Lecture 6 2025 (PDF): Passageways from Cell to Cell and Basal Lamina

Lecture 6 Passageways from cell to cell and Basal Lamina 1  Tight junctions block passageways through the gaps between cells, preventing extracellular molecules from leaking from one side of an epithelium to the other. Ano...

Lecture 6 Passageways from cell to cell and Basal Lamina 1  Tight junctions block passageways through the gaps between cells, preventing extracellular molecules from leaking from one side of an epithelium to the other. Another type of junctional structure has a radically different function: it bridges gaps between adjacent cells so as to create direct passageways from the cytoplasm of one into that of another. These take quite different forms in animal tissues (called gap junctions) and in plants (called plasmodesmata). Gap junctions and plasmodesmata allow neighboring cells to exchange small molecules but not macromolecules. Many of their functions are only beginning to be understood. Here are examples of a couple different types of junctions that connect cells together. Ca2+ and other ions can pass easily through gap junctions, complex carbohydrates or proteins cannot 2  A tight junction is a protein complex between two cells that creates a seal to prevent any leakage of materials between the cells 3  Gap junctions couple cells both electrically and metabolically Gap junctions are present in most animal tissues allowing cells to communicate with their neighbors. The gap is spanned by channel-forming proteins, of which there are two distinct families, called the connexins and the innexins. These are unrelated in sequence but similar in shape and function. Both families are present in vertebrates but connexins predominate. If we have ions moving across gap junctions that essentially represents charge and that allows for neighbouring cells to be coupled electrically to each other. Like in the pacemaker cells of the heart, they w ill have gap junctions that will allow them all to be synchronized with respect to electrical activity. 4  The channels formed by the gap- junction proteins allow inorganic ions and other small water-soluble molecules to pass directly from the cytoplasm of one cell to the cytoplasm of another, coupling the cells both electrically and metabolically. From experiments with injected dye molecules of different sizes, the largest functional pore size for gap-junctional channels is about 1.5 nm. Therefore, cells can share their small molecules (inorganic ions, sugars, amino acids, nucleotides, vitamins), but not their macromolecules (proteins, nucleic acids, and polysaccharides). 5 This is an example of that experiment where we have highlighted 2 different cells and the gap junctions in green linking those cells. Then in the one cell we inject the dyes, the different sizes of dye are different colours and we can visualize the adjacent cell to see what colours have passed through the gap junctions and into the second cell. This will tell us the general size of the gap junctions, here this one tells us that the size of the junction is above 1000 but between 5000 daltons 6  A gap-junction connexon is made up of six transmembrane connexin subunits Connexins are four-pass transmembrane proteins, six of which assemble to form a hemichannel, or connexon. When the connexons in the plasma membranes of two cells in contact are aligned, they form a continuous aqueous channel that connects the two cell interiors. Figure 19-34b Molecular Biology of the Cell (© Garland Science 2008) With respect to their structure, we are going to use the example of connexin to show how they are formed. There are multiple members of the connexin family so you can mix and match when making these channels A hemichannel is when one connexon from one cell does not line up with one from another cell, instead it opens up to the extracellular space. In this case ions can move into or out of the cell (depending on the concentration gradient) through this hemichannel, this essentially creates a pore. Generally hemichannels are kept in a closed conformation because we want to control what goes in and out of the cell. When we are building this gap junction or channel, we can have different forms of connexins in any conformation. 7  Figure 19-34a Molecular Biology of the Cell (© Garland Science 2008) Red is the PM, Gap is between the 2 neighbouring cells. There are examples here of 2 connexons lining up and forming a channel and then on the right we have a hemi channel that will generally remain closed until it is intentionally opened by a stimulus. 8  Gap junctions in different tissues can have different properties because they are formed from different combinations of connexins, creating channels that differ in permeability. Adjacent cells expressing different connexins can form intercellular channels Unpaired channels or hemichannels are normally held in a closed conformation. When we make the channels out of different combinations of connexins we can be more selective about what molecules can or cannot cross and this can be cell type specific. 9  Mutations in connexin-26 are the most common of all genetic causes of congenital deafness. This results in the death of cells in the organ of Corti through the disruption of the flow of ions in the organ’s sensory epithelium. 10 Cells can regulate the permeability of their gap junctions Like conventional ion channels, individual gap-junction channels do not remain continuously open. Gap-junction channels close when a cell’s plasma membrane is damaged causing ions to leak in. This isolates the cell and prevents its neighbors from harm. The neurotransmitter dopamine reduces gap-junction communication between a class of neurons in the retina in response to an increase in light intensity. The reduction in gap-junction permeability helps the retina switch from using rod receptors to cone receptors, which detect color and fine detail in bright light. 