radio active serotonin, which should be transported into the cell through the newly created transporters. 
• Counting radioactivity inside the cells: The 24 well plates are then put in a machine called a Top Counter, which counts the amounts of radioactivity inside the cell, which tells us how well the transporter worked or didn't work. 
• Creating a competition curve from the Top Counter data: A graph of the results will show a curved line on the axies of inhibitor concentration and radioactive serotonin uptake. The curve will shift left or right depending of if the mutation in the transporter is helping or hindering the uptake. If it is being hindered, then we can assume that the place where the mutation was created is most likely a binding site of the inhibitor.

It has all been really exciting to watch my project get to the results stage. I won't have time to run the experiment on all of my created mutations, but someone else in the lab will continue my project after I leave.

I am to the point where I feel accomplished, and I can leave feeling successful. I have learned sooo many lab techniques and computer programs. I have gotten to know my lab mates, and fellow REU students really well, and I am going to miss all of them.

I dream of pipetting

If you read through the protocol for my mutagenesis, you would notice a lot of "add __ microliters of ___ to ___." We work in microliters (uL) in our lab. 1 US teaspoon = 4,928.92159 microliters. Many of the volumes that I work with are any where from 0.5 uL to 1,000 uL. For those volumes we use special micropipettes (Pictured) 
1 uL would only fill the very tipy tip of this pipette. It is so small, sometimes it's hard to see if you even sucked any liquid up. It's crazy to think that one speck of liquid can change my ENTIRE results. It's scary to know that if I forget one pipetting step, or add too much or too little, then my product will most likely be ruined. Most of the liquid I use are completely clear, so for all I know, I could be pipetting water all day long. Everytime I add a new volume to something, I have to use a new plastic tip on the micropipette. After a rough calculation, I figure I have used over 1,000 disposable tips for the 25 mutations I have created so far. I have 65 more mutations to make. Needless to say, a large amount of my time in lab is spent pipetting, and I have recently started to dream about pipetting.  


I told you I would give an update about the mutagenesis once I was further along. Well, as I said in my earlier post, I chose 15 residues to mutate on the protein that makes up the transporter. Each of those residues gets 2 different mutations. One mutation will change the space it takes up, and the other mutation will change the charge that it has. Each of these mutations needs to be inserted into 3 different DNA templates, for comparison purposes. That means I have 90 mutation. So far I have created primers for all of them, but I have only started the mutagenesis on 25. 


The DNA template for the serotonin transporter is somewhere around 4,000 base pairs. I only need to change 2 or 3 base pairs to make my chosen mutations, so we ordered just the tiny segment of the DNA with that mutation in it, called a primer. In order to get the mutation into the template, I ran a PCR (polymerase chain reaction), which is used to amplify a small amount of DNA into a large amount of DNA. Inside a reaction tube I added the un-mutated template, the forward primer, and the reverse primer. During the cycle in the PCR, the double stranded DNA template is pulled apart, and the primers will stick to the template in the place where we want the mutation. The cycle will continue and replicate the DNA with this primer attached, creating a mutation. Once the cycle is over we are left with 20% un-mutated template and 80% mutated template. I then digested the product in an enzyme that cuts up the un-mutated DNA. After that, I transform the DNA into E. coli cells. 

E. Coli is a bacteria that loves to pick DNA up from the environment and start replicating it with it's own DNA. By using bacteria, we can make LOTS of DNA because the cells replicate really fast. I grew the cells up on plates of agar. Once colonies started growing, I moved the colony into a broth for it to grow even more. Once I grew enough E. Coli, I was able to do a miniprep. A miniprep is a procedure used to purify the DNA out of the cells. Basically, we burst the cells, suspend the mutated DNA that we wanted, and purify it out from the chromosomal DNA of the bacteria. At this point, we have to check and make sure we created the correct mutation, and didn't make any mistakes along the way. We use  gel electrophoresis to check, and once we think we have the correct mutation, we send it away for sequencing just to double check. It's a really long process that I didn't think I was going to have to go through. I wish we could just order the DNA that we want and be done with it.....haha.


Hopefully the mutagenesis won't take too much longer....I really want to get started on the actual experiment. 

Long Holiday Weekend

My mutation primers that we ordered last Monday came in on Wednesday, and I started the mutagenesis process. Once I complete the whole mutagenesis, I will blog about it all at once. It will keep me busy for awhile. After completing week 4, I was super excited to celebrate the 4th of July weekend! My lab-mate, Pat, kindly invited our lab out to his family lake house. The lake house was located in Detroit Lakes, Minnesota. We took a half day on Friday, and headed out to the lake. Pretty much as soon as we got there, we put our bathing suits on.  I got to operate a jet ski for the first time in my life. That was really fun. I even drove the stand up jet ski after I got the hang of the normal one.

