Presentation Outline

Hey guys, just an update:

I've finished up my data collection, and I'm finishing my lab report.

As I've been finalizing my presentation, I've been finding that I can rephrase things more concisely, and as a better way to give context to my presentation.

SUMMARY:
I've been working as a volunteer research assistant in the lab of Professor Johnny Fares of the Department of Molecular & Cellular Biology at the University of Arizona. The research team aims to study lysosomal biogenesis and what can go wrong during the process. Improper lysosomal biogenesis can give rise to lysosomal storage diseases such as Mucolipidosis Type IV (MLIV). MLIV affects about 1 in 40,000 people and is caused by mutations in the gene MCOLN1 that codes for the protein TRPML-1. Patients experience neuronal death, deterioration of the retina, and insufficient production of stomach acid. There's no way to study the disease in humans, but it can be modeled in the nematode C. elegans which has the protein CUP-5 (coded for by the gene cup-5) that functions identically to TRPML-1.

In addition to CUP-5, MRP-4  (a transporter protein) is also involved in endocytosis. When mutations only in cup-5 are present, the nematodes present with abnormally large and leaky lysosomes die. Previous research shows that when CUP-5 and MRP-4 are both absent, the nematodes present with normal-sized lysosomes and live. We don't know if the double mutants's lysosomes are normal sized if because they're functioning properly or because lysosomes are leaking more and returning to their normal size. 

I helped conduct biochemical analysis for the localization of CPR-6, a protease that should only be found in lysosomes. I sampled from C. elegans eggs' lysosomes and cytoplasm with membrane fractionation. I helped detect and quantify the amount of CPR-6 in our samples by running Western immunoblots and analyzing the film in the software ImageJ. 

I'll talk more about the results when I present on Thursday, May 8.
 

Hope to see you then!

[Week 10: Apr. 7-11]

The final stretch! Now that things are winding down, I've started to transition into finalizing my lab report. It's harder than I expected. I'm glad I took notes throughout my time at the lab. I learned a lot. Bio in action is pretty different from the biology we're used in seeing in class. 

I won't be able to make any sweeping, ground-breaking conclusions from my results since I only managed to have enough time to do one trial out of several, since we had to wait until we had enough worms, and I've had trouble coordinating other strains; however, I'll be sure to report my findings nonetheless. 

I'd like to once again thank Professor Fares for allowing me to work in his lab, and I'd like to thank Dr. Nadja Anderson for helping get in contact with him.

It's been great!

[Week 9: Mar. 31- Apr. 4]

Hey guys, this week I'm prepping for another Western.

Interestingly, we found some worms with purple coelomocytes. They showed up in the strain when we bought them. Most of them are sterile, but some of 'em can lay viable offspring.

Photo mine

Photo mine

I guess it's not that clear in the pictures, oh well.

This week was quiet for me. I've mainly been keeping up on reading and prepping for my final project and presentation and checking how our nematodes have been doing. Journal articles get pretty challenging. They hit the ground running with their findings. It's good practice to familiarize myself with them. 

I'm aiming to have an outline by the end of this week. I hope everyone's been doing well! 

[Week 8: Mar. 24-28]

This week's been fairly relaxed. I've been doing more reading on some of the papers on MLIV authored by lab members, and I've been working on prepping for another Western. We can't say too much from the results of our previous immunoblot since we don't have anything to compare it to--that's coming up.

We did a Western for LMP-1 which is homologous to Lysosome-Associated Membrane Protein 1 (LAMP1) in humans so we can use our results as a control. LAMP1 is a glycoprotein that helps maintain lysosomal structure and is involved in lysosomal biogenesis. 

[Week 7: Mar. 17-21] Pre-Western Gel Electrophoresis

Hey guys, I hope everyone's been having a nice day. I thought that picture would be nice after that last post which was mostly text. 