11  Control Dopamine Lucifer Yellow Experiment Figure 19-37 Molecular Biology of the Cell (© Garland Science 2008) Here we have a sheet of neurons representative of the neurons in the retina, Dopamine in the retina is allow you to regulate being able to see in bright vs dim light.. When you have a high intense stimulus you need to restrict the amount of activity of the neurons in this area so that the eye is not overloaded with sensory information and that is facilitated by dopamine. In the middle we have a dopamine responsive cell, it is injected with a small dye called lucifer yellow (it can pass through gap junctions). You can see the surrounding neurons are lighting up as well, but they get more dim as they are further away from the neuron that was injected. This is because the dye is moving through the gap junctions and spreading into the surrounding neurons, these neurons are electrically coupled through their gap junctions, their gap junctions are open and they are all exchanging ions so they can respond to a stimulus together. But what we can do is add dopamine and what dopamine does is it makes all those gap junctions close. Now in the exact same setting- we still have all the neurons around this cell, but because the gap junctions are closed the dye stays within that cell. The retina uses this in bright light so that your retina is not overwhelmed with light stimulus and you can still see. 12 We have discussed connections between cells, what about connections between cells and the extracellular matrix? 13  The Basal Lamina Tissues are not solely made up of cells. A very large part of their volume is extracellular space, which is occupied by an intricate network of macromolecules constituting the extracellular matrix. The matrix is composed of various proteins and polysaccharides that are secreted locally and assembled into a meshwork. In our own bodies, the most plentiful forms of extracellular matrix are found in bulky connective tissues such as bone, tendon, and the dermal layer of the skin. Cells will build on this meshwork into a complex, functioning tissue 14  For most animals, the basal lamina (also referred to as the basement membrane) is one of the most important forms of extracellular matrix. It is the underpinning of all epithelia (40-120 nm thick). It surrounds individual muscle cells, fat cells and Schwann cells. Figure 19-39 (part 1 of 3) Molecular Biology of the Cell (© Garland Science 2008) Figure 19-39 (part 2 of 3) Molecular Biology of the Cell (© Garland Science 2008) Basal lamina is a specialized type of ECM In the cartoon we have 2 types, epithelium and muscle, and you can see that the change in tissue gives a change in the organization of the basal lamina. The basal lamina wraps around the cell, you can see in the muscle it is following the striated muscle path, around the basal lamina is connective tissue (fibrous proteins- cells can be present within it but mostly it is protein and polysaccharide based) The epithelium is more polar in organization, you can see the sheath of villi on one polar (apical) and on the basal surface you see it is attached to the basal lamina and below that is where we have connective tissue 15  Basal lamina determine cell polarity, influence cell metabolism, organize the proteins in adjacent plasma membranes, promote cell survival, proliferation, and differentiation. The mechanical role is nevertheless essential. In the skin, the epithelial outer layer (epidermis) depends on the strength of the basal lamina to keep it attached to the underlying connective tissue (dermis). Defects in certain basal lamina proteins cause the epidermis to become detached from the dermis (e.g. junctional epidermolysis bullosa). Polarity – asymmetric shape like the epithelial cells to support their function Metabolism- controlling the directional flow of nutrients If a cell needs to divide and it is trapped in the proteins of the ECM it cannot do so, so the ECM needs to be dynamic. Same idea for differentiation Mechanical role- skin depends on the basal lamina to attach to connective tissue 16  Figure 19-40 Molecular Biology of the Cell (© Garland Science 2008) Here at the top we have epithelial cells, below that we have a fine sheath of basal lamina, below that we have connective tissue. Both the Basal Lamina and connective tissue are considered the ECM. All of these layers of tissue need to be able to interact 17  Laminin is a primary component of the basal lamina The basal lamina is synthesized by the cells on each side of it: epithelial cells contribute one set of basal lamina components while cells of the underlying bed of connective tissue (called stroma) contribute the other. Two main classes of basal lamina: Fibrous proteins (glycoproteins with short oligosaccharide side chains), and polysaccharide chains of the type called glycosaminoglycans (GAG), which are linked covalently to specific core proteins to form proteoglycans. We are going to discuss the proteins that are involved with making up the basal lamina and how they are involved with helping with cell organization and structure. Laminin is the first protein we are going to discuss 18 19  Although the precise composition of the mature basal lamina varies from tissue to tissue, it typically contains the glycoproteins: laminin, type IV collagen, and nidogen, along with the proteoglycan perlecan. These key components are present in the basal lamina of organisms from jellyfish to mammals. It is closely associated with other molecules such as collagen XVIII (the core protein of a proteoglycan) and fibronectin (a fibrous protein important in the adhesion of connective-tissue cells to matrix). Within the families of proteins there are family members and they can differ depending on the organism and tissue type. 20  Green = Protein Pink = GAG Fibrous proteins Proteoglycans (with GAG) Figure 19-41 Molecular Biology of the Cell (© Garland Science 2008) Here we have a cartoon that highlights the different proteins we are discussing. We have 2 main groups, the GAGs and Fibrous proteins. 