CSB327 Lecture 14 Notes

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Cell and Systems Biology
Maurice Ringuette

CSB327 Lecture 14 Notes (November 19, 2012) – Laminins and epithelial cell polarity 2 – A model of the molecular structure of basal lamina • There are two major types of ECM: interstitial ECM and the basal lamina. The basal lamina contains some universal components. • Nidogen can bind to these other partners in the basal lamina and stabilize the interaction. The data indicates it is more complicated. • Laminin is the focus of today’s lecture. Laminin is a family of glycoproteins. Like FN, laminin is a very adhesive molecule. You can form a basal lamina like structure in the absence of all these other components. You have an absolute requirement for laminins to form the basal lamina. • What is special about laminin? What can we learn about its role in development? o It is required for polarization of epithelial cells in embryoid bodies. o We will talk about the disregulation of laminin in cancer.  Laminin is only found in basal lamina. However, in the reactive stroma of cancers, the myofibroblasts can express laminin isoforms that become adhesive for the cancer cells to migrate on. There is a disregulation of the expression and location of laminin. 4 – Laminins • Laminin is a trimer. o It has a coiled-coiled domain. You can stabilize the coiled-coiled structure by disulfide bridges. o The α chain has a globular end attached to it. The globular end is very adhesive to cell surface receptors. o There are rod-like EGF repeats that make up the arms. This is to show how different ECM molecules borrow the same modules to form supramolecular assemblies. o There are several globular domains. I will come back to this when we show the polymerization of laminin. • Laminin is absolutely required for BL organization. • Like almost all ECM components, it affects a broad variety of cellular activities of behaviours like cell adhesion and cell polarity. • There are about 16 different combinations of the α, β, and γ chains. • The α chain is on average about double the size than the β and γ chains. You are forming a molecule that is large in molecular weight. Some isoforms are a million Daltons in molecular weight. This is not a small molecule. 5 – Schematic of 16 mouse or human laminin heterotrimers • This is to show the different isoforms of laminin. • LM-1 was one the first discovered and it is broadly distributed. • You can do a variety of truncations and changes. • There is structural variability that gives you the different isoforms that have different tissue distributions. Different isoforms can be co-expressed by the same cell. They will interact with different combination of integrin receptors and other TM receptors. o You have tissue specific activities of laminin. They make different contributions to different events. • The γ1 chain is the most abundant isoform of that chain. o If you knock out the γ1 chain, you get arrest at the pre-implantation stage. • LM-10 (511) is a very abundant laminin also found in basal lamina, vasculature basement membrane and disregulated in cancer. LM-10 is not as critical as LM-1 in terms of early embryogenesis. o In the literature, they raised an antibody that they thought was recognizing the α1 chain, but it was actually recognizing the α5 chain. This has been corrected but a lot of old literature wrongly assigned LM-10 as being LM-1. o If you knock out LM-10, the mice get to a fairly advanced stage of development before they die. • One of the targets of LM-5 is α6β4 integrins which is associated with hemidesmosomes. o LM-5 (332) is important in the stabilization of epithelia with basal keratinocytes. • All of these laminin are adhesive. 6 – Laminin isoforms, their expression, and receptors • Do not memorize this slide. • The laminins can bind to a variety of different integrins. o They will have a preferential affinity for a subset of the integrins. Even though it may bind to three or four integrins, it may not bind them equally well. • There is broad and overlapping distribution. • There is overlap in the types of integrins that they can recognize. 7 – The mammalian integrin receptor family • α1β1 and α2β1 are highlighted as the prototype integrin that binds to collagens. o The epitope (binding site) for these two integrins is GFOGER as opposed to RGD. This sequence can be targeted reactive breakdown products of glucose. The “O” stands for a modified amino acid. • α11β1 is disregulated and promotes transformation of cardiofibroblasts into myofibroblasts. • Let us look at laminin-integrin interactions and network formation. 8 – Laminin-1 receptor interactions • For the short arms, you have regions of the laminin molecule that contribute to laminin polymerization. • A lot of the signal transduction events that are mediated by laminin are due to the globular domains of the α chain. One of the integrins that binds to a lot of laminins is α6β1. Dystroglycan is another receptor. You have HBSA. You know about FAK. • The contact of laminin with the plasma membrane leads to a whole variety of different molecular interactions ranging from integrins, dystroglycans, HBSA (e.g., syndecan) and scaffolds (e.g., FAK). o Laminin binding to these variety of receptors will induce a lot of intracellular changes ranging from reorganization of the actin cytoskeleton to signal transduction. • Laminin is a powerful molecule in terms of signaling. • Laminin is fundamental for the transformation of mesenchymal cells into epithelial cells. This is one of its major impacts on cellular morphology. • This is to show you that there is an enormous amount of interactions that are promoted by the interaction of laminin to the surface of the cell. • I am not going into the signalling pathways. o I want to show you the molecular assembly of laminin. 