Lecture 8 2025 Cell Junctions PDF

Keywords from lecture 7 Collagen and elastin Fibronectin RGD domain Integrins Outside in and inside out signaling FAK Velcro Principle Anchorage dependance Are Hemichannels usual...

Keywords from lecture 7 Collagen and elastin Fibronectin RGD domain Integrins Outside in and inside out signaling FAK Velcro Principle Anchorage dependance Are Hemichannels usually kept In the closed or open conformation? 1 Many integrin connections = focal adhesion, need to be able to make and break them, FAK is responsible for breaking them 1  Lecture 8 Cell junctions, Cell adhesion, and the ECM 2 2  Of all the interactions between cells in a multicellular organism, the most fundamental are those that hold cells together. Cells cling to one another through cell-cell junctions, or through the ECM. Attachments to other cells and to the ECM control the orientation of each cell’s internal structure. Defects in the apparatus of cell junctions, cell adhesion and ECM underlie many diseases. 3 We are going to continue talking about the interactions between cells 3  How do cells that are small, often motile objects filled with an aqueous medium and enclosed in a flimsy plasma membrane combine into strong massive, stable structures such as a horse or a tree? Two basic building strategies: 1) The strength of the ECM, and 2) The strength of the cytoskeleton within the cell and cell-cell adhesions that tie the cytoskeletons of neighboring cells together. 4 The cytoskeleton of one cell is linked to the cytoskeleton of the neighbouring cell and this provides strength 4  Animal tissues are extraordinarily varied, but most fall into one or other of two broad categories: 1) Connective tissues, 2) Epithelial tissues. Connective tissues such as bone and tendon have abundant ECM and cells are sparse within it. The matrix is rich in fibrous polymers such as collagen and it is the matrix rather than cells that bear mechanical stress. Epithelial tissues such as the lining of the gut or skin, cells are closely bound together into sheets called epithelia. The ECM is scarce, consisting mainly of a thin basal lamina. Cells are linked via cell-cell adhesion and stress is dispersed this way. 5 5  6 Figure 19-1 Molecular Biology of the Cell (© Garland Science 2008) On the top we have our epithelial cells, they are polar, apical side would be on top and the basal side would connect to the basal lamina. Below it we have connective tissue, we have all the fibrous proteins, collagen fibers, fibronectin and other proteins that are secreted by the fibroblasts and mast cells present in the connective tissue. So the actual strength of the layer of epithelia comes from the cytoskeleton that are linked via cell to cell junctions and this is what we are going to focus on today. 6  Physical attachment is critical both in epithelial and in non- epithelial tissues, but junctions between cell and cell or cell and matrix are structurally diverse. Four main functions can be distinguished, each with a different molecular basis: 7 7 1) Anchoring junctions: includes both cell-cell adhesions and cell-matrix adhesions, transmit stresses and are tethered to cytoskeletal filaments inside the cell. 2) Occluding junctions: seal the gap between cells in epithelia so as to make the cell sheet into an impermeable (or selectively permeable) barrier. 3) Channel-forming junctions: create passageways linking the cytoplasm of adjacent cells. 4) Signal-relaying junctions: allow signals to be relayed from cell to cell across their plasma membranes at sites of cell-cell contact. 8  Desmosomes Tight Junctions Gap Junctions Notch Delta 9 Figure 19-2 Molecular Biology of the Cell (© Garland Science 2008) Anchoring junctions anchor the cells to each other and then anchor the cells to the ECM. Like the desmosomes, they anchor intermediate filaments, these are all oriented towards the basal side connecting to the basal lamina or connecting two adjacent cells. Tight junctions are an example of an occluding junction and it is their job to ensure nothing leaks through the epithelial layer, and they are generally found along the apical side of the cell. The channel forming junction, like gap junctions are usually closer to the basal side of the cell, or at least underneath the tight junctions and they allow the transfer of small molecules like ions between the cells. Our last type is the signal relaying junction and the best example for that is the Notch/Delta pathway, where we have a membrane bound ligand on one cell that interacts with the membrane bound receptor on another cell and transmits a signal to both of the cells. 