21  The laminins are the primary organizer of the sheet structure, and early in development, the basal lamina consist primarily of laminin molecules. Laminin-1 is a large, flexible protein composed of three very long polypeptide chains (α, β, and γ) held together by disulfide bonds. The heterotrimers can self-assemble through interactions with their heads. Several isoforms exist generating different laminins creating basal lamina with distinctive properties. Laminin γ-1 chain is, however, a component of most laminin heterotrimers and mice lacking it die during embryogenesis (first essential component) 22  * Figure 19-42a Molecular Biology of the Cell (© Garland Science 2008) This is what that looks like, it is a laminin molecule. There are 3 polypeptide chains present, we have an alpha, beta and gamma chain and each of the domains of this molecule have specific functions. the 3 chains coil around each other, and at the binding domain we have binding sites for integrins which are membrane proteins that are present on cells which shows that this molecule can interact with other cells through their integrins (we will talk more about these next lecture) and then we have binding sites for perlecan and dystroglycan which says we can interact with other ECMs And then at the bottom we have a binding site for nidogen which is a fibrous protein that makes up parts of the ECM. On the N terminal we have another binding site for integrins which essentially means that this molecule can be organized between 2 cells through interaction with integrins on both of them, while still binding the ECM, and then we also have sites for self assembly, which means this laminin molecule can be bound to another laminin molecule while still being bound to other cells and ECM components which gives us that nice sheath lining. 23 Type IV collagen gives the basal lamina tensile strength Type IV collagen is a second essential component of mature basal lamina. It too exists in several isoforms because it consists of three separately synthesized long protein chains. They assemble via terminal domains into flexible, felt-like networks that give basal lamina tensile strength. How do the networks of laminin and type IV collagen bond to one another? The molecules of laminin have several functional domains. 24  * Arrows indicated what each protein can bind Figure 19-43 Molecular Biology of the Cell (© Garland Science 2008) This carton articulates how all these molecules can organize into a sheath. Here in grey is the PM bilayer, so protruding from the PM are these integrins, and they can bind to various components of the ECM, like laminin which we already talked about shown here in blue. But Integrin is also binding to other ECM components, you can see down here what all these components can bind. This global representation is what is happening on one cell, this sheath will cross multiple cells 25  Basal lamina have diverse functions The kidney glomerulus has an unusually thick basal lamina that acts as a molecular filter preventing the passage of macromolecules from the blood into the urine. Mutations in type IV collagen genes result in an irregularly thickened and dysfunctional glomerulus filter (hereditary kidney disorder - Alport Syndrome). Figure 19-39 (part 3 of 3) Molecular Biology of the Cell (© Garland Science 2008) The basal lamina is unusually thick and acts as a filter that prevents the passage of macromolecules from the blood into the urine. Here we see 2 main layers of cells, one facing the blood and one facing the urine, they are separated by that thick basal lamina, it is what is going to allow the passage of some molecules but not others from the blood into the urine. 26  The basal lamina can act as a selective barrier for the movement of cells. It prevents fibroblasts in the underlying connective tissue from contacting epithelial cells. It does not prevent macrophages or lymphocytes from passing through. They use specialized proteases to cut a hole for their transit. It also plays a role in tissue regeneration after injury. The basal lamina often survives injury, creating a scaffold along which regenerating cells can migrate. Defects in components of the basal lamina or in proteins that tether muscle cells to the basal lamina at the synapse are responsible for many forms of muscular dystrophy. It can prevent fibroblasts in the underlying connective tissue from contacting epithelial cells. This connective tissue does have some cells in it, mainly fibroblasts and they can secrete some of the ECM components. One of the big ones they secrete is collagen and they can also secreted fibronectin. The barrier created by the basal lamina doesn’t prevent the movement of specialized cells like macrophages or lymphocytes, they use special proteases to cut holes so they can move around, this is important for the body to be able to mount a proper immune response. 27 Here we have an experiment that demonstrates this. On the left we have a muscle cell (multinucleated cell) it is surrounded in orange by the basal lamina, it is being innervated by a neuron. This neuron is innervated the muscle at a specific place called a neuromuscular junction. In this example we have cut the cell and cut the neuron such that both degenerate, the cell is dead – the shell of the basal lamina remains – the neuromuscular junction is still represented here, in this experiment if you take this shell of basal lamina and you culture it in the presence of a neuron, that neuron will send its extension to exactly where the neuromuscular junction was and it will regenerate that connection. If you add a muscle cell, it will grow inside the shell of the basal lamina, but more than that it will shuttle receptors for Ach (which is the NT the neuron would release) to the exact site where the old junction was, expecting the neuron to eventually be there. So this basal lamina is giving a lot of information to the cells in respect to where to traffic receptors to, where to extend axons to etc 28  True or False. Unlike conventional ion channels, individual gap junction channels remain open continuously once they are formed. True or False. A sheet of basal lamina underlies all epithelia. Certain bacteria secrete enzymes that can digest protein or carbohydrate components of the basal lamina. Why do you suppose they do so? When you wake up in the morning and turn on the light, is dopamine release increased or decreased? How does dopamine alter the neuronal activity in the retina? 29