Extracellular Matrix

·       Describe protein scaffolds that stabilize membranes and connect cells into communities including extracellular scaffolds

·       Identify the features and roles of collagens, fibronectins, integrins, laminins, and our old friend's peptidoglycans.

·       Describe different roles for basal lamina and extracellular matrix, and relate this to some tissue function

Focal adhesion and hemi-desmosomes

Hemi-desomones connected to intermediate filaments.

Focal adhesion plans connected to the actin cytoskeleton (Reason how filopodia movement starts, it moves outward by polimorizing) In the back in the cell the focal adhesion , moves forward and pull on proteins in th ebak of the celel. They are transient.

In general, actin is more dynamic and so the stability is shorter. The they're also pretty good attaching to just plain plastic somehow nobody planned this but the Petri dish plastic is such that these attachment proteins are usually highly specific form cell cell connections cells like grabbing our plastic and so they will crawl out crawl on your garden drawing Petri dish plastic just fine and they will form so those are the structures just a little bit more detail so here's here's reinforcing this idea that the publication plaques are doing something different than they have been tested zones the yes they both involve immigrants although probably immigrants are a big family of proteins also this cartoon structures that look pretty similar chances are they're not the same here then what the focal hegen plaque perhaps onto on multiple components of the exercise of the matrix these proteins called fibronectin or the load bearing proteins called collagens meanwhile having insurance on the evidence aren't really blue threads but they are much more narrowly focused in the region called basal lamina and that's what every different zones attached to OK so now we get to finish this table off what we should have finished before before test 1 so we've got but what we didn't get through where we sell ECM Kashmir and all this table does we summarize exactly what I've just been through in terms of what they're for yes they're both localized but the folks with these impacts are much more of dynamic and where they are and how long they exist so that gets us through the junctions themselves it also introduces us to several of the players in the extracellular matrix so this is the first attempt to look at they're always integral membrane proteins that cross link to some proteins that are purely extracellular and these always forms some sort of web you know the one shown here this is this is the set of proteins that are involved in the basal lamina we'll come back to it but for now the idea that's important is there are all listed integral membrane proteins that are going to organize this whole process so just to get a little bit more real about how these things look this isn't a light micrograph through I'm not sure complex attitude we have reduced the looking with cartoons with one cell layer and the filling out there can be epithelial layers there multi cells thick but they're still anchored by this relatively thin protein structure here that is the basal lamina so this is just this is just orienting and then below that this stuff called connective tissue is extracellular matrix plus all the cells that would be there so just a little bit of a check.

POLL EVERYWHERE:

Comparing the basal lamina and the extracellular matrix:

The basal lamina is more 2 dimensional than the matrix

As opposed to this thing called hibernation it's probably the most versatile of the crosslinking proteins that we've looked at it here has multiple domains and the dimer is held together by myself by prejudice and it finds all sorts of things it's bone collagen fibers and involved in some blood related interactions heparins are are another class of glycosaminoglycan there are some surfaces there's this whole cell surface receptor domain and is that domain here we go with another recognition sequence there is a three amino acid sequence in the fibronectin RGD that's one of the principal things that recognizes that aren't human proteins that are transmembrane proteins but in general there are lots of these domains and the one thing that isn't apparent these this protein is linear highly flexible and linear and it can form these fibers that are more than one cell in length so visual literacy what do you think the blue dots are can you see the blue dot so visual literacy when you're looking at pseudo stained or stained micrographs like this you see a brown blue thing what do you think it is it's a nucleus that there's a stain I can't remember what D API stands for but Dappy staining is the standard fluorescent dye for standard DNA and so you get these nice brown blue blobs that's probably the nucleus and you can imagine that there's a cell standing around it so you can have very extensive fibers of just fibronectin hence the name but each one of the components that is lost that is a major part of extracellular matrix although it's usually represented as being one of multiple types of crosslinkers rather than this looks like this this area of matrix is mostly private and that's not as read that's not as common to other mixes approaches what the fibronectin connects to is the energy so the integrins are the membrane spanning proteins again this is a dimer actually has got three-part because there's adult disulfide bridge and it holds together the alpha subunit membrane spanning 2 outfits utilities right here the alpha subunit that's what recognizes the RGD on fibronectin there are other proteins in the extracellular matrix also kind of there are also other recognition sequences that that allow immigrants to interact with a bunch of different types of proteins and then finally and we looked at this when we looked at total legion plaques and having desserts owns carries a protein scaffold down here on the side so backing off to the more general factor this is This is why we're doing the extracellular matrix first before doing multicellularity is turns out yes we we are we are pile of cells but we are a pile of cells that have to be organized by a bunch of extracellular stuff and that's true for both plants and animals in ways that surprisingly the the breakdown of the types of roles involved in building the extracellular part of organisms the types of roles are similar there are extra sensors we both have extensive extracellular structures it's our basis this exercise matrix cell plans and cell walls there is in both cases of one or a couple of structural fibers the things that take that don't just pull things together but our to load bearing structures the things that make things stand up the animals it's collagens and patiently lassens patient states there was then this thing called the hydrated matrix we've seen proteoglycans before there are similar things in plants one that there's carbohydrates but there are also glycoproteins that are that are that are going to build the space but also fill it with water and another component so then there are the things that hold these water holding things together depending on whether it's the matrix or the basal lamina 5 fibronectin and laminin so one here is there isn't really a protein that works the same way in the plants these are pectins does anybody here have a family member who makes jams and jellies trying to see some dogs the reason that things gel in the Jelly is because of the fact that context so they are adhesive but they are sort of a a a bridge point that I think is useful for us to think about extracellular matrix in that the extracellular space is like a gel it is full of water and it's structured the way that the way and it can be it can be a very dense gel it can be it can be a very loose gel depending on what part of the Organism is functioning in so but so overall in order to be multicellular you've got to have all of these non cellular parts that basically is basically build build up the structure so that so that we aren't all just basic collections of cells within immediate ship that in order to get the shape we have we need all of this so let's let's look at some of the examples of that one of the most interesting things that sort of brings home the point of how important texture cellular matrix infrastructure the way you make known is by cells making a normal carbohydrate and protein based matrix and then dumping calcium phosphate into it mineralizing but the but the scaffolding up that's going to eventually become bone was made-up of the same types of components that every other exercise likewise if it's that are mineralizing you simply lay down lots and lots and lots of collagen of structural reinforcement you get cartilage and then where we don't have anything quite disorganized we're happy to water filled spaces that are what protect and give give support to the actual cellular based organs that's this thing called connect and the difference here you can see where there where there are cells and how they're organized and both voting and partly just cells in connective tissue can be very widely so most of what's here is non cellular and the cells are not the biggest thing the biggest things in this micrograph those are collagen fibers and they can be way bigger than your average so so and normally I just move on from there and just say add it's connective tissue but that's a that's a word that doesn't mean very much to people so I went out and dug out some anatomy Physiology reference material on what kinds of things are connected tissue well here's this loose connective tissue which is mostly extracellular matrix with some with some fibroblasts around it then they're dense collagen fibers but organized not even not particularly aligned state there are again