cc  >> Good afternoon.  Welcome to Frontiers  of Life Science,  our PLATO science class.  Our presenter today  is Allison Lynch, a PhD  student from the UW in the  Department of Genetics.  Her topic is cell adhesion,  how cells stick together.  Allison had her undergraduate  work at the University of  Pittsburgh after having been  born and raised in Michigan.  So Allison, you can tell us  more about yourself and about  cell adhesion.  Welcome.  >> Thank you.  Okay.  Is that on?  Okay. 

 So I grew up in Michigan and I  did my undergrad in Pittsburgh  which is very close to Ohio and  then I came here so I guess the  moral of the story is I like the  Midwest.  And actually really why I went  to Pittsburgh was because they  had a very strong program in  undergraduate research.  And I have known ever since  I was 9 years old that I wanted  to be involved in scientific  research.  I was diagnosed with Type 1  diabetes in October of 1982  when I was nine.  And my mom says it was  pretty much immediate.  I don't actually remember  all this, but my mother tells me  I said it.  She says that I said I was going  to figure out a way to cure  myself because I didn't like  giving myself shots.  I have since gotten used to that  but I'm still fascinated by  research and fascinated by basic  science questions because I look  back when I was in high school  and when I was in college and I  signed up for classes and I  always wondered how am I going  ever going to learn all this  stuff.  This stuff always seemed so  complicated.  But by the end of the class I  understood and so I liked that  sense of I learned something and  this is something that stayed  with me my entire life. 

 So I'm a fourth year genetics  graduate student here at UW  which means that I can see the  light at the end of the tunnel  and some point soon I will soon  be graduating.  So I was glad to be asked to  present my work on a protein  that I work on which is called  MAGI-1 and how that affects cell  adhesion during morphogenesis.  So I'm going to start out with  a little background.  So the lab that I work in is  Jeff Hardin's lab and he's in  the Department of Zoology.  And he's a developmental  biologist.  And so the main theme that  we study in our lab  is called morphogenesis.  And morphogenesis, just by  definition, is the generation of  form and structure in  multicellular organisms.  And I think this is just really  cool.  We all start out  as a single cell.  This is a picture of an ovocyte. 

 It's a mature ovocyte.  Eventually this will be  fertilized and then eventually  it becomes a human being, or  a baby that has arms and legs,  fingers, toes and all different  organs.  Everything that we need to make  unique organisms is in one cell.  Which I find incredibly  fastening.  How on earth does that happen?  And how does that one cell then  divide and become many cells and  how do those cells know what  they're doing?  And how do they know where  they're supposed to go?  So the basic idea that drives  morphogenesis is the fact that  cells can move and change their  shapes.  So in this slide here I just  have some examples of cell  movements that occur during  morphogenesis.  So the first one over here is  something called epiboly where  these cells will stretch out and  eventually cover this entire  surface.  So things like this happen like  when you need to form an  intestinal lumen.  You need to form a cavity.  The cells can come together and  seal off this ring of air, this  space right here. 

 The second movement is  conversion extension.  And so there's these cells in  the middle here that are  slightly different color and  what happens is they converge  together, so instead of having  a single file line now we have  multiple cells in different  lines, and then the entire shape  of the cells extend out.  There's also involution and  invagination, which can be very  similar in the fact that the  cells move in a particular  direction away from the original  plane that they were part of.  And the last movement is  ingression where individual  cells break out of an original  sheet of tissue.  So I'm going to come back  to a couple of these  throughout the talk.  But these five movements,  certainly this is a  simplified version, but these  five movements essentially they  describe what happens during  morphogenesis.  It's amazing how simple it is.  So how does one cell move?  And the answer is each cell has  a cytoskeleton so there's a  skeleton inside the cell that  helps it to move.  So this red is supposed to  describe actin.  So actin is a main component of  the cytoskeleton. 

 And actin accumulates at the  edges of these cells here and  these are the areas that are  moving out.  And so this right here, is where  we have a lot of concentrated  actin branching out.  We have lamellipodium.  We have these sheets and these  ordered arrays of actin moving  like the entire wall of the  cell out, which is also  these ruffles over here.  And then these circular  ruffles, this could lead to  something like part of this cell  branching off from the original  cell body.  So that's one cell.  But all the other movements were  more or less describing groups  of cells moving.  So how do groups of cells move?  Groups of cells move by  attaching to each other  essentially.  So this is a very basic diagram  of cell adhesion.  Invertebrate cells. 

 So at the apex of the cells we  have tight junctions which these  actually form a barrier, so  this is a barrier that  liquid can't get through.  And then we have the adhering  junctions where proteins on the  outside of cells bind to each  other.  The same with desmosomes.  Focal adhesion and hemi  desmosomes attach to a cellular  matrix which I'm not going to  get into much today because my  work focuses mainly on the apex  of cells.  So the interesting thing about  all these adhesion complexes is  that they all attached to the  cytoskeleton.  So if you have cells moving  using actin, you can have groups  of cells moving by essentially  attaching yourself to that one  cell.  So it's like when you move you  kind of drag the next cell along  with it.  And ideally there's also  signaling going on between these  two cells so they both know what  order or what direction they're  going and so they move at the  same time.  >> Allison, can you answer  questions?  >> Sure.  >> Are those three different  types of junctions in all cells  or just some cells?  >> These are vertebrae cells so  in more simplified organisms  they don't have all these  components. 

