Bio 2B03 Module 6 Lecture 1 PDF

BIO2B03 scripts Module 6, lecture 1 Script Notes Slide 1 Welcome back to Biology 2B03, Cell Biology. Today, we will start Module 6 on Cell Signaling, with Lecture 1: Concepts in Signaling. So let’s get started! Sl...

BIO2B03 scripts Module 6, lecture 1 Script Notes Slide 1 Welcome back to Biology 2B03, Cell Biology. Today, we will start Module 6 on Cell Signaling, with Lecture 1: Concepts in Signaling. So let’s get started! Slide 2 The objectives of this lecture are to introduce the topic of cell communication by: illustrating signaling in unicellular Dictyostelium slime mold cells and in human neutrophil white blood cells. We will also identify the general principles of cell signaling and we will describe the mechanisms by which cells in multicellular organisms are able to communicate using signals. Slide 3 The Dictyostelium discoideum slime mold is a eukaryote that transitions from a collection of unicellular amoebae into a multicellular slug and then into a fruiting body. These scanning electron microscopy images illustrate the different stages in the amoebae’s life cycle. Overall, in times when resources are not as readily available, aggregated amoebae will work together to form a multicellular slug that can migrate towards heat, light, and humidity in a search for potential food. The cells of the slug will then differentiate into prestalk and prespore cells. From there, in a suitable environment, the anterior end of the slug will form the stalk and the posterior end will form the spores of the fruiting body which is about 2mm tall. Slide 4 Dictyostelium feed on bacteria, such as E.coli. When food is abundant, the single-celled amoeba divides by mitosis. This is the vegetative growth phase. But, when food runs out, starvation initiates a series of events that leads to the aggregation of the unicellular ameobae. This aggregation occurs in response to the signaling molecule, cyclic AMP or cAMP, which is produced by the starved cells. These aggregated cells then form the migrating multicellular collection of cells, now referred to as the slug. Once the slug finds a suitable, nutrient-rich environment, it stops and the cells begin to differentiate. While anterior cells form the stalk, posterior cells form the fruiting body. The fruiting body contains spores with a hard cell wall, allowing the spore to remain dormant for extended periods. When food becomes available, the spores will finally germinate to form new single-celled amoebae in the environment. Slide 5 Here is a time-lapse movie of the growth of the stalk and the fruiting body. We see the multicellular Dictyostelium change its shape and form the fruiting body containing the spore. This is a fascinating organism that can be studied from many perspectives. In this module, we are going to focus upon the cell-to-cell communication between the single-celled amoebae to illustrate the concept of signaling. Slide 6 We see here, a collection of single–celled Dictyostelium amoebae in culture. Just out of focus is a pipette that contains the signal for aggregation, the signaling molecule cyclic AMP. This pipette is adding the cyclic AMP to the dish. So how do these cells perceive this signal? Well, the receptor for cAMP is a transmembrane protein called a G-protein coupled receptor or GPCR. The extracellular domain of the receptor binds specifically to cAMP, activating the receptor. In response, the cells reorganize their intracellular actin cytoskeleton network to move towards the sources of the signal. In this video, cyclic AMP is being added to the petri dish. As you can see, every single cell on the petri dish is moving towards that single source of cyclic AMP on the dish. Slide 7 In this Nomarski image of a single Dictyostelium cell we can see the nucleus and the cell membrane. Again, a pipette containing cAMP is just out of focus. The cell moves towards the source of cAMP. This time, the researcher moves the pipette. Every time the signal moves, the cell responds by changing its direction of movement. Dynamic filopodia extend outwards to allow movement. Signaling initiates actin reorganization including nucleation, polymerization, and depolymerization to enable movement. Slide 8 In this three-dimensional reconstruction of a Dictyostelium cell, we see the amoeba as it is moving towards the cyclic AMP signal that is provided at the left side of the field of view. These images highlight the formation of the finger-like filopodia at the leading edge of the cell, in the direction of movement. Slide 9 In contrast, in this three-dimensional reconstruction, the Disctyostelium carries a mutation in the gene for the clathrin heavy chain. This means that the cells are unable to form the vesicles necessary for transport of proteins to the cell membrane. Here, the signal, cAMP, is still at the left side of the field of view. Now, while the cellis are still forming filopodia, there is no net movement towards the source of the signal. Why is this happening? Well, remember that the cyclic AMP signal will be detected in these cells by the transmembrane GPCR proteins. So, in the absence of protein transport