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Lecture 18

BIO241 Lecture 18

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Jennifer Harris

Thursday, March 12, 2008 - We ended last day talking about collagen, which is one of four types of fibrous proteins, so now we’ll talk about these other three. These fibrous proteins run through the extracellular matrix providing structural support similar to how cables would run through reinforced concrete. - This is the concrete with cables. - We learned how collagen could form these long bundled structures running through tissues, but collagen, its organization can vary with tissue type. You can imagine that for example in a tendon, you may want a whole bunch of collagen fibres to be in parallel to form that tendon structure. Underlying our skin however where it is more of a plane, you would want more of a meshwork of criss-crossed collagen molecules to support our skin. - Different tissues call for different organization of collagen and the organization of fibrous collagen is regulated in large part by these fibril associated collagens so there are different types listed. - Here is our large fibril of collagen and these here are the non-fibril collagen molecules attached to the side. These will decide whether these fibrils will run in parallel or criss-cross structure.  Propeptides after secretion  Fibrils  Surface of collagen fibrils - These fibril associated collagens are similar to real collagen that forms fibrils except these ones retain their propeptides after they are secreted from the cell. We saw for collagen, these collagen molecules that form fibrils have propeptides on them that blocks their formation into larger fibrils before they go outside the cell. Then those propeptides are removed to make these fibrils but for these fibril associated collagens, they are not removed. These ones do not form fibrils and instead they bind to the surface of the collagen fibrils and reshape and organize them in different ways. - That is one way that the extracellular matrix can be remodelled and organized in a specific way.  Protease - Another way is that collagen and other extracellular matrix molecules can also be remodelled is by extracellular proteases. This allows for cells to migrate through the extracellular matrix. - This is a picture right here discussed last day about how this is a fibroblast which is creating these collagen molecules and these fibroblasts and white blood cells and other cells in the connective tissue must be able to crawl around within this tissue. - How do they get through this network of ECM molecules? They can make use of proteases to actually cut these molecules, chop them so they can go through. The action of these protease, this has to be highly regulated so you should note that these molecules, the proteases are secreted from cells in an inactive form, and they’re normally secreted along with inhibitors so you secrete the proteases and also inhibitors of those proteases. Generally you don’t want these proteases to work except in very specific circumstances. If you sent out active proteases into the ECM, it would digest the entire ECM and it would be gone. These inactive molecules are sent out into the extracellular space and normally their activity is controlled by binding to the surface of the cell that wants to use them. These inactive molecules will bind to the surface of the cell & therefore be locally activated & very specifically controlled. You have very specific digestion of the extracellular matrix where you need a fibroblast to crawl through it or a white blood cell, etc.  Proteases - In addition to these cell types, this is also relevant to cancer progression. As we’ll learn about and as we’re all aware, we will have a primary tumour and then as cancer progresses, the cells will start to migrate away from that primary tumour to other sites in the body. To migrate from the primary tumour, those cells also have to travel through the extracellular matrix. They also make use of proteases to do that. - It is important to know about these proteases because there is the potential to develop drugs to block the ability of cancer cells to travel through the extracellular matrix so you might be able to slow down the progression of cancer, stopping them from spreading through the extracellular matrix. - That is just shown in the cartoon. There is a tumourous cell that is growing and metastasizing, moving through the extracellular matrix. It is doing that here because it has these uPA receptors being expressed so therefore it is recruiting this protease called uPA to the surface of the cell and activating it there. Now this cell will have active proteases coating its surface so when it comes into contact with extracellular matrix, it can chop those proteins up and travel through that extracellular matrix. - On the other side, there is an inactive form of the protease that is being expressed, this is competing away the normal protease so most of these receptors on the surface of the cell are now bound to inactive proteases. You can imagine in this case, this is a mutant form of the protease that outcompetes the normal form but you could also imagine that these yellow molecules could be a drug that could be given to a cancer patient. So you can add a competitor of these active proteases that could compete with those proteases for the receptor, basically bump them off the receptors & now this cell here, it may still grow but it will be much more difficult for it to travel through the ECM becoming more contained in the body, preventing metastasis.  (e.g. blood vessels, lungs) - Now onto elastic fibres. These fibres also provide structural support but as the name implies, they are elastic. These are abundant in highly flexible tissues like blood vessels or lungs so that when your lungs expand, they will snap back to their original position; when the blood vessel expands, it will snap back to its original position. - A number of our organs have this elastic property so elastin plays a key role in this because it can recoil after tissue stretching. - In the cartoon, we have the elastic fibre that can be stretched out and then snap back afterwards.  (e.g. blood vessels, lungs) - The main component of this is a protein called elastin shown in green. That is one polypeptide there in the green. The soluble elastin precursor is secreted from cells. This is similar to collagen where you can’t assemble these huge protein cables inside of a cell b/c they're actually larger than cells so they have to be secreted out as the subunits & then form the fibre outside of the cells. - In this case, to form this fibre, these individual elastin molecules are cross- linked together at lysine residues shown in the red dashes. So there are covalent bonds between lysine residues that are connecting these subunits together making a really strong structure which can then stretch and relax. - What we talked about so far is this picture right here & he really likes this picture b/c you can touch your hand & imagine what is under the skin, imagine what is making up the structure of the body, imagine the outer epithelium, the skin, below that you have the epithelium, then you’ll go into the ECM, you may hit a the capillary which is a blood vessel with another epithelial cell layer or you might hit another body organ, you might get to the stomach, you go in & hit the epithelial layer of the stomach, the lumen of the stomach inside, so here you have an epithelium with an apical surface as well as a basal surface. There is another epithelium with apical surface facing the lumen & the basal surface facing this connective tissue. - The molecules we’ve been talking about are these collagen fibres, the elastic fibres, which provide tensile strength to the space in b/w cells & also the GAGs & the proteoglycans that act as space fillers to fill up the tissue & provide resistance to compression. When you look at the cells here too, we’ve looked at the fibroblast, they are the cells that secrete the ECM molecules. We just talked about now how both microphages & fibroblasts can remodel the ECM with proteases to chew it up & travel through the ECM. - For these larger assemblies of cells, the epithelial cells & also muscles not shown in the picture, how do they connect to the ECM? There is a very specialized layer called the basal lamina that sits at the basal surface of these cells, the basal surface of epithelial cells & surrounds muscle cells. This is a specialized thin & flexible ECM, it underlies epithelial sheets or tubes & surrounds cells such as muscle cells & is formed of these components in the slide. - Here is an electron micrograph showing what this structure looks like. Basically an epithelium has been peeled away so that here we see the edge of an epithelial cell right in the slide. This whole thing up there is the epithelial cell, this thing that looks like a sheet is the basal lamina. The epithelial cell sits on it and underneath this basal lamina, we have these collagen fibrils so this is the protein network, the connective tissue underneath the basal lamina. - Now it has been found that if there are defects between these cells and the basal lamina, this leads to blistering diseases in epithelia. The epithelium will lift off the basal lamina creating blisters and defects here also lead to muscular dystrophy where you have detachment of the muscle from the extracellular matrix. So the muscles detach are therefore weakened. - How do cells connect to the extracellular matrix?  Type IV collagen, perlecan and nidogen  Integrin - Here is where we can talk about the last two fibrous proteins and they're intimately associated with cells. - This one, laminin is a link between the ECM and cell receptors. Laminin is a major component of the basal lamina that we just talked about, so that sheet that is sitting underneath cells is full of this protein laminin. - When we look at the protein structure, we can see that it is a heterotrimer, so there are three polypeptide chains here, one red and blue and green. They are wound around each other in this coiled coil domain. As you can see there are sub domains of these polypeptides that can bind to other things. Two sub domains in the slide can lead to further self assembly of the lamina to form larger assemblies of lamina. - Laminin can also bind to collagen, perlecan, nidogen, so these are all components of the ECM. It can also bind to integrins which we’ll talk about and these are receptors on the cell surface. Here is a molecule that can connect proteins of the ECM to receptors on the cell surface.  