Here's what it looks like right before you finish running a gel when you're doing a Western. I took this last Monday. The black lines are how we marked the bottoms of the wells to make it easier to pipet in our samples without poking through the wells. You can see in bands in fifth lane; that's the molecular weight ladder that helps us find the size of our protein of interest later on. There's a total of four clips; we run two gels at a time; the second one's on the other side.

Feel free to ask if you have questions, comments, or if anything's unclear. 

That's all for now! 

[Week 7: Mar. 17-21] Western Blots

Proteins, proteins everywhere!

I've been learning how to do Western blots, also called protein immunoblots, a two-day process use to detect the presence of a protein in a sample. 

From the previous post, we have the samples listed in decreasing density: EGG, PELLET, and SUPE for each strain, so let's start off on what to do next: Western blotting.

Western Blotting 

We used Novex® Tris-Glycerine Express Protein Gel Electrophoresis Kits! The gels themselves are sticky, as I learned after getting them all over my gloves.

1. The first step is gel electrophoresis so we can separate the proteins by size with an electric current and have all the protein of interest in one place so it's easier to detect. We have pre-made gels so we don't have to pour our own. Don't forget to pipet a ladder into a well on each gel! 
2.  Then the proteins are transferred onto nitrocellulose, a kind of blotting paper that proteins stick to so your proteins will keep their pattern from gel eletrophoresis.
3. Afterwards the nitrocellulose is incubated in a buffer with genetic proteins (we used some from milk) so the antibodies won't get stuck all over the nitrocellulose that's sticky to proteins and mess with detecting our protein of interest. 
4. Next, we add our primary antibody. Remember how our strains of interest were created to have GFP or mCherry in their genome? The strains produce the protein of interest and the GFP or mCherry, linked together by several amino acids. The primary antibodies here are anti-GFP and anti-mCherry so they can tag the GFP or mCherry attached to the protein of interest. You might be wondering why the antibodies aren't anti-[protein of interest]. My understanding that it's easier and cheaper to make anti-GFP or anti-mCherry rather than to make an antibody with an antigen-binding site specific that has to be customized to the antigen associated with the protein of interest. Using the primary antibody lets us tag where our protein is.
5. After incubating for usually overnight, we then wash off our primary antibody and add our secondary antibody which is anti-primary antibody. The second antibody has horseradish peroxidase of another kind of protein or dye that we can use to see our sample later.
   
   You may be thinking: why two rounds of antibodies?
             Primary Antibody: Tags where our protein is (cannot be seen).
             Secondary Antibody: Helps us see where our protein is, how large it is compared                                                 to other proteins, etc..
   
6. Afterwards, the nitrocellulose blotting paper the protein are on can be visualized with a special method such as autoradiography and the intensities of the protein bands can be quantified. 

That's been my week, along with prepping for some other things, so I've been pretty busy. Hope you guys are doing well!

Reference:

• Davidson College, Department of Biology. (2001). Western Blot Procedure. Retrieved from Davidson College, Department of Biology website: http://www.bio.davidson.edu/genomics/method/Westernblot.html

[Week 6: Mar. 10-14] Membrane Fractionation

Hello everyone! I've been able to focus my project more and here's an update. 

Mucolipidosis type IV (MLIV) is caused by mutations in the gene MCOLN1, coding for the protein MCOLN1, also known as TRPML1. MLIV is a lysosomal storage disease, meaning that patients with MLIV have improperly functioning lysosomes which leads to neuron death and deterioration of the eye. In this case, the lysosomes have trouble breaking down whatever's inside them because of defective lysosome biogenesis which is how cells get ready to break down materials ((endocytosis→) early endosome → late endosome → lysosome). Not much is known about the disease so we're hoping to find out more. 

We can use C. elegans to study MLIV since the nematode has the orthologous gene CUP-5 which when mutated results in a phenotype with defective lysosome biogenesis, like in MLIV. In worms, mutated CUP-5 results in death of their embryos since their intestinal cells die and they starve. If worms with mutated CUP-5 also have the gene MRP-4 knocked out, then their embryos survive (called "rescues"). The defective lysosomes in both organisms are abnormally large and ineffective at breaking down their contents. 

