[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  

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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!