 And even within some vertebrate  cells they don't have these  types of junctions.  But this is the typical  epithelial cell, and I work on  epithelia, so all vertebrate  epithelial cells have all these  components.  But if you have something like a  red blood cell, that's an  individual cells so you really  don't want junctions on that  cell because you don't want your  blood sticking to the inside of  your blood vessel.  That would be bad.  Yes?  >> And when you say vertebrate  cells does that mean bone cells  or the cells have bones?  >> When I say vertebrate cell,  I mean like your skin cells.  You're a vertebrate.  Any other questions at this  point?  Okay.  Okay, so you know the picture  that I start off with I would  say going in and forming a baby  and the babies are cute but at  this point you know none of us  are quite that young anymore so  why do we care about these  things now?  And there's a couple of  applications, first of which is  wound healing.  So any time you have a wound on  your skin you need to, cells  need to be able to grow and they  need to move and fill in the  area that was wounded.  This is kind of like a reverse  invagination so instead of the  cells buckling in, the cells  buckle out to fill that original  gap. 

 The second big area that I'm  going to come back to is cancer  metastasis.  So cancer is obviously a very  broad reaching problem.  And it's more a problem when it  metastasizes because then it can  travel to different parts of  your body and create secondary  tumors.  And in order for cancer to  metastasize, cells on the  original tumor need to modify  the way they adhere to other  cells so they can leave the  original tumor and go travel in  the bloodstream and find another  place to attach to and make  another tumor.  So this is similar to an  ingression movement during  development.  How can we study how cells move  during morphogenesis?  So I talked about the fact that  you need the cytoskeleton to  move or you need the  cytoskeleton in order for cells  to be able to move, and cells  need to attach to each other so  they can move in an organized  fashion.  So really what I focus on is  studying proteins that connect  adhesion complexes and the  cytoskeleton.  So these proteins are kind of  linker proteins because it  connects two different areas of  the cell.  I first gave you a very  simplified diagram of the  vertebrate adhesion system.  This still isn't all  the proteins that are involved  but this is a more expanded view  of the proteins that are  involved. 

 So if you want to study a  protein that connects adhesion  protein to the cytoskeleton,  this is very complicated.  There's a lot of things in this  diagram and so what I use  is a model organism called  C. elegans.  So C. elegans is a free-living  nematode.  It lives in the soil.  I always tell everybody  if you've ever eaten dirt,  I guarantee you've eaten  a nematode.  They're not dangerous,  for the most part,  or C. elegans isn't anyway.  They're a great model organism  for a number of reasons.  They're small so they can be  easily kept in the lab setting.  They can also be frozen down so  we can store them which is nice  because you can just thaw out a  tube of worms and start an  experiment with those worms.  They can survive space shuttles  exploding. 

 So when the Columbia exploded  there were actually worms on  board that fell down to the  earth in part of the debris and  the worms survived.  Which is kind of incredible so  they're very durable organisms.  They have a short generation  time which means we can do  experiments quickly so I'm going  to be here for five years as  opposed to like 20.  Not that I don't like Wisconsin  but I'd like to get my degree  before then.  They're optically transparent  which means we can make movies  of them, we can image parts of  the worms, we can image parts of  the cells.  So this worm actually right here  is expressing a protein that's  causing it to be green.  But probably one of the best  reasons that C. elegans is a  good model organism is because  there's something called  homology between worm  and human genes.  As a human your DNA, you have  a certain number of genes.  Worms have about 17,000 genes  and about 80% of those are  similar to genes that are found  in humans.  >> And we have?  >> Well, the number varies. 

 Somewhere, give or take, 30,000  or 35,000.  Something like that.  The number of C. elegans varies  too, because people are  constantly setting these genomes  and figuring out  what actually is a gene,  and that kind of stuff.  So it's a smaller genome for  sure but the fact that 80% of  these worm proteins or genes  are similar to human genes,  that means that we can take  what we learn in C. elegans  and apply it to what happens  in humans.  Which is obviously the model  system that we are most  interested in because we are  humans.  So I'm a genetics student  so, yeah genetics,  genetics is awesome.  And something that really is  attractive about C. elegans is  they have powerful genetics.  And a lot of the basis of that  comes from a technique called  RNAi.  So RNAi stands for RNA  interference and it's a  technique where you silence  expression from any gene in the  gene specific manner.  So essentially what happens is  you have a piece of double  stranded RNA and you have this  enzyme called dicer. 

 It comes and essentially chops  up your double stranded RNA into  small pieces.  Those small pieces then go out  into the cell where they either  bind to an mRNA that's being  made and that causes the mRNA to  be destroyed.  So you don't get any protein  made from that.  Or if they bind to the mRNA  and they prevent translation  from happening.  So again, you don't get protein  made.  So if you have this gene in that  you're interested in studying,  one of the first questions you  typically ask is what happens if  I don't have that gene anymore?  And C. elegans, it's very easy  to silence any gene in the  genome through this technique.  So a little bit about C. elegans  morphogenesis just so we're all  oriented on the same page.  There are two movements that  I'm going to be talking about  specifically.  The first one is called  enclosure, the second one is  called elongation.  So these are just still images  right here. 

