CSB331 Lecture 2 Notes

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Cell and Systems Biology
Katherine Sodek

CSB331 Lecture 1 Notes (January 11, 2012) – Cell-Cell Adhesion: Adherens Junctions (Cadherins) 5 – Two basic tissue types: epithelial and mesenchymal  Tissues are made up of cells (aqueous bags), but how do they give strength?  Two strategies: o Connective tissue: Cells make a lot of the ECM that bears the stress (for tissues like bone, tendons, dermis) o Epithelial tissue: Cells themselves through their cytoskeletal filaments that bear the stress and those cytoskeletal filaments must be anchored between cells so they cross the whole epithelia (their mechanical role is one of many roles though) 6 – Cytoskeleton and cell junctions in polarized epithelial cells  Three types of junctions:  Occluding junctions o Tight junctions – regulate what can pass through epithelia  Cell-cell anchoring junctions anchors between cells o Adherens junction – link actin filaments between cells o Desmosomal junction – link IF between cells  Cell-matrix anchoring junctions o Hemidesmosomes – anchor IF o Integrin – anchor actin filaments  Note that hemidesmosomes are a special kind of integrin. 7 – Resolving power for light versus electron microscopy  Every window is 10 fold more magnified.  20 mm is the size of thumb.  20 μm is the size of average animal cell (1000x smaller than thumb).  20 nm is the size of cell-cell junction (1000x smaller than animal cell).  Can see down to a certain size with LM (subcellular organelles, mitochondria, bacterium) because the resolution is limited due to the wavelength of light.  Cannot see cell-junctions with LM, you need EM. 8 – Cell junctions can be visualized with electron microscopy  Desmosomal junctions seen well in epithelium because dense and distinct.  In some cells, junctions aren't seen because they are smaller but it doesn't mean they are not important. Must use molecular biology and biochemistry techniques to study functions. 9 – Cell junctions in vertebrate epithelial cells  Cell-cell junctions are symmetrical meaning that in one cell, the actin cytoskeleton of one cell connects to the acting cytoskeleton in the next cell. This is the same for intermediate filaments. You never see intermediate filaments on one side and actin on the other side. This is because of the specificity of the transmembrane proteins for the cytoskeletal filaments and what they are binding outside the cell. 10 – Four types of anchoring junctions  The cadherin family looks after both types of cell-cell junctions. The cadherin family is a big family. We are looking at the members of the cadherin superfamily that mediate adherens junctions called the classical cadherins. 11 – Schematic representation of the cadherin superfamily  The two cadherins we are doing are not the only members of this family. The two cadherins are well studied and important in cell-cell adhesions. A lot of the work that has been done on these adherens junctions have been done in Drosophila because they have adherens junctions and can be visualized with fluorescence microscopy. The classical cadherins mediate adherens junctions. 12 – Some members of the cadherin superfamily  E-cadherin is a hallmark of epithelial tissues that strongly regulates the epithelial phenotype.  N-cadherin originally found in neurons but expressed in many cell types. 13 – E-cadherin expression confers the polarized epithelial phenotype  E-cadherin gives epithelial cells their characteristics (apical and basal polarity). Because E- cadherin is so important for the epithelial polarity and the formation of other junctions, if you remove E-cad then it can cause EMT. If you express E-cad in mesenchymal cells by overexpression then they can adopt epithelial phenotype. 14 – Structural organization of the ectodomain of classical cadherins  For all classical cadherins, they all have 5 extracellular cadherin repeats.  Each have Ca2+ binding domains where several Ca2+ bind.  The region where they bind is called the hinge region.  The Ca2+ bind between E-cad repeats in hinge region. 15 – Classical cadherins show homophilic binding  The classical cadherins mediate homotypic adhesion.  They are homophilic.  T each type only wants to bind its own kind (E-cad only binds E-cad, not N-cad or any other cadherin). 16 – Knob and pocket model for cadherin interaction  How is homotypic adhesion possible?  EC1 is the furthest out cadherin repeat.  EC1 mediates selective binding.  Each outer domain new the N-terminal have a knob and pocket. They are arranged so that when they interact with another classical cadherin in trans, the knob of one fits into the pocket of the other.  The hinge region is shown in detail where the Ca2+ is binding.  Ca2+ binding in hinge region is thought to give a structure that subtly affects the structure of EC1 domain. If you remove Ca2+, the structure of outermost domains change and lose affinity for each other. 17 – Cadherin-mediated adhesion is calcium dependent  If you have less than 0.05 mM Ca2+, it will collapse. Normally, we know Ca2+ is higher extracellularly at mM. Ordinarily, the rigid structure is maintained because there is plenty of Ca2+ outside the cell.  Intracellularly, however, Ca2+ is lower because cells use Ca2+ as a signalling molecule. They regulate how much is in the cell and keep it low until a signal needs to happen, they let let Ca2+ flood in to get intracellular signalling. This does not happen outside the cell. Cells get rid of Ca2+ by sending it outside the cell. Extracellularly, Ca2+ concentration is high enough for Ca2+ always to be bound.  