The next day I got to SLEEP IN :) Ate some delicious pancakes, and headed out on the speed boat for some water sports. I gave water skiing a shot. I was really scared, because I attempted water skiing years ago, and hurt my leg. This time I got right up. And then proceeded to fall. The second time, I was going for awhile, then as I tried to go outside the wake, I wiped out. That was enough for that day. When we weren't out on the boat, we were playing board games, watching movies, enjoying the hot tub, slack lining, or just laying in the sun. After dinner, we went out on the lake again to play jet ski frisbee. Some one a jet ski zooms by the boat, while someone on the boat sends a frisbee off into the lake as far as he can. Then the jet ski rushes to catch the frisbee. I only caught 2 frisbees out of maybe 20...haha but it was fun either way.

Sunday morning I slept in again. When I got up, we went out on the boat for more water sports. I watched some people wake board, which looks really hard. Then I went tubing. Then I learned a new sport called wake surfing. I had never heard of it before, but it was awesome. You start out behind the boat hanging onto a short rope. You put your feet up on the mini surf board, and then stand up as the boat speeds up. Once you get to the outside of the wake, you pull yourself in towards the boat, until you are riding the wake directly behind the boat. Once you are stable, you throw the rope back to the boat, and just surf along until you fall :) Wake surfing shown in picture.


The forth of July monday wasn't all that spectacular. I practiced my skiing, surfing, and slack lining. Watched Tangled. Packed up, and headed back to Grand Forks. The plan was to be back in time for the fireworks show, but a severe thunderstorm trapped us in Fargo for about an hour. The tornado sirens were going off, and we were instructed to abandon vehicles and find shelter. Once the stormed passed, we continued on our way to Grand Forks. We may have missed the fireworks, but watching the massive lightning storm over the prairie was equally cool. Back to reality in the lab. 

Mutagenesis

Today will be an educational blog post. This past week I started the beginning of my own experiment. Now, the big picture of the experiment is to learn more about the binding site of anti-depressant drugs. Not a lot is known about HOW these drugs work on a molecular level. We know they bind to the transporter in the cell membrane (see earlier post), and plug the gate, so that neurotransmitters cannot get back into the cell. We are not quite sure where the drug actually binds though. These experiments will test different areas on the transporter protein to see if they are involved in the binding. Before we go any further, let's work our way down to the molecular level.

• The neuron cell is enclosed by a cell wall
• The cell wall has transporters embedded in it. (rainbow colored thing in picture)
• The transporter is a large coiled protein.
• Protein is made of a long string of amino acids. (The numbers in the darker image are used to identify specific amino acids. So the 320th amino acid on the protein chain is F for phenylalanine. There are 20 different amino acids)
• Amino acids are coded from the DNA sequence.

Now let's work back up:

• If we change letters in the DNA, a different amino acid will be coded.
• If we have a different amino acid, the shape, and or chemical properties will change in that section of the protein.
• That change in shape and chemical properties may or may not effect the ability for the transporter to do it's job.
• If the mutation causes more neurotransmitters to get through the transporter, than we know that the mutation effected the binding area of the inhibitory drug, and is there for an important section of the protein. 

By making many different mutations, we can begin to see what area the drug is actually binding to, and what areas it could be forced to bind to. Mutagenesis basically means that a mutation is created at a defined site in the DNA molecule. I have found 15 locations on the transporter that I would like mutate. I figured out which amino acids are in those locations. I decided what amino acid I wanted to change it to. I then found the triplet on the DNA that codes for that specific amino acid. For example, on the DNA strand TGT would code for a cystine amino acid. If I want to mutate that into tyrosine, I would change it to TAT. 

It's as simple as that. After hours of preparation and research to figure out what to mutate, where to mutate, and what to mutate it to, we just delete a triplet and add a new one. Bam. 

Nerd Herd

So my section of the dorm is full of nerds. We are all interns for either neuroscience or chemical engineering. I feel right at home :) I can be myself. I can tell science jokes and everyone understands! I can actually talk to them about the research I am doing in my lab and they actually understand the terms I use. No offense to my family, but it is really hard to explain what I am doing when you aren't fluent in biology talk. 