 So this is a C. elegans embryo.  It's kind of lima bean shaped.  And I'm concerned with  epithelial morphogenesis.  So all of the epithelial cells  or all of the epidermal cells  of C. elegans are born  on the back side of the embryo.  And then what happens during  enclosure is that the two  outermost rows come around the  sides of the embryo and meet in  the middle, where they seal.  So that's what this is right  here.  This is a cell, this is a cell  and they're coming in they're  meeting here right in the  middle.  And then what happens is the  embryo starts to elongate.  So the enclosure seals the  embryo in an epithelial sheet.  So you're all contained in one  place. 

 And then the embryo will turn  and it starts to elongate.  And it eventually becomes about  four-fold its original length.  So this is one, two, three,  four, before finally hatching.  And elongation is kind of like  squeezing a tube of toothpaste.  You start off with a big mound  of stuff and then you squeeze it  out you get something that's  much thinner.  So this is a movie of a single  embryo developing.  So right now here this is  enclosure happening the cells  are meeting in the middle.  Now the embryo turns.  And it starts to elongate.  So these contraction forces are  driven by rings of actin that  surround the embryo. 

 >> How long is that?  >> That was...  >> I mean, how many minutes?  >> That was about eight hours.  >> And it was divided from one  cell?  >> Well, it actually didn't  start at one cell.  It already had multiple cells at  this point.  So total development embryo  genesis takes about 20 hours for  an average C. elegans embryo.  So this is only part of it.  It spends a fair amount of time  in the four-fold state before it  actually hatches and then we've  missed a fair amount of  development by this point.  We've missed gas relation,  but were not talking about that.  Any other questions right now?  The fact that we can film  individual embryos like this,  it's pretty incredible.  >> Is there growth group of  cells or are all the cells  divided?  >> At this stage not all the  cells are dividing, some of them  certainly are. 

 But by the enclosure stage the  cells have been born, or a lot  of cells have been born that are  going to eventually form  specific organ systems.  The reproductive organs, they  have not developed yet at this  point so those cell divisions  would still occur.  But most of your basic cells  have already been formed at this  stage.  Yes?  >> Is that optical microscopy?  >> So this technique is called  namarsky.  Essentially what we do  is we cut open a bunch of worms.  We collect the embryos.  We stick them on a mount.  We stick the mount on a  microscope, and there's a camera  mounted and it's just  time lapse microscopy.  We take focal planes slicing  through the embryo and then we  compress those images and run  them for however long we took  the movie for.  Okay, so I'm interested  in adhesion. 

 The nice thing about using a  model organism, and I kind of  touched on this, C. elegans  has a smaller genome.  So as you might have expect, it  has a smaller adhesion system.  There's not as many proteins  that appear to be involved  or appear to be essential.  So this is still a fair number  of proteins though.  So I'm interested specifically  in the cadherin-catenin complex.  Which is this band right here in  the middle.  So a little bit more close-up.  The cadherin-catenin complex  in C. elegans has three  essential proteins.  The first protein is this  HMR-1 cadherin.  So this cadherin molecule binds  to cadherins on neighboring  cells. 

 So this is how they anchor cells  together.  Intracellularly, cadherin binds  to this HMP-2 beta-catenin.  And beta-catenin recruits  alpha-catenin and alpha-catenin  binds actin.  Now the actual mechanism as to  how this occurs is kind of a big  question in the field right now.  It's one of those we thought we  knew how worked and then  somebody went along and actually  tested that idea and determined  no, that's not actually how  it works.  There's a little more work to do  in figuring out how these arrows  actually work.  But the idea is alpha-catenin,  we know is a mediator between  this adhesion complex and the  cytoskeleton because  alpha-catenin interacts  with actin.  So as one might expect, if you  mutate either cadherin or either  of the catenins, you get fairly  disastrous results.  So HMR-1 is the cadherin  which the cadherin was the cell  that bound to cadherins on  neighboring cells.  So that actually what's  responsible for holding cells  together. 

 So if you mutate cadherin, these  embryos cannot enclose.  So what's happening here, you're  trying, the embryo is trying  enclosure but there's kind of  that gap there and the embryos  never seal the epithelial sheet  so when it contracts to drive  elongation instead what you get  the guts spilling out of the  head region.  Kind of gruesome.  Kind of cool.  (LAUGHTER)  It's a worm.  >> And what you've proven is  that you stop the production of  that substance?  >> Right.  The cadherin is no longer being  made so when an embryo tries to  enclose it doesn't have  cadherins on the outside of its  cell so it can't stick together.  In contrast, if you mutate  either of the catenins, HMP-1  or HMP-2, these mutants fail to  elongate.  So these embryos still have a  functional cadherin.  So when the cells try to stick  together, they can. 

 But because you have disrupted  this connection to the  cytoskeleton these embryos can't  elongate.  So this guy has already enclosed  and it's turned and it's going  to start to elongate and what's  going to happen is it's going to  immediately retract and develop  these characteristic dorsal  humps, hence, HMP-1 and HMP-2.  They have HMPs.  So, as you might expect, if you  mutate HMP-1 you've also  disrupted this connection to the  actin cytoskeleton, so the  cytoskeleton is no longer  organized.  So this is, these are embryos  that have been stained for a  protein using a protein called  faloidin.  So faloidin binds to actin  filaments and then we use  a fluorescent molecule to  determine where the faloidin is  and you take images.  This is just a wild type larva.  Or wild type embryo.  So this is pretty much fully  elongated.  You can see there are these nice  ordered arrays of filaments. 