Cells growing on a dish and you want to lift them off the dish. If you artificially remove Ca2+ by adding chelator, then cells will be released from each other. When cadherins are collapsed over, because it is not their natural structure, they are also more susceptible to being cleaved by proteases which helps lift the cells off the dish.  If you remove Ca2+, the hinge regions collapse and fold over. The whole molecular which used to be a rigid rod ready to interact) collapses and unable to make interactions. 18 – Lateral (cis) interactions stabilize cadherin associations  Before we were looking at a monomer. This is a model for how cadherins are believed to make their interactions. They interact in trans for sure. It has been proposed that to be more stable, they first interact laterally (cis interactions) and upon clustering, they are more stable and able to interact in trans.  More than just the EC1 domain is interacting here. In this model, EC1 domain is needed for selectivity because of the knob and pocket model. It decides whether the interactions are made in the first place. It is possible that later on, they settle into more overlapping interacting structure. 19 – A typical junction, many cadherins are arranged in parallel, functioning like Velcro to hold cells together  Each one of the interactions is not very strong. You need lots of cadherins to come together and make bridges between cells to get a strong adhesive structure. It has been compared to Velco. Cells are well tethered together and has a strong structure if you have lots of cadherin molecules together making the adherens junction.  The distance between the plasma membranes is approx. 20 nm. 20 – Transmembrane adhesion proteins use intracellular anchor proteins to link the cell cytoskeleton to exterior structures  We just talked about the ectodomain (outside the cell) of classical cadherins.  For most cell-cell adhesion processes, the intracellular or endodomain does not interact directly with actin or IF. They have intracellular anchor proteins that connect cytoplasmic tail of transmembrane receptors (classical cadherins) or endodomain to cytoskeletal filaments. 21 – The linkage of classical cadherins to the actin cytoskeleton  What does the tail associate with?  For classical cadherins to associate with actin cytoskeleton, they need to have linker proteins. They need β-catenin which links to another anchor protein called α-catenin. It goes from tail to β-catenin to α-catenin and other anchor proteins to the actin cytoskeleton. 22 – The cadherin-catenin complex  Showing the ectodomains interacting.  β-catenin links to α-catenin linked to actin.  Technically, α-catenin can interact directly with actin, but when it is bound in an adherens complex to β-catenin, α-catenin binds to actin through the other proteins. Vinculin is thought to be involved in mechanosensing.  When a cell's adhesion molecule like E-cad or when it is associated with another molecule, the stress is sensed. It can tell whether it is attached and transmit that information to the cytoskeleton on the inside and vice versa. 23 – The linkage of classical cadherins to the actin cytoskeleton  If force is exerted on these cells, another molecule gets activated and phosphorylates β-catenin that causes it to dissociate and go to the nucleus and turn on genes involved in making colon cancer cells more invasive. It also goes backwards as well. When epithelia are remodelling, there are actin rearrangements. That information by one cell rearranging its actin and pulling on the junction will affect the cell beside it and that is how you get coordinated behaviour between epithelia.  When forces from the outside are sense, they are transmitted to the endodomain. There are signalling events that cause actin rearrangements so the cytoskeleton remodels. The information is transmitted inside the cell. Whatever actin conformation is on the inside (actinomyosin contraction) and that from the inside will be transmitted to the outside to the other E-cad molecule bound. When cells tighten their actin cytoskeleton, that information is transmitted across epithelial layer. There is communication between the endodomain and ectodomain.  In general, β-catenin not only plays a role in part of adherens junction by tethering α-catenin to E-cad, but also plays a role in the Wnt signalling pathway. If β-catenin is released from the tail of cadherin, it is free to go to the nucleus and acts as a transcriptional co-activator for binding partner in the nucleus. Signals can get transduced, including mechanosensing.  If there is a lot of force or other signalling, β-catenin can be caused to dissociate from E-cad where it goes to the nucleus and causes alterations in gene transcription. Outside the cell, that information can be transmitted and used to change gene expression. β-catenin goes between those two processes.  β-catenin is involved in EMT. 24 – Adherens junctions connect to bundles of cortical actin filaments in polarized epithelia (e.g., epithelial cells in small intestine)  Adherens junction is shown where E-cadherin would be. There are a lot of actin filaments bundled that connect to cadherins from one cell to the next cell.
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