[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!

[Week 5: Mar. 3-7] Still learning, but Starting to get into the Swing of Things

Hey guys, how've you been? 

Remember how last time I mentioned that nematodes could have their eggs frozen? This week I was able to thaw some strains. One of them was NP1579. They keep frozen strains in the freezer at -80 ˚C ( -112 ˚F), so cold that it can start to feel like a burning. 

I've thawed other strains like NP1678 and NP1662, and I'll be using them to run a Western blot to figure out the location of particular proteins. The two strains contain genes (for the proteins) F11E6.1 and CPR-6 (C25B8.3) which're homologues to Glucosylceramidase and CATHEPSIN B in humans, respectively. Those're two proteins that are thought to be involved in mucolipidosis type IV (MLIV), but we don't know very much about them. 

I'm still learning about the process and I've been doing plenty of reading. 

Wishing you guys the best!

[Week 4: Feb. 24-28] Nematodes

I've mentioned C. elegans several times, so I haven't talked about them in detail yet. 

Here's a picture I took with my phone through the microscope

So, what's a C. elegans? Here we go: 

Caenorhabditis elegans is a type of nematode that's about 1 mm long as an adult. It's a free-living (not parasitic)  hemaphroditic roundworm that's normally found in soil where it eats microorganisms such as some bacteria. 

Why do people use them in labs?  

• Similarity to other organisms
• Eukaryotic, useful as a model to see how other multicellular organisms (like humans) work
• Depending on your bioinformatics method of assessment, 60-80% of C. elegans genes are homologues with human genes.
• Simplicity
• Adults have >1000 cells, but their tissues are well defined and have been studied extensively
• They have a relatively small genome and it's been sequenced completely (9.7 * 10^7 base pairs, by comparison, the human genome has 3 * 10^9 base pairs)
• Since there're so small, there's not much anatomic variation between individuals. They're eutleic, so all of them have the same number of cells. Their neurons are also eutelic so scientists can reconstruct their nervous systems and study them.  
• They're transparent so you easily see dyes if you're tracing something and you can see individuals in different stages in life which is really important for identifying L4s. L4 individuals are also called "virgins" and are useful in crosses and making strains of nematodes.

L4s have a clear spot in the middle of their bodies. It looks kind of like an eye here.
This is from a book called The Nematode Caenorhabditis elegans

• Their biology
• You can easily grow them on petri dishes with agar with a bacterial lawn of E. coli. 
• You can freeze their eggs for decades and healthy individuals will hatch so you can store strains for over thirty years 
• They have brief life cycles: 2-3 days/ generation.
• You can study genes using RNAi (RNA Interference) to your advantage. When the worm encounters a double-stranded RNA segment that corresponds to one of its own genes, it treat it like a virus and down-regulates its gene so you can control gene inhibition which can be useful if you're studying the effects of different genes. 

That's all for now.

If you're curious for more feel free to ask. Here're my sources:

• Kaletta, Titus and Hengartner, Michael O. (2006). Finding function in novel targets: C. elegans as a model organism. Nature Reviews Drug Discovery, 5, 387-399
• Waksman Student Scholar. (2000). C. elegans as a Model System. Retrieved from the Waksman Institute of Biology of the University of the State of New Jersey website: http://avery.rutgers.edu/WSSP/StudentScholars/project/introduction/worms.html
• Wood, William B. (Ed). (1988). The Nematode Caenorhabditis elegans. Long Island, NY: Cold Spring Harbor Library Press 

P.S. This wasn't one of my sources, but I stumbled across this website if you're curious to know more. 

• Worm Atlas. (2006). INTRODUCTION TO C. elegans ANATOMY. Retrieved from http://www.wormatlas.org/ver1/handbook/anatomyintro/anatomyintro.htm

[Week 4: Feb. 24-28] Mucolipidosis type IV

Here's some info on mucolipidosis type IV, the disease my lab's resesarching. Feel free to ask questions if anything's unclear.

Introduction
Mucolipidosis type IV is a genetic disease with autosomal recessive inheritance pattern. The disease affects nerves and the eyes. Patients with it frequently present with "delayed development and impaired vision" (U.S. National Library of Medicine®). There are two forms: typical mucolipidosis type IV(the severe form) and atypical mucolipidosis type IV (the less severe form). Around 90% of affected patients have typical mucolipidosis type IV.

Symptoms

• Psychomotor delay (taking longer to develop mental skills and coordination)
•  Problems with controlling muscle movement that can worsen over time
• Impaired vision which can develop into blindness
• Clouding of the cornea, the outermost layer at the front of the eye
• Death of the light-sensitive cells of the retina
• Achlorhydira
• Reduced production of stomach acid
• Results in abnormally high levels of gastrin the blood 
• Doesn't really cause much else in this case
• Iron deficiencies which can result in anemia (not enough red blood cells)

Patients with atypical mucolipidosis type IV have less severe symptoms.

Causes
Mucolipidosis is caused by mutation(s) in the gene Mucolipin 1 (MCOLN1) on chromosome 19. MCOLN1 codes for the protein MCOLN1 which is involved in transporting materials in and out of endosomes and lysosomes. Its regular function and the specifics of how it mutated MCOLN1 leads to developing symptoms of mucolipidosis type IV  is not entirely understood. Individuals with mutated MCOLN1 have abnormally large lysosomes and their neurons die.  

In the lab
The nematode C. elegans has an orthologue to MCOLN1: the gene CUP-5. Nematodes with mutated CUP-5 also present with abnormally large lysosomes. Those with mutated CUP-5 can lay eggs, but their eggs never hatch because the embryos die; however, if they're given MCOLN1 (from humans) then they can survive and become what's called a "rescue."

------------
Sources

1. U.S. National Library of Medicine®. (2013). Mucolipidosis type IV. Retrieved from U.S. National Library of Medicine® Genetics Home Reference website: http://ghr.nlm.nih.gov/condition/mucolipidosis-type-iv
2. U.S. National Library of Medicine®. (2009). MCOLN1. Retrieved from U.S. National Library of Medicine® Genetics Home Reference website: http://ghr.nlm.nih.gov/gene/MCOLN1