 This arrowhead is pointing to  the underlying body wall muscle.  And this arrow is pointing to  the adherence junction.  And you can see that these lines  are fairly straight and are all  anchored at the junction.  So in panel B here, this is a  HMP-1 embryo that is just  before it's started to elongate.  So it managed to do enclosure,  and this is before it starts to  elongate.  So again the arrowhead is just  pointing to the body muscle  here.  And this arrow is pointing to  the adherence junction.  And, as you can kind of tell,  you can see nice ordered bundles  here.  You're kind of missing that  here.  This looks a little messier. 

 And you've got this kind of big  disconnect right here.  And so it's no wonder that  elongation fails because you  have a disconnect and you have  disordered actin.  And in panel C here this is an  embryo that's actually tried  enclosure so you can see that  there these HMPs outlined here.  And you can actually see that  there's actin filaments that  have completely detached from  the junctions they're supposed  to be at.  So this is just a schematic  diagram.  This is where the junction is  supposed to be.  And you have actin fibers and  you have actin that's just not  organized and so you don't have  an attachment at the junction.  >> If you do that over, to say  what you just said,  that that is what happens?  >> It depends, of course, on the  procedure.  So a lot of the protocols that I  will do when I'm looking at  specific proteins,  I will do them--  On an individual slide, you  can get hundreds of embryos.  But not all of them are stained  to the same level. 

 Not all of them look pretty,  essentially.  So I do the experiment at least  three times.  And I make sure that I have  replicable results each time.  So this is something that's been  done with a lot of care.  At that stage you've looked at  thousands of embryos.  And looked for consistent  phenotypes across them.  So my research question is, how  is the cadherin-catenin complex  modulated during morphogenesis?  So all that stuff that I talked  about, all of these pictures,  these were done before I joined  the lab that I'm currently in.  So we know what the three  essential proteins are.  We know what the phenotypes are.  We mutate those genes. 

 My question is, it's a little  bit more, how can you modulate  how this complex functions?  I'm looking more at subtleties  as opposed to phenotypes.  The major tool or one of the  major tools that I use is a  hypomorphic allele of a HMP-1  alpha-catenin.  So alpha-catenin is this linker  protein.  So we have this hypomorphic  allele.  So hypomorphs are alleles that  have a mutation in them but they  still retain some of their  function.  They only retain some of the  function, it's not perfect.  So it's a single mutation  in the binding domain.  It's about 70% lethal but about  30% of these guys live which  that's decent odds, all things  considered.  So this is a wild type larva.  So you can see this guy is  nicely elongated. 

 Uniform size.  This is an embryo, or this is a  larva that is homozygous for  this hypomorphic allele called  HMP-1, FE-4.  There the same age.  That filming experiment was done  on the same day.  And as you can see, this guy  doesn't look anything remotely  like what this guy does.  You can also see that there are  HMPs.  Which is again characteristic of  the HMP-1 -- phenotype.  But this guy is still alive.  Amazingly enough.  So the question really is can we  make this any worse?  It's already pretty bad. 

 70% lethal.  That's not great.  But can we make it worse.  And the reason behind that is so  when I first introduced this  diagram, I only talked about  three proteins.  I talked about cadherin,  beta-catenin and alpha-catenin.  And that's because those of the  only three essential proteins.  So we know that there are these  other proteins.  So like this JAC-1/P120 catenin.  We know that it's part of  this complex.  So the question is, what does it  do?  And so we can study that using  this hypomorphic allele of  HMP-1, FE-4. 

 So if we knock down JAC-1 using  RNAi to specifically silence  that gene, that's going to,  that increases the lethality of  HMP-1 FE-4 homozygotes to  100%.  And interestingly and  importantly, this pheno copies  the HMP-1 -- mutation.  So this is a HMP-1, FE-4 embryo  before it's hatched and I don't  even remember if this guy did  hatch.  So this is the final stage of  elongation.  You can see this guy is not  four-fold.  It's got these mild HMPs here.  But if you knock down JAC-1  RNAi in this background, this  guy hasn't elongated at all  and you've got this very  characteristic dorsal HMP.  It's a highly penetrated  phenotype.  I think the official number  is 99.8% lethal.  And because HMP-1 is the  protein that mediates the  interaction of the cytoskeleton,  when you knock down JAC-1 in  the HMP-1, FE-4 background  you enhance the disorganized  actin phenotype. 

 So this actually might be a  little clearer picture, but you  can actually see these nicely  ordered bundles of actin.  And this is what it looks like  in HMP-1 FE-4's.  So you can kind of see there are  these little globby areas.  But overall it's not too bad,  it's not great, but it's not  awful.  If you knock down JAC-1 RNAi  and the HMP-1 FE-4 background,  that's pretty awful.  You've got these clumps.  There's a complete lack of  organization.  You don't actually see bundles  except for the clumps.  All you can see are those nice  clumps over there and kind of  just fuzzy stuff in the middle  which is actin that's not in any  sort of organized manner.  That initial observation led to  an idea. 

