CSB327 Lecture 5 Notes

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Cell and Systems Biology
Maurice Ringuette

CSB327 Lecture 5 Notes – Basal lamina and embryogenesis (September 24, 2012) 1 – Lecture topics • We talked about getting out of the cell. We talked about the classification of collagens. We used type I collagen as a prototype. We followed the fate of an alpha chain as it assembles into a triple helix to the formation of fibers. We looked at pathologies that were associated with events that occurred in the ER. For example, the lack of hydroxylation translated into the lack of stability of the triple helix. Even though a mutation might occur and interfere with an event in the ER, such as the formation of a stable trimer, it can be translated into abnormal fibrils outside as a downstream effect. We looked at the basal lamina because the basal lamina (specialized extracellular matrix sheet) communicates with interstitial matrices. You will see examples today of how other types of collagen bridge the interstitium to the basal lamina and how the basal lamina is part of some of the components that are involved in the communication of the basal lamina and the anchoring to epithelia. • I want to talk about different types of states of cells and how powerful the ECM is in the absence of growth factors. When we look at embryogenesis, you will see that the formation of ECM is coincident with early embryonic development. We are going to come back to this as the course continues because as I introduce new molecules, I want to show you their relationship to embryogenesis and other cellular events. It is often difficult to study mammalian development because the embryos are internal. We can observe embryogenesis with eggs of Xenopus laevis, zebra fish, and fruit flies. We are restricted in being able to visualize embryogenesis without disturbing them. There are ways around this. If I talk to you about development, let’s get straight about some terminology so that you’re not confused when we move on. 2 – The major differences between an epithelial cell and a mesenchymal cell • An epithelial cell has polarity. It has an apical surface, basolateral surface, and basal surface. Here is the basal lamina underlying connective tissue. When people think about ECM with respect to epithelia, they are talking about the basal lamina of the cell. However, there are clear examples of apical secretion of ECM components. It is not just basal, but by large, this is where we will focus. You have a free surface facing the lumen. You have your basal lamina that might have a muscle layer or a connective tissue layer. • In a normal state, the basal lamina is critical for the stability of this epithelium. If you knock out the basal lamina, then you start to lose the apical surface. The cross-talk between the ECM and epithelial sheets is fundamental. • You have two basic cell types. There are epithelial cells and mesenchymal cells. Mesenchymal cells are like fibroblasts. During early embryogenesis, they are naive cells until they are told to differentiate or go through some lineage cascade. Usually, this is a complex series of events. A naive mesenchymal cell, which has this morphology, doesn’t have a beautifully designed epithelium (not that they don’t have internal polarity like the Golgi). Mesenchymal cells are these spindle shaped cells. If they are migrating, then they have this filopodia. We are going to look at these structures because cells moving on a matrix are going to use integrins to a large degree to cross communicate with actin filaments. Mesenchymal cells reside in the ECM and may be motile. The Golgi complex is usually at the leading edge. • What is a major difference between a mesenchymal cell and an epithelial cell? This mesenchymal cell is embedded within matrix. Mesoderm is not mesenchymal cell. There are two cell types in terms of polarity. Mesoderm can have a combination of epithelial and mesenchymal. Mesoderm is a germ layer. 3 – The cycle of events during which epithelial cells are transformed into mesenchymal cells and vice versa • The details here are irrelevant at the moment. I only want you to remember one thing from it at the end. • Here is the epithelial cell. Epithelial cancers have to go through a basal lamina. In EMT, you start breaking junctional complexes from the apical end to the basal end. This is not the way it occurs in nature. It goes the other way. During embryogenesis, mesenchymal cells can go back to epithelial state. During MET, you reassemble the complexes. During embryogenesis, the embryo can do EMT or MET without any effort. In adult cells, MET is much harder than EMT. Cancer cells originate from epithelial tissue and go through this transformation. What are some of the ECM molecules associate that help direct that? Fibronectin has an intimate relationship with collagen. You cannot form collagen fibrils without fibronectin. • I want you to remember that by in large, it is easier to go to the mesenchymal state. Which one is motile? Do epithelial sheets move? Mesenchymal cells move. Well, this is absolutely not true. Epithelial sheets do move. They move during embryogenesis. What is a major event that can occur in our bodies where epithelial sheet moving is critical? Wound repair. You have to re-seal the wound. 4 – Epithelial to mesenchyme transformation (EMT) and vice versa • The piercing of the basal lamina requires some help from matrix remodelling metalloproteinases. They are losing their epithelial character. Not all transmigration cross an epithelia are dependent on metalloproteinases. Our immune cells are able to squeeze themselves through the basal lamina that lines the vasculature to take care of an infection. For epithelial cancers, they have to destroy the basal lamina. The size of epithelial cancers does not permit them to go through. • In somitogenesis, EMT occurs all the time. Somites are spheres of epithelia surrounded by basal lamina. • In kidney development, you get signalling in the mesoderm to form a tube. You see the mesenchymal cells will become polarized, but in the process, they form a basal lamina. They cannot reach epithelial state without formation of a basal lamina. Laminin is responsible for this. • Some basal lamina promotes epithelia. Some basal lamina promotes mesenchyme. • In neural crest cell migration, almost all the connective tissue in your face is derived from neural crest cells. The neural crest cells undergo EMT. • The ECM and neural crest cells can promote path finding. The behaviour of the neural crest cells is somewhat similar to that of the growth cone of your axon. You are going to see that nature makes use the same logic for what appears initially to be really different cellular event. They borrow the same molecules. The fine tuning comes in isoforms and some differentiation. • I am trying to get you prepared for when we talk about embryogenesis. 5 – Summary of experiments testing the effects of a variety of solubilised ECM and other molecules on the corneal epithelial cell surface • If you look at the cornea epithelial cells on a Millipore filter, at the basal surface, you have these blebs. This means, without a basal lamina, you begin to lose epithelial polarity. The basal surface is compromised. Albumin or IgG cannot rescue the basal lamina. We are using Millipore filters because they are porous so the blebs can go through. If you put a native corneal explant with its basal lamina attached on a Millipore filter, you see beautiful epithelial state maintained. The basal lamina is safe guarding epithelial polarity. The signaling events or the cross-talk that is occurring between the basal surface of the cell and the ECM is maintaining this state. The information encoded by ECM molecule is acting to maintain and required to establish epithelial polarity. In some organisms, the primary epithelium does not have a basal lamina initially until later in development, especially in organogenesis. • What did they learn? If you add an intact basal lamina or you put in constituents of the basil lamina, then you can rescue this. If you put it in with basal lamina, then it stays. In this diagram, it is placed on a Millipore filter and you are putting collagen I-IV, and you maintain it. Laminin is universally found and restricted to basal lamina. Fibronectin is abundant in our serum, tissue, and part of embryonic basal lamina. • You can see that there is something about these molecules that allows them to promote this stage. Now we understand this better. There were surprised that no growth factors being added. They were adding purified components in the absence of any growth factors. By itself, they saw the ECM has some kind of regulatory function that is not just correlated with structure. As further evidence of this, they did these experiments to support the idea. 6 – Fate of cells derived from adult or embryonic lens explants placed on either the surface or suspended in type I collagen • You put an embryonic lens explant on a 3D type I collagen gel. The embryonic lens explant has an epithelium with a lens capsule, w
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