9  10 Table 19-1 Molecular Biology of the Cell (© Garland Science 2008) When we are anchoring cytoskeletal elements between 2 cells, the 2 main filaments we are anchoring are actin filaments and intermediate filaments. Today we are going to talk about the adherens junctions. With IF we have desmosomes or hemidesmosomes – if one on each cell connect to each other there are 2, but if connecting to the basal lamina there would only be one (hence the “hemi”) 10  Cadherins and cell-cell adhesion The structures of cell-cell adhesions are most clearly seen in mature epithelia that are held together by strong direct anchorage of cell to cell. 11 Figure 19-3 Molecular Biology of the Cell (© Garland Science 2008) Cadherins mediate a specific type of cell to cell junction – these are called adhernes junctions, this is the junction that link the actin cytoskeleton between 2 cells. Notice the placement of each of these types of junctions relative to each other and the apical or basal surface. Linking the cytoskeleton to the ECM is always done at the basal side of an epithelial cell, actin through the integrin signaling and IF through the hemidesmosomes 11  Anchoring junctions At each of the 4 types of anchoring junctions, the central role is played by transmembrane adhesion proteins that span the membrane, with one end linking the cytoskeleton inside the cell and the other end linking to other structures outside it. The cadherin superfamily mediates attachment of cell to cell. The integrin superfamily mediates attachment of cell to matrix. 12 12  13 Table 19-2 Molecular Biology of the Cell (© Garland Science 2008) Desmosomes mediate their interactions using a special class of cadherins but this gets confusing, so when I talk about desmosome interactions I will say that and when I talk about cadherins I am referring to the classical cadherins that are associated with the adherens junctions Same idea with the integrins, when I am talking about integrins I am referring to the proteins that link the actin cytoskeleton of one cell to another cell. The hemidesmosomes use a special class of integrins but I will not refer to them by this name and just use “hemidesmosome” when I am referring to the intermediate filaments of one cell being linked to the basal lamina. 13 Cadherins mediate Ca2+-dependent cell-cell adhesion in all animals Cadherins are present in all multicellular animals. Other eukaryotes including fungi and plants lack cadherins. They are also absent from bacteria and archae. Therefore, cadherins seem to be part of the essence of what it is to be an animal. 14 14  The cadherins take their name from their dependence on Ca2+ ions: removing Ca2+ from the extracellular medium causes adhesions mediated by cadherins to come apart. Virtually all cells in vertebrates, seem to express one or more proteins of the cadherin family, according to cell type. 15 If Ca2+ is present they all stick together, if absent, they all fall apart 15  The cadherin superfamily in vertebrates includes hundreds of different proteins, including many with signaling function The first 3 cadherins that were discovered were named according to the main tissues in which they were found: E-cadherin on epithelial cells N-cadherin on nerve cells P-cadherin on cells of the placenta. These, and other classical cadherins, are closely related in sequence. Their adhesive functions are complemented by signaling functions that relay information to the cell interior. 16 These aren’t exclusive- E-cadherin is not the only cadherin on an epithelial cell, you have an N cadherin on an epithelial cell (just in lower abundance) The shift in relative abundance is important for differentiation and for organization in the cell 16  Cadherins mediate homophilic adhesion Anchoring junctions between cells are usually symmetrical: if the linkage is to actin in the cell on one side of the junction, it will be to actin in the cell on the other side. 17 Figure 19-8 Molecular Biology of the Cell (© Garland Science 2008) Like binds like This is depicting with one cadherin linked to actin bound to an identical cadherin linked to actin. On the right we have 2 cells expressing different types of cadherins and this does not happen. And this specificity allows for the organization and formation of tissues because like-binds like. 17  18 Figure 19-9a Molecular Biology of the Cell (© Garland Science 2008) The dependance on Ca2+ comes from this flexible hinge region. We have 2 cells, we have the PM on the right and left. Connecting them in between we have 2 cadherin molecules. Focusing in on the hinge region we can see these Ca2+ binding sites. In order for this hinge to be in the locked conformation, Ca2+ has to be present, 18  19 Figure 19-9b Molecular Biology of the Cell (© Garland Science 2008) That is represented here in this cartoon, when we have calcium present at greater than 1mM it promotes this rigid structure by binding to those hinge regions. But if it drops below 1mM we lose binding to all these sites and the cadherin folds in on itself and it cannot interact with the neighbouring cell 19  20 Figure 19-9c Molecular Biology of the Cell (© Garland Science 2008) We employ a Velcro principal here, instead of having one really strong interaction, we have multiple interactions that are lower in affinity. Benefit- this cell, which could be in a blood vessel, may get injured or may need to migrate, it needs to be able to make and break these connections readily. 