MCDB 423 Lecture 32: Lecture 32

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Molecular, Cellular and Developmental Biology
MCDB 423
John Kuwada

Lecture 32 Slide 1 There were 3 main kinds of alternatively spliced mRNAs that were identified 1 form called the Y+Z+ included 2 axons -The Y and Z axons are the inserts Another form had the Y but not Z A third where there was no Y or Z insert He showed that proteins that were made from the Y+Z+ had aggregation activity but the other two didn’t Since this is the case, the Y+Z+ must be specifically made by motor neurons and not muscles -This is exactly what he found Now you want to knockout the Y+Z+ agrin Slide 2 What he did was he specifically formed a mutation that knocked out the Z axon and thus you won’t have the Y+Z+ form anymore -This had an affect on the motor axons as they are distributed in a wider portion but there are no AChR clusters in the muscle If you looked at mice where you knocked out the Y axons, they don’t have any aggregating activity -The NMJ also forms pretzel like shape -But the aggregation of the AChR was not distrupted slide 3 The next question was to find out what is the receptor for agrin? One of the candidates is a protein called MuSK -It’s a protein made by skeletal muscles -If you make a knockout in MuSK, the mice are paralyzed Looking at distribution of receptors in a wild type muscle, they are localized to a thin strip down the middle but in MuSK -/- you don’t see that localization So it’s possible that MuSK might be the receptor for agrin Slide 4 If MuSK is the receptor for agrin, then agrin must affect MuSK somehow Instead of using muscle cell, they used a special skeletal muscle cell line called C2C12 -They are immortalized muscle fiber (not real muscle fiber cells though) You could take C2C12 cells and when they grow initially, their AChR are not aggregated -But if you put the Y+Z+ agrin onto the C2C12 cells, just like normal muscles, the AChR become aggregated (they aggregate all over because the agrin is in the solution; if you just did it at one spot, they would aggregate at that one spot) After you do this aggregation of AChR, you can use antibodies against MuSK to immunoprecipaite MuSK -When you do this, you can take the antibodies that are bound to MuSK -Purify the beads with anti-MuSk -Dissociate the MuSK from the antibody and run the MuSK on a gel When you do this and probe with an antibody against phophotyrosine -The Y+Z+ form or any form that has the Z+ form is phosphorylated -Any form that doesn’t have the Z form isn’t phosphorylated This demonstrates that MuSK is tyrosine phosphorylated by the action of agrin So MuSK is part of the signaling pathway initiated by agrin -It doesn’t prove it is the receptor of agrin If you take muscle fiber from a MuSK -/- mouse and add Y+Z+ agrin, you get no aggregation So phosphorylation of MuSK is required in this pathway But it turns out that MuSK doesn’t bind agrin so it can’t be the receptor for agrin Slide 5 Answers 1: B 2: A slide 6 So what is the agrin receptor? It could be another protein called LRP4 (subject of paper 7) -LRP4 is a member of a large family of membrane bound receptors -It has a LDLa repeat which is a signature for the family of these proteins The first hint that it could be the agrin receptor is that if you mutate the Lrp4, the terminals spread out more than normal and there’s no aggregation of AChR -This is what you would expect from a nerve derived agrin slide 7 Various experiments were done -First were done in vitro -Take a group of mammalian immortalized cells -They were transfected with Lrp4 -If Lrp4 is the receptor, it should bind agrin One assay for this was to take cover slips and coat them with recombinant agrin -Apply the cells that are expresing Lrp4 and see if they’ll bind to the substrate -They do but they don’t bind to a cover slip that’s coated with BSA or laminin -So it’s specific for agrin This is called internal reflectance microscopy -Main point about IRM is that it highlights tightly proximity of a membrane to the cover slip -So the binding is pretty tight If you take agrin cover slips and apply Lrp4 transfected cells, they’ll bind to the agrin -But if you take cells that are transfected with MuSK, they don’t bind If you transfect them with LDLR which is a member of the large family of receptors, they don’t bind So this binding in this assay between agrin and Lrp4 is specific Flag is a short amino acid sequence for which we