 So we have this protein that was  part of the cadherin-catenin  complex that enhanced this  HMP-1 FE-4 phenotype.  So the question was, can we find  anything else in the genome that  did the same thing?  Can we build up that complex so  you know it looks as complicated  as the vertebrate one.  We didn't think we'd get that  far, but we thought we'd find  additional proteins that  influence how this complex  functions.  Remember how I said RNAi  was easy?  Well, it is fairly easy.  So essentially all you have to  do is you have to go grow out  bacteria that have a plasmid,  that are carrying a plasmid  that has a piece of C. elegans  DNA.  You induce transcription of  double stranded RNA from this  piece of DNA, and you feed this  to worms.  So you feed them, you put the  worms on a plate, you transfer  them after 24 hours then you  remove all the adults and you  look for phenotypes in the next  generation.  So it's fairly simple.  The worms eat the bacteria.  You knock down a specific gene. 

 Just imagine doing this 16,000  times.  So my lab, in collaboration with  the Pettit Lab at the University  of Aberdeen in Scotland,  did just that.  We went through the entire  genome of C. elegans  and did this experiment.  We were looking for things that  greatly enhanced the value of  HMP-1 FE-4.  They're about 70% on their own  so we wanted something  that was close to 100%.  And we wanted something that  didn't have a phenotype  in wild type worms.  And the reason for that is we  wanted to look for things that  specifically influenced the  adherence junction, and so  there's a worry that if  something was lethal to wild  type worms, of course it's going  to be lethal to HMP-1 FE-4  worms, and we're not going to  learn anything interesting in  that particular case.  And then we've been going  through and we been filming to  characterize all the terminal  phenotypes, seeing if they  pheno copy the HMP-1 node.  Do we see dorsal HMPs?  Do we see a different phenotype  and what is that possibly going  to tell us?  So the first summer that I was  actually in Madison, I spent  every day in the lab working on  this screen.  The other thing about screens  are you got something to do  every single day. 

 When I finally came down to it,  I screened one chromosome all by  myself and so when I was finally  done with the screen I came down  with a list of proteins that  were true enhancers of the  hypomorphic phenotype but didn't  really have much of an effect  in wild type worms.  And so the next question is,  I sat down and I discussed this  with my adviser, and the  question is what proteins, what  gene do you want to work on?  What gene is going to become the  basis of your thesis research?  So you go down through this  list.  The first gene is a protein  involved in a planar cell  polarity.  Planar cell polarity can be  interesting.  It's a signaling molecule so  cells do have to signal to each  other so they can coordinate  movement.  But I wasn't sure.  The next protein was worm  specific that had unknown  function.  So those are always exciting  because you never know what  you're going to find out but you  could find out something really  cool.  Or you could find out something  that's maybe not so cool.  So it's a bit of give-and-take. 

 Things like this protein that's  involved in chromatin  remodeling, the ribosomal  subunit, those are probably more  things that were concentrated in  the nucleus as opposed to  actually at cell junctions.  So I'm going through this list  and there is obviously one  protein that kind of jumped out  at me.  This protein called MAGI-1.  It was previously shown  in tissue culture in vitro  to be a protein scaffold  at cell junctions.  So the idea, cell junctions,  yeah, that's exciting.  >> Where did that list come  from?  >> That was from my screen.  So I did the screen.  >> You did all 16,000?  >> Not personally.  Chromosome 4 had, let's see,  if I remember the number...  It had about 4,000 genes on it,  3,000 to 4,000 genes. 

 So I screened all of those.  So worms have five autosomes and  one sex chromosome so a total of  six chromosomes.  So I screened one of them  personally and I also screened  another one with another person  in my lab.  So it was kind of split up.  People in the lab had their own  chromosomes to work on.    Mine just happened to be  chromosome 4.  It took a while but you find  something like this.  Protein scaffold at cell  junctions.  I was looking for something that  was at cell junctions that could  be influencing this complex.  Exciting for me. 

 So this is just going back a  little bit of the wild type  phenotype, the embryo encloses  and elongates four-fold.  The FE-4's, they will enclose  just fine.  But when they elongate, they  come up with these HMPs.  When I knocked down this  protein, MAGI-1 in the  hypomorphic background, again I  didn't get really any elongation  and I got these dorsal HMPs.  So it pheno copied the HMP-1 --  phenotype so I was really  excited at this point.  And so the first thing you do is  you typically go to the  literature and you look and see  what other people have  discovered about your favorite  protein.  So at this point MAGI-1 was my  favorite protein, it still is.  So but I wanted to go see what  other people found out.  So there's this big collection  of papers called Pub Med,  and so I searched for MAGI-1.  And 48 papers came up. 

 Usually if you put in a protein  you get like thousands.  So this is a relatively  unstudied protein which is also  kind of exciting for me.  And so what is MAGI-1?  It's a protein scaffold at cell  junctions.  So what does that mean?  So MAGI-1 has all these protein  domains, these -- domains, these  WW domains and these PZ motifs.  And presumably all of these are  motifs that can interact with  other proteins.  So I just pulled this picture  from the web.  You've got scaffolding, you got  people that are at different  levels of the scaffolding so if  you imagine the scaffolding is  MAGI-1 and the people are  in fact proteins, that's kind of  how this was thought to work.  There's all these places for  proteins to bind on this  protein, on MAGI-1.  So MAGI-1 could organize  proteins at cell junctions.  That's the general idea. 