20  Unlike receptors for soluble signal molecules, which bind their specific ligand with high affinity, cadherins typically bind to their partners with relatively low affinity. Strong attachments result from the formation of many weak bonds in parallel. Many cadherin molecules packed side by side collaborate to form an anchoring junction. The strength of this junction is far greater than that of any individual intermolecular bond, and yet it can be easily disassembled by separating the molecules sequentially, Velcro Principal 21 Just like integrins and focal adhesions 21  Selective cell-cell adhesion enables dissociated vertebrate cells to reassemble into organized tissues Cadherins form specific homophilic attachments, and this explains why there are so many different family members. Cadherins are not like glue, making cell surfaces sticky. Rather, they mediate highly selective recognition, enabling cells of a similar type to stick together and to stay segregated from other types of cells. This phenomenon occurs when disassociated cells from two embryonic vertebrate organs, such as the liver and the retina, are mixed together. The cells in the mixed aggregate gradually sort out according to their organ of origin. 22 If you need tissue to be strong and bound to each other, but you don’t want your liver to merge with your pancreas, there has to be different family members to give distinction 22  Studies with cultured cells support the suggestion that the homophilic binding of cadherins controls these processes of tissue segregation. Experiment: In a line of cultured fibroblasts called L cells, cadherins are not expressed and the cells do not adhere to one another. When these cells are transfected with DNA encoding E-cadherin they become adherent to one another, and the adhesion is inhibited by anti-E-cadherin antibodies. Since the transfected cells do not stick to untransfected cells, we can conclude that the attachment depends on E- cadherin from one cell binding to E-cadherin on another cell. If L cells expressing different cadherins are mixed together, they sort out and segregate. 23 23  Disaggregation – Reaggregation experiment Red – Ectoderm, Green – Mesoderm, Blue - Endoderm 24 Figure 19-11 Molecular Biology of the Cell (© Garland Science 2008) This is a representation of what we just talked about, but we are going to use the context of development. We have E and N cadherin expressing cells mixed, they will sort themselves and completely separate (because like binds like), but what if we have a group of cells that express high levels of e cadherin and the other group of cells has only a little bit of E cadherin? This is reality, as I mentioned most cells express a few different cadherins but will have a “dominant” one that is expressed. What will happen is that the tightest connections will form when there is the most amount of cadherin- the more we have, the tighter the binding, like the Velcro principle – the more connections, the stronger the bond. So the cells with the strongest connections through E cadherin will form tight bonds and come to the center, while the cells with less E cadherin will form a ring around the center ball. This is essentially a very fundamental principle that helps with gastrulation of the 3 layers. The different layers will have different expression of various cadherins. It is the respective expression that allows us to generate this pattern of gastrulation in development. But you do not want the tissues to be completely separate not able to adhere or communicate at all, this about mesoderm – giving rise to muscle and ectoderm giving rise to skin and the nervous system, you cannot have tissues of the skin completely disconnected from the muscle, they need to be able to interact. They achieve this by expressing different levels of specific cadherins. 24  25 Figure 19-11 Molecular Biology of the Cell (© Garland Science 2008) This is the developing neural tube- gives you the central nervous system -spinal cord and brain. On the top we have the neural crest- it gives rise to the peripheral nervous system. These cells that will become the neural crest cells need to migrate out of the neural tube, so they will migrate out, they will proliferate, then they will begin to migrate and differentiate 25  Cadherins control the selective assortment of cells The appearance and disappearance of specific cadherins correlate with steps in embryonic development where cells regroup and change their contacts to create new tissue structures. 26 Figure 19-12c Molecular Biology of the Cell (© Garland Science 2008) What happens during development you start with this layer of ectoderm that has high expression of E-Cadherin, from this layer we get an infolding of the ectoderm that will eventually develop into the neural tube, once the ectoderm folds in we see a change towards higher expression of N-cadherin in those cells (down regulate E- cadherin). At the junction of the fold you see a different expression of cadherins, you see an upregulation of cadherin 6B so what this does is it lets this inward invagination actually fuse (because like binds like) so what we can do is have the neural tube close, and yet be somewhat separate from the cells above. We still maintain a separate population of cells (in green) that can migrate out, differentiate and change their cadherin expression to have high levels of cadherin 7 to form the neural crest cells and the peripheral NS. By regulating the up and down regulation of different cadherins allows us have this differentiation during development. 