sheets of collagen that form in dense connective tissue there is connective tissue that's made out of collagen but the lassen more time on that when we get circulatory systems and then of all things adipose tissue fat fat cells are another part of the connective tissue and then there's articulated tissue that gives a more rigidly structured space for things like blood cells either in screening the bone marrow for either storage including development of blood cells in the case of bone marrow so something not to memorize but if you want to look up for your own satisfaction just what all these things are that are connected tissue which is to say a bunch of things that are mostly made of extracellular components with the occasional cell living unit these are all the types and if you go through the list of all of the different organs and tissues and joints and skin that all of these things are involved in that that's how important extracellular structures to the way we function so so rather than rather than just a gas connection yeah it's a lot of other roles for extracellular matrix go beyond the structure this is a tissue culture story of some mammary gland epithelial cells that have been taken out of their normal context in the memory glands grown on a dish that's been stripped of any reliable physical landmarks to grab onto I don't do much they're small they're not they're not very happy on the other hand if you put them in a dish that has had not specifically mammary gland extracellular matrix coded on the base but just any old generic exercising matrix that's enough to stimulate these cells to divide and multiply of but also to differentiate the shape and become secretory glands cells so there's information that's the take away message information in exercising the makers and in the very vague sense this is another seven months or seven months was if you don't have exactly the right protein for one cell interact with it doesn't differentiate properly into its final role so here it's not just the one it's not one protein but it's the whole collection of proteins that make up extracellular matrix that provide the information to these cells as to yeah you should differentiate and adopt the function that you would so information is also a function of expectation come back and show some marketplace another function of extracellular matrix particularly basal lamina they can act as important permeability barriers for things bigger than your technical solutions so this is a basal lamina yellow with the basal lamina occurring between the Disney will vary with between the bloodstream and collecting ducts and eventually we're going to concentrate during so and what that tells you is what doesn't penetrate the basal lamina are big big proteins of salt sugar water will pass through this basal lamina but things like proteins and particularly that blood cells will not pass through but so this allows for it removal of small molecules and eventually their concentration and separation between things like the real mature concentrated in these areas and things like repairs and and ions issue returns department that is the filtration process starts with not a fossil of the viral membrane thought with a with with the basal lamina so and one of one of the indicators of kidney malfunction is a bloody urine or large proteins in the brain which they shouldn't get through this they selected they slowed down in certain types of so that's another function of the exercise so here in place actually what I really should do and I'll do this after class is go through this slide and just put big X's through the things you don't have to keep track of there are names for a bunch of these proteins like apparel and declaring and advertising OK but they're also pretty locked in the only one I really is we're going to spend some time on how we ever gets there because it's so big volume the fibrous proteins yeah you got to know your college or at least these two type 4 collagen is in basal lamina fibrillar collagen matrix glycoproteins again I'm going to go through the nitrogen is an important crosslinker but its role only matters after the laminates fiber reactants hub gumbo really work of setting up and so so this is this is more vocabulary than me but it certainly is it does organize things as the premium black and but and plutonium bike in the fibrous proteins and glycoproteins glycoproteins are also smaller and particularly this is a single collagen trimmer it's going to assemble into a fiber way bigger than any of these privacy and that's all that we're seeing now so here it is enormous so here's our our initial public 3 polypeptide chains that will never reach 1 college and fiber that collagen fiber optic gets in gets assembled into should call these volumes because the biggest thing is a fiber the intermediate stage or fibrils which are collections of individual collagen triple people and one of the interesting things about these they're always shown with these stripes that's not just an artist getting bored if you take electron micrographs they have differences in electron density and actually the the diameter of the fiber changes just a little bit sometimes there are sometimes wider that all comes from the fact that these fibers like intermediate filaments have overlapping assembly so we have one college molecule assembling into one of these fibroids the fibrils are made-up of lots of molecules and they all are stacked offset So what the starting point is so you never see collagen as a single polypeptide functionally by the time that the we're changing what's done with it it is a triple Helix nothing about that Helix it's not a typical alpha Helix because it has protein in it and as you might remember from way back in August pro doesn't form out felices well so instead it of the Helix has to bend around the building and the best amino acid for bending is glycine because it's only got a hydrogen as its argument so this is your triple healings of of the individual collagen molecule few more and what happens to addition to assemble the triple Helix that all happens in the ER then it gets created let's get trimmed off and we'll see why in a minute and then you have all the copies these college military ready to be assembled it assembles in these offset fashion with the people that particular regular spacing