 So what does MAGI-1 do?  Because it organizes all those  proteins, it stabilizes  junctions.  And if you destroy the scaffold,  you kind of destroy, you move  all that stability.  And so MAGI-1 is a known target  of cancer causing proteins.  Presumably for that reason.  If you knock down the  scaffolding, then these cells  have an easier time to assemble  in their junctions, to go in  and metastasizing elsewhere.  Which is bad.  We want to figure out why.  So how does MAGI-1 stabilize  junctions?  And the answer is most likely  through its binding partners.  And so this is when I really  started to get excited about  this protein because MAGI-1 has  been shown in vertebrate tissue  culture cells to bind  beta-catenin.  So beta-catenin is part of the  cadherin-catenin complex. 

 So I had a connection to a cell  adhesion complex that was there  in vertebrate cells  and tissue culture and in vitro.  The question now is,  is that also in vivo?  So the model could be that  cadherin does what it normally  does.  Beta-catenin does what it  normally does.  But beta-catenin also recruits  MAGI-1 to junctions.  So this other binding partner,  this PDZF-1.  Kind of a weird name, kind of an  annoying name but that's what  it's called.  So what is that protein?  That's a protein that activates  another protein called RAP-1.  So RAP-1 is interesting because  it promotes junction maturation  and it influences actin  dynamics.  Another name for this protein,  or the C. elegans name  for this protein is PXF-1.  So maybe MAGI-1 binds this PXF-1  protein and then activates RAP,  which influences cytoskeleton  dynamics at cell junctions. 

 A theory.  And it's a little bit more  nicely written in this diagram.  So the top half we've already  seen.  Cadherins bind to the catenin  which influences actin.  And so I initially hypothesized,  after all my literature  searching, was that maybe there  was a pathway that acted in  conjunction with the top pathway  that involved MAGI-1.  MAGI-1 binds this PXF-1 which  activates RAP, which influences  actin downstream.  So that's the idea.  This is the model.  And I have since proceeded to  destroy the model.  So the remaining questions,  all this work on MAGI-1  has been done in vitro. 

 That means it is not done  in living organisms.  And so we want to know what  MAGI-1 is doing in a living  organism.  How is it localizing in a living  organism?  What is it doing at junctions?  Is it influencing actin?  Because they can replicate  living conditions as best as  they can, but the bottom line,  all the work has been done  in a petri dish.  We want to know what happens  when there's other things  inside.  So the first question is  does MAGI-1 localize at cell  junctions in vivo?  This is kind of basic.  We're kind of assuming the  answer is going to be yes, but  you can't assume anything, you  have to actually do the  experiment.  So a couple of tools that I've  used to do this.  I used fluorescent proteins.  So mostly GFP and RFP.  So GFP stands for green  fluorescent proteins, a protein  that is originally found in  jellyfish and something that  is fluorescent green. 

 And you can see it in whole  organisms like in the these GFP  expressing mice.  I'm not entirely sure why you'd  want glow-in-the-dark mice.  But more importantly, you can  look at specific cells or  specific parts of cells in an  organism.  So this is actually part of  a C. elegans embryo or a  C. elegans larva and you can see  that there's these RFP rings,  these fluorescent rings and  they're kind of these dots.  And over here GFP, this is like  what the cells look like and GFP  is specifically marking certain  proteins in those cells.  So the idea that we can look at,  you can look at whole cells, you  can look at whole organisms and  you can look at specific  proteins.  And I'm also using antibodies in  a very similar fashion.  So the GFP or the fluorescent  proteins are something that you  physically tag to your protein.  Antibodies, they bind to your  protein all on their own.  And then you tie the fluorescent  molecule onto something that  recognizes the antibody. 

 So that's a long way of saying,  yes, C. elegans MAGI-1  does localize to cell junctions.  So these are actually antibody  staining pictures.  So the diagram here in A is a  schematic of the worm epidermis.  So these pink cells are a  ventral cells so this would be  like the belly of the worm.  These yellow are seam cells,  so that's its side.  And the blue cells are dorsal  cells so that's the back of the  embryo.  And we can see that MAGI-1 does  localize quite nicely to cell  junctions in embryos.  Furthermore, it localizes in  other epithelial tissues.  So things like your intestine,  your pharynx, those are all  of epithelial tissues.  This is the C. elegans pharynx,  its intestine, and then  its developing renal cell  or rectal cell, sorry. 

 And MAGI-1 localizes along all  of those cells.  So MAGI-1 is part of epithelial  cell junctions in C. elegans.  And so I also use the GFP to  look at this dynamically.  So this is showing enclosure.  So we're kind of watching the  cells come over here and they  meet in the middle and you can  see that there's GFP imaging  on lines that show where cells  normally are.  And so this movie is elongation.  So just before I even start this  movie, you can see that there is  GFP localized at cell borders.  So these experiments are pretty  convincing.  Yes, MAGI-1 is at cell junctions  which is what we expected but  it's good to actually see that.  So the next question I am  interested in is what part of  junctions is MAGI-1 associated  with?  And I'm predicting that MAGI-1  is going to localize with the  cadherin-catenin complex. 