But other than the fact that they can relate to me, it's just nice to have friends here. I was a little hesitant coming here, because it was like starting freshman year all over again. It was easy to make friends with the Neuroscience interns, because we all lived together, we all eat at the same times, and we have scheduled events that we all attend together for the program. It was also pretty easy to make friends with the chemistry people too, mostly because we live with them, and because we share that nerd bond. (if any of you guys read this, take this post as a compliment). 


So other than hanging out in the dorms together, eating meals, and talking about science, we do other fun stuff together. Last weekend we all went to a movie, and then bowling (but bowling was closing soon, so we left). On Wednesday we all went to this documentary showing about grizzly bears. On Fridays we have what is called "networking" in our schedule, where we all meet and talk about what we are doing in lab. We basically get paid to talk to our friends for an hour and eat food. Last night after dinner someone decided that it would be fun to all play basketball. Now, imagine a group of 8 science nerds who all suck at basketball. It was hilarious. Probably the best game of basketball I have ever played. It ended in blood. After that we had a movie night in the lounge. Someone brought their big screen tv out, and we moved all the couches, and we connected to netflix. It's been fun getting to know these people. We have known each other less than 2 weeks, and we are already acting like we have been friends for life. It should be a fun summer.

What do people DO in North Dakota?

This is a question that I have asked a variety of people. It is also a question that people from back home have asked me. Being surrounded by fields, I would assume people have to get creative. Grand Fork, ND, home to the University of North Dakota is 75 miles north of Fargo, and is located right on the eastern boarder, next to Minnesota. It's a small college town, but it has a lot to offer (more so than Waynesburg that is). Whenever I get a chance to talk to locals, I asked them what they do for fun. Here are some of the responses I have gotten:

• Sleep
• Watch movies
• Catfish noodling
• Drink
• Frisbee golf
• Go to Fargo
• Go to Canada
• Go to Minnesota

Since this list was a little boring, we decided to look into local events. These are the options we were given for this weekend:

• School bus races
• Art festival
• Stargazing
• Movie theater
• Bowling
• Laser tag
• Annual rhubarb festival

Of all these suggestions, we chose to attend the Art Festival. They had hundreds of booths set up with a wide variety of art from local artists. There was ceramics, paintings, photography, basket weaving, wood works, jewelry, clothing, and all sorts of food and even some live music. It was the perfect day for an outdoor festival. After a cold week with the temperatures not reaching above 60, it finally was sunny and 70! I have been freezing all week long, so this was wonderful. It was 35 degrees when I walked to work Wednesday morning! I am so glad I brought two blankets that I didn't think I would need. Let's just say we haven't had to turn on the air conditioning at all yet. I really hope it will not be like this all summer. Boy am I glad that I am here in the summer instead of winter. They said it gets down to negative 40 degrees here!!! I can't even imagine. 

Hopefully we won't run out of things to do here. I am thankful I have a car, and I am able to get off campus whenever I need to. So far I have used it to go to Walmart (for and ICEE), Target, the movie theater, and church. I pretty much walk if I am going some place on campus. The school of medicine, where I work, is about a 15 minute walk from my dorm. I have walked every day because I didn't get my parking pass until the end of the week. I will probably start driving though, so I can sleep in, and so that I don't waste 30 minutes of my lunch break walking to the dinning center and back. 

Stay tuned for more exciting adventures.

First Week Down

9 more to go. This week has been mixed emotions for me. The first day in Grand Forks ND was Sunday. I got in around 4:30, and it was sunny and warm at about 80 degrees. As I drove through the college town, I was just eager to NOT drive any further. I found the parking lot in front of my dorm-to-be, and just sat there for a minute taking it all in. I was praising God that I had gotten here with no car trouble, no traffic jams, and no bad weather. It was seriously a problem free road trip. I walked into my building, with NO clue what I was supposed to do. The door was locked, but I saw a phone number on the door. I called the number, and an RA came and let me in. I signed a couple papers, they gave me my keys, and sent me on my way. They didn't even show me where my room was. I wandered around and eventually found 316B and got into my room. I began to wonder how on earth I was going to move all my stuff from the parking lot to the third floor all by myself (I brought a lot of stuff, and some of the boxes were pretty heavy). Well after my first trip, the RA showed me a cart I could use to roll my stuff in. That was much easier, and after about 4 or 5 trips, all my stuff was in my room. My roommate wasn't getting in until 8 or so, so I had some time to unpack and settle in.  A little while later I met my suite-mates, who share a connected bathroom with us. 