and and that offset has places where it's thicker and where it's thinner and so you end up with these stripes of either more dense protein or less dense protein going from A5 grill to an entire college environment so these things that really really big once they get outside the cell now why outside of the cell because these fibers can be bigger than individual cells so you wouldn't want to ask the poor angioplasty in particular to have these things assemble and polymerize to the point where they poke out the ends of this thing so that's why as far as it gets it in the ER is the pro collagen pieces this is true for most of the types of collagen mostly long fibrils sometimes they're interrupted sometimes they're feeds vote but wherever you find them on the one we're interested in basement membrane matrix basal lamina hardly growth plates and then there are some minor ones like this one in the skin that's transient you have to memorize when you're asking yourself how do you end up with all of these think about all of the different twists and turns and different ways and organs come together and have different joints come together how do you get them all to fit in the shapes they are because there are multiple types so for more detail on how college should assemble it it is this triple T links with license and proteins and also hydroxyproline so the hydroxyproline is there two reasons one that turns this nonpolar amino acid into at least a slightly polar one but it also adds back the hydroxyl group that is chemically active and when one collagen fiber assembles into into a bigger project structure they're struggling and that cross linking goes through these cycles and there's one for one further amino acid modification that happens extracellular events also there's hydroxylysine so here here's here's the aspiration why you don't assemble college and completely inside of them but here's a fiberglass here's here is a bundle up here's a collagen fiber bundle and you can see it's longer than the cell it would be even longer than this these this micrograph as a whole the slice of tissue that was made accurately followed this Bible that it would continue out this way and out the other way multiple settlements so these things are beautiful they're also shown here they're also oriented in multiple directions occurred a collagen fiber that's that is oriented this way that has been sliced through and you spend you see all the individuals liberals in cross section so these things are really big which is why the assembly process works this way in the ER review for the last Test everyone that you have signal hypothesis assembly of the protein you have 2020 constellation happening here you're actually problems here you have been you have protein non protein disulfide mesoloras you eventually get a fully folded protein that has the triple Helix and but also these and those end pieces prevent the assembly of the larger fiber structure so as long as these end pieces you can see they're disorganized they're also captured so that they can't interact with then once they're secreted you get out into the extracellular matrix several things happen the end pieces get trimmed off that allows the collagen to begin to fold up into a full fiber it also is where the crosslinking and so several important enzymes exist extracellularly 2 proteases the names of which you don't need to know that these scissors are indicating these terminations that turn off all the excess annual assets on both ends now allowing for the individual college molecules to first cross them by drills and then ultimately the fibers of that process has both cross linking within a single triple Helix and cross linking between them and this also shows this offset assembly so that the overlaps are incomplete and that causes the structure of collagen and it becomes a fiber to have this strange character the cross linking the capacitor is the hydroxyproline and hydroxylysine so but if you're fan of ending with the Pirates of the Caribbean movies remember anybody getting called the scrutiny dog anybody know what disturbing is yes sometimes it's something that everybody because they have a deficiency yes it's it's a it's a vitamin C deficiency syndrome and this diagram explains pretty much all scurvy is a lack of vitamin C leading to the inability of the extracellular collagen to have it lysine converted to hydroxyzine if you don't have hydroxylysine you don't get the proper cross linking that means bone self corn properly the lining of the organs don't assemble properly I've never I'm sure one can look up on the Internet dreadful medical photographs of people with curly but this is this is the case where you it's traceable but so ascorbate also known as 100CC ascorbate donates the electrons that carries out the reaction that turns light into hydroxy mice and also proline to hydroxyproline and that that allows the those two amino acids to cross them with each other so it was interesting when you have a global organismal level phenomenon that you can trace back to exactly 1 chemical and it is this process that is missing in individual serving because of the laptop 1st the pepper and green is the original fibronectin illustration but it's I've not seen any really good visualizations of exactly where fibronectin is in the extracellular matrix this is a good cartoon doctor 55 and active because it has RGB sequence that causes it to be bound by integral does tend to be fairly close to the plasma membrane surface but then because it has all these other interactions it connects outwards into the extracellular matrix and the other thing that's not shown here is fibronectin connects to itself and so there are there can be longer polymers and fibronectin interacting all the way out to the outside edge of the exercise the other thing shown here are the proteoglycans and collagens and this thing called elastin which we're going to spend more time on when you get to the circulatory system but the idea is a part of the extracellular matrix at least in some types of connectivity so here we're back to this track of fibronectin so it looks like there are fibronectin fibrils here and it looks like cells are aligning themselves to those fibers that can be experimentally validated so we have some cells and tissue culture here that somebody decided to run more