 Based on the fact that  vertebrate MAGI-1 combined  vertebrate beta-catenin which is  part of the cadherin-catenin  complex.  So this is a little bit more  simplified diagram comparing  vertebrate and C. elegans  junction organization.  So vertebrates have, I'm showing  three components of junctions  here.  Tight junctions, -- adherence  domain and desmosomes.  C. elegans have more  concentrated junctions  at the apex of cells.  But they still have distinct  domains.  So at the very apex of cells you  have something called the --  membrane domain.  And then you have the  cadherin-catenin complex and  then you have something called  the DLG aging complex which is  right below the cadherin-catenin  complex.  This is the top of the cell and  this bottom of the cell.  So I'm predicting MAGI-1 is  going to be here. 

 And it's not.  So all that lead up, and yeah.  This is science.  Things don't always turn out the  way you think they're going to.  So what I did was I stained for  that protein JAC-1 which we  know is part of the  cadherin-catenin complex because  it's a catenin protein, and  I stained for MAGI-1 in embryos,  and I looked at where  they were localized  relative to each other.  So when we do this, we stain the  embryos and we set up a mount on  the microscope and we take all  the focal planes and so this is  a compressed series of about  12 focal planes and it's kind of  hard to tell what's going on  just because you've taken  all these focal planes and  compressed them.  So what I've been using, I've  been using software to do  three-dimensional junction  extractions.  So this is kind of like you're  looking at it from the bottom of  the embryo down towards the top.  And so this red signifies MAGI-1  and the green signifies JAC-1.  And so there is a little bit of  yellow there, but for the most  part I got two very distinct  bands. 

 So I was like, okay, what next?  So then if you remember the DLG  aging complex was just below the  cadherin-catenin complex.  We're saying that MAGI-1,  because we're looking from the  bottom to the top, MAGI-1 is  below the cadherin-catenin  complex.  So does MAGI-1 localize with the  DLG aging complex?  So again this is an antibody  stain looking at -- which is the  green protein and MAGI-1 which  is my red protein.  And I did the same thing where I  did the three-dimensional  junction extractions although  you're probably a little far  away to see it, but you can kind  of see the same thing.  What you see, is you see green  and you see red.  And you see green and then you  kind of see a little bit of  empty space and then you see  some red, some green, some red,  some green.  I've got my own version of  Christmas lights here.  So this is very interesting.  It was interesting mostly  because they don't appear to be  occupying the same space.  They appear to be in the same  plane. 

 But they're not in the same  space.  I do, I have like colorful beads  on a string here.  So what that means,  not entirely sure yet.  I'm working on that.  So we know that the DLG aging  complex has been firmly  established.  So MAGI-1 is probably acting as  part of a different complex.  So not entirely sure what other  components of the complex are  going to be or how that's going  to influence cell adhesion.  So that was all exciting.  So the next question that I was  interested in is how does MAGI-1  localize?  We know that it junctions, how  does it get there?  And initially I would have said  it involved HMP-2 beta-catenin  but considering the fact that  MAGI-1 doesn't localize with the  cadherin-catenin complex,  I'm not so sure at this point.  So actually I have  a collaborator. 

 Right now he's in the  Netherlands but he's moving to  Basil in December.  So he did an experiment where he  did something called  the GST Pull Down.  So essentially what he did, he  grew up a liter full of worms.  And he -- all those worms,  collected protein,  he essentially ran that over a  column that had HMP-2  beta-catenin with the GST tag.  So the GST tag caused HMP-2 to  bind to the column and he dumped  these worms over the column  and looked for a presence of  MAGI-1.  This is just a lysate  on its own.  This is the GST tag on its own.  So these are our controls.  So this is really interesting  band here.  GST HMP-2. 

 If we have HMP-2 bound to GST  and we run a lysate over that,  HMP-2 can interact with MAGI-1.  So now again I'm not so sure  how this is going to work.  And it could just be that  they're interacting because this  is an in vitro situation.  He lysed a bunch of worms  and ran them over a column.  It's not a living organism  anymore.  Not so sure about that.  So I tried to take this to a  living organism and see what  happens.  So what I did is I used RNAi to  specifically target and knock  down beta-catenin HMP-2.  So last panel here is just my  control panels saying that yes,  there are still junctions in  these embryos.  And the question is whether or  not MAGI-1 is there. 

 So this top panel here  is a wild type embryo.  So as you can see in the control  and in the MAGI-1 staining,  there is staining at junctions.  So both of these embryos are  depleted of their HMP-2 by  RNAi.  So there are still junctions  there and if you look at this  embryo down here at the bottom,  you pretty much see a whole lot  of nothing.  But in this embryo right here,  so it is kind of faint, but you  can see that there is these  lines.  And what are these lines?  They kind of look like the  remnants of junctions.  So you can kind of still see  MAGI-1 at junctions.  So it's like, all right,  how do I interpret this?  It does physically interact but  if I knock down HMP-2 and a  living organism, sometimes I see  MAGI-1, sometimes I don't.  Granted this is not normal  localization and this doesn't  look anything like the picture  of the wild type embryo.  But it's still clearly at  junctions. 