Monday was the first big day. All the research students met in the morning for a meeting. There are 9 of us in the program that I am in (REU neuroscience). There are 3 other research programs running at the same time though, so we all met together. Most of the other programs are for students from the University of North Dakota, but our program is mostly people from out of state. We sat through a welcome meeting, several lectures, a few training seminars, and things of that nature. I had to pass a test about the proper treatment of lab animals. At the end of the day I got to meet my professor, Keith Henry, who I will be working with in the lab. He has his only lab where he studies neuroscience physiology. Working in the lab for him are 2 grad students, 2 medical school students, and 2 lab technicians. I am the only undergrad, so I'm the baby of the lab. The first day I was only there for an hour, but Dr. Henry just lectured me about all the research they are doing. It was a lot to take in, and it felt a little over my head. I have only learned the very basics of the nervous system and the functioning of neurons, so he had to basically start from square one and explain everything before he could get to the point of what their research is actually testing. After a week of asking questions and slowly getting the big picture, I am more confident in what they are actually doing, and I feel like I really understand it! For the sake of this blog though, I think I will try to put it into terms you will all understand. And maybe through some big words in just for fun. 

This is just the most basic diagram I could find to show you the important steps of neural functions that I am specifically researching. So you have neurons (nerve cells) that communicate to each other with chemical signals called neurotransmitters. These neurotransmitters are release out of the cell and into the synaptic gap between two neurons. Some of the neurotransmitters attach to receptors on the next cell, and the signal is passed on to that cell, and so on and so forth. The neurotransmitters that are floating around in that synaptic gap are recycled back into the cell that they came from. In order to get back in the cell, they are taken in by a re-uptake transporter in the plasma membrane. This transporter has an active site that the neurotransmitter will chemically bind to, and when calcium and sodium are also present, the transporter will open to the inside of the cell, placing the neurotransmitter back into the cytoplasm to be reused. Ok, so if the transporter is structurally changed, the neurotransmitter may not fit properly, or may need to bind in a different area. If the DNA that codes for the proteins that make up these transporters is mutated, then the structure will change. We are testing different DNA mutations in the lab to see which ones work best, and which ones don't work at all. 

I'm starting this blog for 7 reasons

1. I have nothing better to do tonight
2. I want Angie to have more than one follower, and I have to have a blog to follow someone I think
3. I am 1500 miles away from my friends and family, so this may help them to stay connected with me
4. Andrew Juntunen deleted his facebook and needs to know what's up in my life
5. I do a lot of cool things
6. Both of my sisters have a blog, so I should too
7. I'm not sure if many people will care enough to read my blog, but I know my grandma will, and that's enough for me

So as some of you may know, today is my 3rd day in North Dakota. I just bought my first car 3 weeks ago, and it is here with me. I packed my car full of all my belongings, because I am going straight to college after the summer here. I drove over 1,500 miles to get here (my first official roadtrip). 


The roadtrip consisted of 3 stops: Pittsburgh (Brandon), Danville IL (BOP), and Rochester MN (Random people that my parents went to college with). The trip was fun. I drove in NY, PA, OH, IN, IL, WI, MN, and ND. I tried to take a picture at every state boarder welcome sign, but I missed PA and MN. The coolest one was Wisconsin. All the rest were boring. Impressions of each state: (And if you haven't noticed already, I love lists)

• Ohio-long and boring
• Indiana-the only cool part was seeing Indianapolis
• Illinois-REAL flat. Ridiculously high gas prices 
• Wisconsin-Too many billboards about cheese, cool rock formations though
• Minnesota- Crossing the Mississippi was the coolest looking place I have every seen, and then after that the state got real boring real fast. I did stop by the Mall of America though. Got lost walking around with out a map. And who knew they would have 10 roller coasters!?
• North Dakota- Flat, pretty sunsets, and A LOT of farms, and this is my home for the next 10 weeks

Okay, now we can get to the point of why I am here in this crazy boring state. I applied for this research internship at the University of North Dakota back in February. There are hundreds of REU (Research Experience for Undergraduates) internships at various colleges across the country that are funded by the National Science Foundation. I was overwhelmed by the choices and had no idea how to narrow down the list of places to apply. Don't ask my why I chose this one, because I don't have an answer. I also applied for one in Boston. I had planned to apply for several others, just to up my chances, but I got lazy and figured I wasn't going to get one either way. Applications were due in March, so I figured I would hear back sometime in April. I didn't hear back from either school by the beginning of May, so I had given up hope. Well 3 days before heading home for the summer I received an acceptance letter from ND. I was in utter shock. Of course I jumped at the opportunity, and here I am. 

I have so much more to say, but I am going to save that for another day. Stay tuned for posts about the internship, the research that I am doing, the things I am learning, and the adventures I am having. Thanks for reading.