9 – Receptor-facilitated laminin assembly and laminin-cortical network formation • The sticky ends help promote assembly of laminin into a polymer on the plasma membrane. This will result in the clustering of the integrins and dystroglycans. This will lead to intracellular signalling and reorganization of the actin cytoskeleton. • It is similar to FN in the sense that you cannot polymerize FN into a network without the cooperation of interactions with cell surface receptors. • Laminin can stick together, but to form a proper network, it is a cell-mediated event. • It is Ca2+ dependent. It can be a reversible dissociation. • The coordination of cell surface association, binding to receptors and reorganization of the actin cytoskeleton. During embryogenesis, as laminin is made, you promote epithelial morphogenesis. 10 – Embryoid body morphology at different stages • If we look at embryoid body development, you take stem cells from the inner cell mass. You allow them to differentiate. Eventually, they form these embryoid bodies, which is a facsimile of what you see during normal mammalian development. • We are focusing on the epiblast component inside the embryoid body. You are going from a mesenchymal state and you gradually polarize this into an epithelia. • If you immunostain for the γ1 chain, you see the expression and formation of the BL. Eventually, you get a nice BL separating the epiblast from the endoderm. 11 – Polarity defects resulting from laminin mutations • What is the consequence of generating these embryoid bodies derived from inner mass cells from a mouse that is null for the γ1 chain in comparison to type IV collagen? • In the WT, you have the classical features of an embryoid body. The endoderm secretes laminin and type IV collagen that will make up the BL. • When you take cells derived from the homozygous null for the γ chain, you knock out a range of different LM. This results in failure of epiblast polarization and cavitation. o When you immunostain for junctional complexes that are hallmark of epithelia, you find there is no epithelia formed and these key elements (e.g., β1 integrin, β- catenin, etc.) of epithelia are all around the cell surface. They are uniformly distributed as opposed to being localized and polarized like in an epithelia. o It is not that these don’t contribute to the BL. These components tend to contribute to stability and more downstream events than laminin. • If you knock out the γ1 chain in mice, you have embryonic arrest. This is a great model how knocking out the most abundant chain of the laminin family results in the loss of epithelial morphogenesis or epithelial polarity. o If you knock out γ1, the signalling and biological activity of basal lamina is disrupted. You absolutely need laminin to form a BL. 12 – Polarized ectoderm (epiblast) • The junction complexes are associated with a polarized epithelium. o If you knock out LM, the components that make up the junctional complexes are uniformly distributed. 13 – Laminin is required for basal laminae assembly • Knock-out of nidogens, perlecan, agrin, type IV and XVII collagens does not prevent assembly of basal laminae, but lead to defects in stability and signaling 14 – Abrogation of epithelial development in embryoid bodies by a dominant-negative fibroblast growth factor receptor • Growth factors are intimately linked with the assembly process. One of the key growth factors involved is fibroblast growth factor. • The dominant-negative form of FGFR means you could be co-expressing an isoform the receptor (e.g., receptor tyrosine kinase) that as no kinase activity. If you over-express the Dn form, it will out-compete the wild-type receptor. You are seeing a loss of signaling because instead of having only normal receptor that is capable of signaling, you are binding the growth factors to a receptor that cannot go signaling downstream. o Dn is a null in terms of further signaling. o In this case, you would have some normal receptor, but when you force expression of a kinase-dead FGFR, it will silence the signaling because it is out- competing the normal receptor for binding to the ligand. • The nidogen-1 component is divorced from FGFR signaling. You are not knocking down the level of expression of nidogen. Nidogen is not localized because there is no epithelial polarity. The BL is absent. • However, you see a complete loss of LM-1. When you stain for laminin, you see a complete absence of staining for laminin in the Fn-FGFR. FGFR signaling is critical for the expression of these two components of BL: LM-1 and collagen IV. • The nidogen-1 component is divorced from FGF signaling. You are not knocking down the level of expression of nidogen. Nidogen is not localized because there is no epithelial polarity. The BL is absent. o The expression of these two components (LM-1 and type IV collagen) of the BL in the embryoid body is are dependent on FGFR signaling. 15 – Regulation of laminin and collagen IV expression in embryoid bodies • I am showing you the entire pathway as a teaching aid. • How is FGFR signa
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