by painting of the fibronectin protein on this patch that's that's delineated by these two lines so within these two lines somebody came along with basically no actually paintbrush and painted on her fiber napkins on an otherwise conventional cell culture plate and pointing the cells the cells clearly are interested in forming connections with fibronectin so fibronectin is as would be suggested by this micrograph fibronectin is a track for cells to follow if they're trying to migrate to a specific location during development or response to some sort of insult so having mentioned the lesson for those types of connective tissue that need to be you need to be able to bounce back and need to be able to stretch and recover the last thing is a major component it's a protein that I like those proteins where from where dynamically the folded up state is far far more stable than an unfolded state Alaskan can exist in two states that are only slightly different from what I'm doing if you put just a small amount of torsional stress on the last thing they will stretch out into therefore they're still crossing but if you can take that force away they will recover they they will relax by scrunching up and that's an important capacity for a tissue that needs to be able to go to stretch and recover and one of the places that's and it says we'll get back to that so that was the fibronectin glassons we now we're going to move close to cells and look at the structures of the basal basal land based labor organizer muscle muscle tissue in surrounding connective tissue here's how old scrapping the aphelia layer here's the kidney code Mary list that but they're all relative to the size of the cells that he organized with their relatively thin and and relatively prudential why that is has to do with the principal protein that assembles them this thing called laminate it's another primer that has multiple arms that come off with these arms have different binding sites or extracellular matrix interactions they also has this hooked tail which is how lavens interact with integrates so this is not an RGD binding site it's another set of amino acids but it does form a highly specific orientation between the integrants coming up through the membrane and the laminates extending sort of an angle out away from them but not one laminate at a time but lots of laminates that are going to cross link with each other through some of these arms of the lemons so that you're going to get a sheet coming off the surface itself and here and that she is going to be principally made-up of immigrants connecting the laminates you're going to add in some of the type 4 collagen and other small connecting molecules that the basic structure all the basal lamina comes from the structure of the land and the fact that it's rigid the factors planner and what kinds of interaction it creates with other laminates and within groups so that the way that the reason the basil lamina is not very wide and it's a flattened sheet is because of the nature of the land and that's in contrast if I were accounts where these things are incredibly flexible they can bend all sorts of directions that could be around for a whole bunch of different things that's why they are the component that is organizing the extracellular matrix that's not the basal lamina so this is the sort of thing that leads directly to a wonderful compare and contrast questions about telling me why the structure of laminins is is gives lamina the properties the properties of basil lamina has and why fibronectin are an essential component to why there are extensive extracellular matrix can be much thicker and also much more varied incomplete construction so so compare and contrast OK So what but back to the base of Labor itself this this is another graph showing that the filial cells that have been peeled away from the base over here this thing looks solid there are those are actually proteins there are probably at a level we can't see there probably are holes that are permeable but overall this thing really is a much more will hear and sheet of macromolecules as opposed to out here we have extracellular matrix that's mostly collagen fibers and and there's much more spacing between them so is this another vacuum is it is this the electron micrograph making its own version of a bad artist moment there's water in there that this is one of the most hydrated spaces that that and also probably in the process of preparing the specimen some of the smaller carbohydrate structures got lost but in general exercise of the matrix is more loosely organized just like paper napkins lemons are a source of information this is a this is a basal lamina that that's at the edge of an epithelial layer and along that edge there are cells so they somehow so they could crawl in and we're like on this micrograph where they're crawling on an exposed basement so obviously there's information in in the lab in the lab and proteins and the other proteins and proteins and one of the things is captain come back to you umm most of these proteins like the the huge list of colleges these are a gene family there are multiple laminations and so there could be multiple paths in the organization this is from every genesis where there's the embryo proper and then there's this embryonic cell body and here at certain points in development some of these cells migrate into the embryo the erythroid cells in other words the cells that are going to make those those the cells that make up the blood they're going to migrate through bone marrow some of these the thymus the lymphoid cells that are part of that they're going to make up the lymphatic system and they've been screening and refine us and other parts of the of and this this other parts of the lymph system and the procedural germ cells is one of the strangest things that hit early in development for whatever reason cells that are going to preserve their capacity to do meiosis they pack up and they get out of hand and they hide in this actually embryonic space until the gonads begin to differentiate and then they migrate but each one of these colors of cells migrates along a different type of laminate together so just like if I remember the laminate provides information and organization or the surroundings so yeah we're not going to try to take up all of the all of footage