ECM components  Integrins via an Arg-Gly-Asp (RGD) motif - Fibronectin is also a link between cells and the ECM. - Fibronectin is shown in the cartoon in the slide. It is a heterodimer so there are two polypeptide chains, shown in the slide connected by disulfide bonds between cysteine residues. - This is like other ECM molecules, it is normally fibrous so the fibrillar form will interact with cells but it is also present as a soluble form too. This soluble form is found in our blood serum & it plays a key role in blood clotting with injury so if there is an injury to a blood vessel, the fibronectin that was soluble will convert to the fibrillar form, this will start to block the bleeding & will also allow cells to repair the damage in a wound repair response. - This is a really complicated gene, it has 50 exons and the alternate splicing of those exons can give you many different isoforms of this with many different binding properties. - Similar to laminin, it can bind to ECM components as well as to integrins. Here is another connection b/w the ECM & receptors on the cell surface. When you look at the structure here, we can see the protein is folded into different domains, we see the polypeptide chain and within this region of the chain, it folds into a specific subdomain and in this case, it is important for self-association of the molecule, the next one down is important for binding so collagen and the domain is there that can bind to cells. - We may have heard this before, but there is a relationship between exons and these domains in proteins so here he mentioned that the fibronectin genes have 50 exons so these exons will contribute to this domain structure here, so there may be specific exons then that will encode for this domain in the slide, a different set of exons that will code for another domain, and another set for another domain etc. Through this alternative splicing, the cell may be able to pull out one of those domains and therefore remove one of the binding abilities of the collagen and that is how the collagen can have different properties. If you removed the exons that encode the domain for self association, it won’t self associate as a result. - To follow up on this, just a little bit more, so how would you actually identify what these different domains do? How do you identify the binding partners for the different domains of the molecule? - One way people have done it would be you could purify the whole molecule, and then you could treat this with a very small amount of protease and the protease, because you have this region here that is all folded and protected, the protease will tend to cleave these small little linker regions in between where there is a loose and exposed polypeptide so it will break these domains apart. Now you have these broken domains, you can then isolate those domains and then start to test what they interact with in vitro assays. So for example if you mixed a domain with itself, you can see that it self- associates; if you mixed another domain with purified collagen, that domain will bind to collagen, but another domain will not bind to collagen. Then you can start to see all the different binding partners for this molecule. - An alternate way of doing that is instead of purifying the protein, chopping up the protein, the other way is you can start from the DNA so you can take these exons & clone the exons that only encode this domain & express that domain in bacteria, purify only that small piece of the protein & then test what it can bind to so that is an alternate approach. In this sort of way then, you can walk through the molecule & see all the different binding partners for all the different regions of this molecule & then see that parts of it can bind to ECM components, other parts can bind to receptors on the surface of cells. - You can start to see that this is an important molecule for connecting cells to the ECM. Once you characterize the component that binds to cells is shown in the slide, you can go into the structure. If you have this gene cloned then you can do site directed mutagenesis & delete AA residues. Then you can identify that those 3 AA residues, see how they form a loop out there, this RGD sequence. You can see that those 3 are critical for binding to the cells so if you convert these 3 to alanine, then this protein would no longer bind to cells & through this sort of way, you can identify this sequence binds to cells & that there is a synergy sequence that helps the binding of that loop to cells so synergizes with this loop to promote cell binding. - So those sorts of experiments suggest there are links between extracellular matrix and cells. We saw that we have a molecule that can bind to ECM as well as receptors in the cells. - Here is a picture where we’re looking at fibronectin which is this ECM outside of the cell. In green we are looking at actin so it is inside the cell. What do you notice about the distribution of actin and the fibronectin? They are all parallel. You can see this same sort of pattern between the slide, there are all these little v shapes of the actin and these similar sort of v shapes of the fibronectin. The actin filaments inside the cell are mirroring the fibres of collagen outside the cell. This implies a connection between the extracellular matrix and the inside of the cell. - These connections to the ECM are similar to connections between cells. The cell adhesion complexes that do this had similar properties. - There are interactions with multivalent ECM molecules. All of these ECM molecules are fibrous, multiple binding sites all the way along the fibres so that will promote clustering of receptors along those fibres. This will increase the binding strength because of avidity like we discussed before. - Second, these complexes also have linkages to the cytoskeleton. These interactions, they’re mediated by integrin receptors. Integrin receptors connect to the extracellu
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