 I was looking to see if there  was anything else that could be  responsible for localizing  MAGI-1.  And so what I was focusing on is  these proteins called claudins.  So this is the last molecule in  this diagram that I haven't  explained and I'm going to now.  So this protein is actually  called -- C. elegans.  Claudins are membrane spanning  proteins that have these four  regions that pass through  membranes and then these loops  interact with other proteins.  So it's not that these loops on  the outside here interact with  claudins on other cells and  then this intercellular loop can  interact with proteins inside  the cell.  So it's possible that instead of  this arrow coming from  beta-catenin, maybe there's an  arrow that comes from -- from a  claudin molecule.  And you get the same results.  MAGI-1 localizes at junctions  where it possibly influences  actin through downstream binding  partners.  And the other alternative, of  course, is that there could be  arrows coming from both of these  proteins. 

 So I looked at the claudins.  So C. elegans has five  claudin-like molecules.  Which actually is vastly  simplified compared  to vertebrates.  So knocking down CLC-1,  CLC-2 or VAB-9 individually  using RNAi has no effect  on MAGI-1 localization.  They look wild type.  And that's when we do something  called functional redundancy.  So what is functional  redundancy?  So there are these five  claudin-like molecules and so  I've given them all different  colors but essentially they all  have the same structure.  And a lot of them are expressed  in the same kinds of tissues.  So if I knock down VAB-9, I've  still got four good proteins  that could possibly do  what VAB-9 normally does.  So it's like I need to keep  knocking down proteins  maybe before I actually see  a phenotype. 

 So what can I conclude from this  part?  So as I showed you, MAGI-1  localization at junction is  reduced if I knock down HMP-2  but it's not always completely  eliminated.  And the hypothesis that I'm  favoring right now is that  that's probably due more to a  dissolution of junctions.  So HMP-2 is obviously a crucial  component of junctions because  if you don't have HMP-2, then  your junctions are destabilized  and MAGI-1 as a scaffolding  protein is one of the first  proteins to go.  It's one of the first proteins  to leave junctions because it's  normally there to organize them.  And they're already disorganized  at that point.  And individual claudins don't  seem to be responsible for  MAGI-1 localization but that's  not to say that pair-wise or  triple combinations of these  claudins won't be responsible.  So that's actually what I'm  working on right now and I did  that experiment this morning  before I came here.  So I don't have the data yet.  It takes a little bit longer  than two hours.  So in the future--  I talked a lot about MAGI-1  being a scaffolding protein. 

 So why is that important?  You have all these different  domains and so I want to know  what domain is important for  these various roles of MAGI-1.  What domain is required for  localization?  What domain is important for  function?  So with my collaborator, we've  generated all these constructs  that are GFP tagged.  And so one of the nice things  about worms is we can take this  DNA and we can inject it into  the gonads of C. elegans,  and then the resulting embryos  in that particular worm  might express our particular DNA  of interest.  You don't get 100% expression  but you do get some level,  typically.  So we can see which of these  constructs are going to  localize.  And then I'm hoping to build a  strain that doesn't have MAGI-1,  and it has this homozygous  or this hypomorphic allele  of HMP-1.  So ideally 25% of these guys are  going to be dead.  And so if I add this construct  to the mix, which of these  constructs can rescue that?  Which can restore complete  viability?  So I like to illustrate this  because this is a large diagram.  There's a lot to process in  here.  So really the question is much  simpler. 

 Why do we care about individual  domains?  So if you guys ever play Jenga  you have these building blocks  and you build a tower and the  idea is you start taking away  those blocks.  So this is how I'm kind of  visualizing specific domains of  MAGI-1.  These are blocks that I'm taking  away.  And how many can I take away  before total meltdown?  And that's really the question  that I want to know the answer  to.  And a second question is I want  to know what proteins interact  with MAGI-1 in vivo.  So in a worm, specifically.  So I've talked about this PXF-1  PDZ-GEFprotein, I want to know  if that's a physical interaction  in living cells,  in a living organism.  And then there's this other  protein.  This protein is called SAX-7.  It's a L1-CAM. 

 So it's a cell adhesion molecule  which, cell adhesion,  that's kind of what I study.  So it was identified  in a hybrid screening as being  an interacter with MAGI-1.  And we know that SAX-7  genetically interacts with  HMR-1 which is the cadherin  molecule during gas relation  which is a step prior to the  enclosure and the elongation  that I was talking about.  So cell adhesion molecule,  that alone would get me enough.  That's enough for me  to look at this protein.  And with that, my advisor,  as I mentioned earlier,  is Dr. Jeff Hardin.  He's been instrumental  in helping me get all this data  and get it all together.  Various people in the lab,  so Drs. Abbi Cox,  and Theresa Grana,  and Dr. Jonathan Pettit,  and Annabelle Couthier,  those were the other people  who were involved in it --  of the entire genome.  And these are current members  of the lab.  I'd like to give a special  shout out to Jess Vera  and Sarah Pedracine. 

 They're undergrads who work  in our lab.  They make a lot of the stuff  that we use on a daily basis,  and they don't get  a lot of glory for it.  Thank you.  (applause)