26  https://www.youtube.com/watch?v=1b9LLvlt MNk 27 Here we have a cell that is expression E cadherin-GFP, You can see the adherens junctions making and breaking as the cells move 27  Adherens junctions are an essential part of the machinery for modeling the shapes of multicellular structures in the animal body. The prototypical example of adherens junctions occur in epithelia where they form a continuous adhesion belt. 28 28  Cadherin 29 Figure 19-14 Molecular Biology of the Cell (© Garland Science 2008) The cadherin links to the actin cytoskeleton through accessory proteins – particularly some cadherins. 29  30 Figure 19-15 Molecular Biology of the Cell (© Garland Science 2008) When we have a big cluster of cadherins, each one linking to an actin filaments and that linking these actin filaments linking to the neighbouring cells actin filaments, that creates a structure that we call an adhesion belt, this is a bridge of actin across all this epithelial layer that helps give it strength and helps it move in unison 30  31 Figure 19-16 Molecular Biology of the Cell (© Garland Science 2008) The adhesion belt is very important in forming anything that has an epithelial sheath, like the ones lining a blood vessel. You can see here This is multiple epithelial cells all linked through adherens junctions (actin), you can have a pressure that causes an invagination of this layer, all of these cells will still remain linked together. And with the expression of the appropriate cadherins we get pinching off of this fold to form a separate structure, an epithelial tube- this is how we make the neural tube but also how we make blood vessels. 31  Cell-cell junctions send signals into the cell interior. The making and breaking of attachments are important events in the differentiation of cells and provoke large changes in their internal affairs. There is complex crosstalk between the adhesion machinery and chemical signaling pathways. β-catenin 32 32  33 Figure 15-77 Molecular Biology of the Cell (© Garland Science 2008) If you need stable beta catenin to be involved in specific junctions with cadherins, it make sense that WNT would active genes involved with cadherins (like the cadherin itself) 33  Desmosome junctions give epithelia mechanical strength. They are structurally similar to adherens junctions but link to intermediate filaments instead of actin. The particular type of intermediate filaments attached to the desmosomes depends on the cell type: they are keratin filaments in most epithelial cells and desmin filaments in heart muscle cells. 34 34  35 Figure 19-17a Molecular Biology of the Cell (© Garland Science 2008) They work in a similar manor, you can see IF clustered to this dense localized anchoring proteins and then a similar linking structure that uses the Velcro principle to link to another cell 35  36 Figure 19-18 Molecular Biology of the Cell (© Garland Science 2008) What you end up with is the joining of these strong, rope like structures. But what is different here compared to the adherens junctions is that this does not create a belt across cells because these are not polar structure, they are rope like structures, they can go in multiple directions across the cell 36 1. Define: Anchoring junctions that connects actin filaments in one cell to those in the next cell. 2. True or False. Cadherins promote cell-cell interactions by binding to cadherin molecules of the same subtype on adjacent cells. 3. Overproduction of cadherins such as E-cadherin … a. is often found in cancers originating from epithelia. b. is induced by the transcription regulatory protein Twist. c. induces epithelial–mesenchymal transition. d. leads to stronger cell–cell adhesion. e. All of the above. 37 37  8) Sam, the undergraduate working with you in your lab, has just asked for help. You and Sam are investigating the role of the b-catenin protein at adherens junctions. Your adviser has asked you to clone the b-catenin gene by PCR. When Sam took the PCR clone you produced and sequenced it, he found a mutation that would change a conserved arginine to a methionine. When Sam ran the b-catenin protein through some web-based protein domain-finding algorithms, he found that this arginine was a conserved residue in a predicted nuclear-localization signal (NLS) and thus figured it did not matter that much because the b- catenin at adherens junctions must be a cytoplasmic protein. To Sam’s dismay, your adviser was horrified at this news. Explain why a protein localized at an adherens junction might need an NLS. Are Hemichannels usually kept In the closed or open conformation? 38 Beta catenin has a dual role, it is not just in this cell cell junction, it is also involved in WNT signalling and in the nucleus it activates the transcription of cadherin genes 38