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Lecture 15

BIO241 Lecture 15

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University of Toronto St. George
Jennifer Harris

Tuesday, March 3, 2009 - This week we’re going to be discussing cell junctions & adhesion b/w cells. - They connect cells together or they connect cells to the matrix that underlies cells.  Mechanically attach cells to cells or the extracellular matrix  Seal the contacts b/w the cells  Allow chemical or electrical signals to pass from cell to cell (through these channels) - Today we’ll focus on anchoring junctions. - Anchoring junctions hold cells together mechanically – it attaches them to one another or to the matrix. - On this diagram, he’s drawn to scale a typical size of a mammalian cell, ~50 microns wide & the tiny little dot/speck is a 50 nm long cell adhesion molecule. How can these individual molecules deal with these relatively massive cells? - We heard about this a little bit when we talked about the actin cytoskeleton & how actin molecules, which are also very small, similar to the size in the diagram, we saw that they could impact cell structure by assembling huge networks inside the cell & pushing against the plasma membrane, pushing the plasma membrane outwards to change cell shape. Here that same principle applies. - These cell adhesion molecules, these tiny cell adhesion molecules, assemble larger complexes & these complexes have 2 features that overcome this challenge of size (as listed in the slide). - Diagram: Here is one single cell & here is a cell-cell adhesion complex – these would be a large number of cell adhesion molecules clustered together in that complex & they connect to the cytoskeletal networks. - So you can follow protein-protein interactions b/w the cell adhesion complex through the cytoskeleton to another cell adhesion complex through the cytoskeleton of that cell, through another cell adhesion complex & so on so you have a huge assembly of proteins within these cells supporting the tissue support. - How can clustering cell adhesion molecules increase the strength of their binding?  The binding strength b/w 2 molecules via a single binding site  The total binding strength b/w 2 molecules or complexes involving multiple binding sites - So you might think that this total binding strength here in the avidity would be just all of those individual interaction affinities added together to create the overall binding strength. So if there were say 5 interactions here & you knew the affinity of each individual one, you could add those 5 together & that would give the total. This is not the case.  The binding strength b/w 2 molecules via a single binding site  The total binding strength b/w 2 molecules or complexes involving multiple binding sites - You might imagine that there would be one interaction interface, if this was sitting on its own, it might have a tendency to break apart at a certain frequency so it may have the tendency to break apart but if a neighbouring interaction is still holding on at a different site, that’s going to hold that first one in place & it’s going to reduce its tendency to break apart so this is this co-operativity. - If you have 5 interaction sites all beside one another, if one is about to break but the other 4 aren’t about to break those 4 sites will keep that first one in place & keep it bound. So that is why this avidity is higher than the sum of the individual interaction affinities so you get this real boast of binding strength b/w the molecules. - Another feature of this is that the pulling forces are distributed among many proteins & this is significant if you think about 2 cells that might be trying to crawl away from one another, or move away from one another or being pushed by physical stress to separate from one another but these many molecules will hold those cells together. - This is where you can see the analogy of a Velcro type of effect where you’d have these tiny little loops, right here would be one single cell adhesion molecule, one thread over here would be the other single cell adhesion molecule so if those 2 interacting with one another, they’re not going to be very strong at all but then when we have them all clustered together into this large cluster here, those 2 Velcro patches will adhere to one another very strongly. - So that is one key aspect of cell adhesion complexes is this clustering of receptors. The other aspect is the linkage to the cytoskeleton inside the cell.  Adherens junctions (cell-cell)  Focal adhesions (cell-matrix)  Desmosomes (cell-cell)  Hemidesmosomes (cell-matrix) - There are linkage sites for actin & microtubules. So actin & microtubules can interact with adherens junctions which are cell-cell adhesion complexes & also with focal adhesions which are cell-matrix adhesion complexes. - Then intermediate filaments, they can attach to specific cell adhesion complexes – the desmosomes that mediate cell-cell & hemidesmosomes which mediate cell-matrix interactions. - These are the basic properties of any adhesion complex that are going to be clustered together & they’re going to be attached to the cytoskeleton on the other side of the plasma membrane of the cell & this allows those adhesion complexes to adhere cells together into a larger tissue. - Now he’s going to talk more specificity about adherens junctions. - These epithelia are sheets of cells that line our body compartments so it’s the most common architecture in our body, it lines all of our organs, it forms our skin. - Diagram: can see 2 epithelial cells – what you can think of here is these individual cells are sort of like bricks in a wall, they add up together to form this sheet of cells & this separates one body compartment from the other. Here, for example, would be one body compartment, say the lumen of the gut & on the other side of this would be the underlying tissue. The adherens junctions are forming right b/w these cells – this cell is cutting cross-section so this adherens junction is actually forming a ring right around this cell & connecting that cell to all of its neighbours so you can think of these adherens junctions as acting as mortar in b/w the bricks so they’re found all around the cells, connecting all the cells together in the tissue.  Clustered cadherin adhesion receptors Actin cytoskeleton - If we look close up at the molecular structure of these adherens junctions we can see these 2 basic properties of a cell adhesion complex as listed in the slide. - Diagram: So we can see that right over here in this diagram so this is one cadherin receptor in green so it has 5 blobs, a transmembrane domain in the cytoplasmic tail so you can see this one cadherin forms a dimer with another one which interacts with another dimer from the other cell & another one & another one. This is just in 2 dimensions here but this will form a 3D plaque of all of these receptors interacting with one another in a cluster. Then these adhesion receptors then run through the plasma membrane, on the other side they interact with adaptor proteins here in blue & these adaptor proteins link the adhesion complex to actin cytoskeleton shown by these red filaments in the diagram.  