Right now I'm working with two strains of worms:

• NP1678: unc-119(ed3); mrp-4(cd8); KxEx148(F11E6.1a::mCherry; pRF4(Rol-6D); pw1s50[lmp-1::GFP, unc-119(+)] 
• NP1662: mrp-4(cd8); cup-5(zu223) unc-36(e251); KxEx148(F11E6.1a::mCherry; pRF4(Rol-6D); pw1s50[lmp-1::GFP, unc-119(+)]

That's a lot of notation that I'll summarize:

• unc mutations change how worms move so when put in a plasmid with the genes of interest, we can see which worms are expressing those genes
• pRF4(Rol-6D) also changes how worms move. Worms with that mutation are "rollers" that travel in a circle rather than slither across a petri dish 
• mCherry and GFP (green fluorescent protein) will be used to visualize where the genes of interest end up
• lmp-1::GFP is a lysosomal membrane protein that's been tagged with GFP and that'll be used to visualize membranes later as part of the Western blot which is a way to detect if a protein's there or not. 

What we're focussing on is that the worms have F11E6.1 (GBA-3) and CPR-6 (C25B8.3) which are homologous to Glucosylceramidase (a glycoside hydrolase which is involved in breaking down carbohydrates) and Cathepsin B (a protease), respectively, which are in lysosomes. Mechanisms of cell death in MLIV and the nematode CUP-5 mutant phenotype are not understood, and we think that the presence of lysosomal enzymes outside lysosomes may be responsible.  

Recalling that mutations in cup-5 result in defective lysosomal processes in nematodes, the mutation cup-5(zu223) results in phenotypes where nematode embryo's intestinal cells die. The mutation mrp-4(cd8) knocks out MRP-4 so it isn't expressed; when MRP-4 is expressed it transports lipophilic molecules which build up in lysosomes and exacerbate lysosomal problems in worms with no CUP-5 ;mrp-4(cd8) by itself isn't known to result in any adverse phenotypes but normally. Remember that mutated CUP-5 in the presence of knocked out MRP-4 results in rescues (viable embryos that don't starve to death during development). 

TL;DR so far: 

• I'm interested in seeing where F11E6.1 ends up depending on whether CUP-5's present or absent. We have to knock out MRP-4 in NP1662 otherwise the embryos will die and won't be useful to the experience and we have to knock out MRP-4 in NP1678 to keep things consistent. 
• CONTROL: NP1678 has no MRP-4, but has F11E6.1 
• EXPERIMENTAL: NP1662 has no MRP-4 and no CUP-5, but has F1E6.1  

________________________________________________

Membrane Fractionation: The Plan

1. The worms we're interested in are all "rollers." Identify NP1678 and NP1662 rollers, keeping them separate. We can identify them by how they move and we can pass them onto multiple petri dishes with food and nutrients. Then we wait for them the lay eggs, since we're interested in where the proteins localize in the embryos. 
2. We collect NP1678 and NP1662 individuals (worms and eggs) and  bleach them, so only the eggs are left. 
3. Wash with appropriate buffers to remove bleach, break apart outer membranes, prevent sample degridation, etc.
4. Centrifuge nematode samples to get: 
    -Supernatant 1 (cytosol)
   -Pellet 1 (membranes and membrane-bound organelles) 
5. Centrifuge supernatant 1 from both strains to get:-Supernatant 2 (materials inside membrane-bound organelles, such as lysosomal enzymes)
   -Pellet 2 (membranes such that those that make out the outer parts of some organelles)
6. Treat samples with appropriate buffers and store at -80 °C until Western where we'll compare ratios of the amounts of F11E6.1 in pellets and supernatants. 

I'm off the bleach worms right now. See you soon!