have good antibodies for -So this is used to fuse a marker to the protein and use it to find the protein (myc is also another marker) This suggests that Lrp4 was the receptor Slide 8 This is a cell aggregation experiment -What you do is take cells, put them in a little dish, put it on a mechanical shaker that shakes it in a circular fashion, and if they’re sticky they’ll stick and form clusters If you take regular cells and do this assay, they don’t bind to each other -However if you take those cells and transfect them with Lrp4 that’s been fused with Mcherry (red fluroescent proteins) they do form clusters -If you assay for the mcherry, the clusters are expressing mcherry -So these are the cells that are expressing Lrp4 This suggests that Lrp4 binds Lrp4 If you transfect those cells with MuSK that’s fused with GFP, they don’t cluster -So MuSK doesn’t bind musk Take some cells that express Lrp4 and some that express musk, you do get large clusters and those clusters contain both mcherry and GFP positive -So they are expressing Lrp4 and GFP If you take a close look, you’ll see that each cell is expressing one or the other and each aggregate is a mixture of the red and green -Therefore, Lrp4 binds musk and vice versa Final thing to demonstrate binding is a biochemical binding assay -Transfect cells to express Lrp4 mcherry and musk GFP -Then do a immunoprecipitation (IP) where you take an antibody, couple it to a bead, put it in a column and pass proteins through the bead -The beads will sequester the protein that’s being recognized by the antibody This is co-immunioprecipitation -So if protein X is pulled down and protein Y is bound to protein X, then protein Y and protein X will be pulled down at the same time So here, Lrp4 was precipitated -Then the protein was released from the beads, ran out in a gel, and used an antibody against GFP which should indicate musk -They get a nice bad at the predicted size This demonstrates that Lrp4 and musk bind each other Slide 9 Since lrp4 binds lrp4, the receptor would have to be a dimer of lrp4 It also binds musk so each of the lrp4 must bind musk This leads to two lrp4s and two musk Upon binding of agrin to the lrp4, you get a change in the conformation of the receptor such that the tyrosine of the musk is phosphorylated -Musk then goes on to tyrosine phosphorylate downstream proteins which ends up in the aggregation of AChR slide 10 At embryonic day 13.5, there are lot of red spots that aren’t contacted by green presynaptic terminals 5 days later, you have the collection of presynaptic axons in terminals -Every red dot is contacted with a presynaptic terminal This suggests that there are aggregates of AChR that form aggregates even before they’re contacted by the presynaptic terminal -There are nerve independent accumulation of AChR slide 11 When lrp4 was knocked out, you also don’t get the aneural accumulation of AChR Slide 12 Musk is a critical participant in the aggregation of AChR -So maybe musk is also important for the clustering of aneural clusters of AChR This was examined in zebra fish -Injected an antisense MO -MO is just a specific form of oligonucleotide that is stable -This has a sequence that’s complementary to the 5’ end of the mRNA -This forms a duplex onto the mRNA which then prevents translation from occurring by preventing the binding of ribosomes So they injected musk antisense MO into the fish AChR are shown in red In green are the axons of neurons When you inject the MO to prevent the translation of musk, you see that the clusters including the aneural clusters of AChR are missing So musk is also required for aneural AChR clusters So lrp4 and musk are needed for both neural and aneural AChR clusters Given that agrin receptor is required for aneual clustering and given that they are aneual and don’t bind neural agrin (Z agrin), what might be the actual ligand for this receptor that mediates the clustering of AChR independent of agrin? -There must be another ligand slide 13 The other ligand is Wnt11r If you knock down wnt11r, you can greatly decrease the aneural AChR clustering Doing the co-IP again, you fuse myc to musk and wnt11r that’s tagged with flag -The musk was pulled down with the antibody and then assayed for wnt11r by assaying for labeling with flag -You get flag labeling as well as myc that you pulled down -So this shows that biochemically, the two are interacting together In the
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