Five extracellular domains  A transmembrane domain  A cytoplasmic tail - Here is the structure of the cadherin receptor in more detail, at the molecular level. - So for an adhesion molecule or adhesion receptor to mediate adhesion b/w one cell & another, that receptor has to reach out from one cell & make contact to another cell in the extracellular space. So these molecules, they have to be a transmembrane proteins, they have to cross the plasma membrane so they can reach out into the extracellular space & contact other cells. - Diagram: Here in the cadherin structure, there are 5 of these extracellular domains so here again is one individual cadherin receptor, this is a 2 receptor so this is a dimer of the 2. So if we look in the extracellular space here, we can see 5 of these repeats & each of these repeats has a specific folded structure of the amino acid residues. So this would be the N terminus of the protein, this folds into a specific structure making up that domain & then the C terminus of this then connects to the N terminus of the next one & this is all one continuous polypeptide all the way through to the cytoplasm where the C terminus is. So to run through to the cytosol, there has to be a transmembrane domain then there is a cytoplasmic tail on the inside.  Five extracellular domains  A transmembrane domain  A cytoplasmic tail  Dimers b/w cadherin proteins along the plasma membrane of the same cell - There are 2 key features of this molecule for it to build larger complexes. One feature is that these receptors bind to Ca & they do so in b/w each of these extracellular repeats & that’s what these red dots are in the diagram. The other thing is that they form cis homodimers so these are dimers b/w cadherin proteins along the same plasma membrane of the same cell. So cis means it’s on the same PM. In trans is b/w the cells – it runs across, b/w 2 cells & then the homo here is b/c it is the same molecule. So we have the same molecule interacting along the same PM forming dimers – that is a cis homodimer. So this Ca binding & the formation of these cis homodimers plays a key role in making larger clusters of these adhesion complexes or of this adhesion receptor. - With Ca binding this straightens the receptor up so if there is no Ca present, people have done electromicroscopy studies & have seen that the adhesion receptor is folded over on itself so it’s not reaching out to other cells, it’s not available to reach out & grab hold of other cells. But upon Ca binding this will straighten this molecule up & this then will promote trans homodimerization so if it’s straight & reaching out towards the other side, this will promote a trans interaction b/w the cells. - Diagram: Here we have our cadherin dimer that’s extended out, it then starts to interact in trans b/w its extracellular domains so then we have this one dimer interacting with another dimer & now another cis homodimer can come in & insert right here & then another one from the other side can insert here & so on & walk along & then basically you can view this cis dimer, you can see it almost like a tooth in a zipper so you have the teeth from the zipper zippering up along the plasma membrane producing a large clustered array of cell adhesion complexes. - So this is the idea of creating these clustered cell adhesion complexes from the individual molecules, this increases avidity of these interactions, increases strength of binding b/w the 2 huge cells. - So that’s the basic molecular & cellular mechanism associated with cadherins in terms of receptor binding. Now we have to shift into the cell. - We just talked about the clustering that occurs out here but they also link to actin filaments. This is mediated by these adaptor proteins so they’re called the catenins, so there’s beta-catenin, there’s another called alpha-catenins & these catenins can bind to the cytoplasmic tails of the cadherin & they can also link to actin so that is why they are called adaptors – one part of the complex binds to the cadherin & the other part of the complex binds to the actin so they can connect the receptor to the actin. - In this sort of model right here, this looks rather static but in fact, these adaptors can be quite dynamic & recent work is shown that it’s not really clear how this is working & this is a subject of active research right now in the cadherin field. **Don’t need to know the details of this** - So for example we can see this all connected up to actin, but recent papers have suggested that one of these key adaptors, alpha-catenin may not actually bind to the cadherin & the actin at the same time & it might have to release from this & then bind to actin separately. - One thing that this does highlight is that these complexes can be quite dynamic so you can have a protein releasing from it, binding to actin, maybe binding back to the complex so there is a lot of molecular dynamics going on. - Another thing is that in a developing organism or in our bodies, the cells that are there, we often think of epithelia or tissues as being quite static, however in development these sheets of cells move quite dramatically so it’s a question of how these cell adhesion complexes can deal with that sort of movement. - Diagram: Here we’re looking at a developing Drosophila embryo. We’re looking at individual epithelial cells. And we can see how this sheet of cell moves around in a very orderly way, they’re maintaining contact with one another, it looks like an intact sheet but it’s shifting through a process that we’ll talk about in the last couple of lectures during our discussion of morphogenesis & development. So it’s amazing, it’s being held intact but it’s undergoing this dramatic movement so what’s holding this all together? If you take these embryos & you remove adherens junctions from them by looking at an adherens junction mutant, you see something quite different. - So this is the wild-type embryo, the normal one. - In the mutant, it will look like this to start with but something very different will happen after that. Here is an adherens junction mutant – it starts off relatively normal & it starts to try to extend & then basically the tissues start exploding, the cells start separating from one another, rounding up & that overall organized tissue structure is lost, you don’t see this orchestrated normal movement of the tissue. Basically all these cells just lose contact with one another & are all wiggling around. This illustrates a key role for adherens junctions in maintaining tissue structure & in this case, during development. - Here we’ve taken a normal embryo & experimentally removed adherens junctions from it by looking at a mutant & we see a loss of this epithelial str
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