with this clathrin heavy  chain mutation found in these cells, the transmembrane G- protein coupled receptor is not transported to the cell surface. As a result, if there is no receptor for cAMP on the surface of the cell, the cell is unable to respond to that signal. Slide 10 Just like the Dictyostelium cells, single cells within our own bodies are also able to move towards chemical signals. For example, white blood cells called neutrophils are able to respond to a signal produced by bacteria that have invaded our bodies. In this movie recorded using bright-field microscopy in the 1950's by David Rogers,a single neutrophil is shown following a bacterial cell while in a petri dish. The neutrophil is the irregularly shaped cell in the middle,surrounded by red blood cells. The bacterium is the dark, bi-lobed cell in the middle. As you can see, the bacterium is moving around, and the neutrophil is following the bacterium. This happens because the neutrophil is responding to a chemical that is produced by the bacterium. This is possible because a receptor on the surface of the neutrophil binds to this chemical signal, activating a series of internal changes that facilitate directional movement. Eventually, we see that the neutrophil is able to capture and engulf the bacterium in a process of endocytosis. So what signal is being detected by these neutrophils? Slide 11 Well, the signal is actually being produced unintentionally by the bacteria. It is a protein containing the tripeptide, formylated- methionine, leucine, and phenylalanine. On the part of our neutrophils, they have a cell-surface receptor that specificallyrecognizes this fMLP peptide. Once again, the receptor is a G-protein coupled receptor (or a GPCR). In this short movie, the pipette contains just the fMLP peptide and the neutrophil responds to the signal by moving towards the source. Slide 12 After watching these movies, we can form a definition of the term “signaling”. Signaling is the transmission of information from one cell to another that induces a change in behavior. Keep in mind that the signal on its own is not very useful; it is only useful if there is a response to that signal. Overall, our definition of cell signaling must include production and release of a signal, the perception of the signal, interpretation of that signal inside the cell, and a resulting change in behavior. Let’s take a closer look. Slide 13 To start, a signaling cell produces and releases signaling molecules. The recipient or target cell has a receptor that specifically binds to that signal. Binding of the signal activates the receptor, and this initiates a cascade of chemical events, inside the target cell. This cascade of events will interpret and transduce the signal in what we refer to as the signal  transduction pathway (or STP). In the end, this culminates in some change in target cell behavior. Responses include changes in transcription, cell movement or growth, cell differentiation, and changes in metabolism corresponding to enzyme activation and inactivation within the cell. Ultimately, the signal must be removed to terminate the target cell response. But it’s important to keep in mind that within our bodies, while many cells might be exposed to a signal, it is only the target cells with the appropriate receptor that will be able to respond. Slide 14 Signal- receptor interactions are very specific, and follows the same principles of molecular complementarity as any protein-ligand interaction. This figure illustrates the interaction between a growth hormone (here, the signal) and its receptor. The hormone is shown in the space-filling diagram in red. Only the extracelullar domain of the receptor is shown in the ribbon diagram. Here, the amino acid residues in the receptor that are necessary for protein-ligand interaction are highlighted in yellow. There is a specific and high-affinity interaction between the receptor and the signal that is determined by molecular complementarity between the interacting faces of the molecules. O v e r a l l , complementary shapes allow the interacting surfaces of the two molecules to come close together and the collection of non-covalent interactions provide specificity and high affinity. Essential amino acid residues on each molecule are those amino acids that are necessary for the receptor-signal binding. Within the growth hormone signal, we see pink highlights showing the essential amino acid residues in the hormone that are interacting with the yellow highlighted essential amino acid residues in the receptor. It’s these interactions that facilitates the ability of this signaling hormone to interact with its receptor. Keep in mind though, that a single amino acid change at any of these amino acid residues can reduce or eliminate signal binding and therefore disrupt signaling. As a general rule, a receptor will only bind to one natural ligand or closely-related molecules. On the right, we see the hormone-bound receptor. As a result of this interaction, there is a conformational change in the intracellular domain of the receptor. That change in conformation in the intracellular domain of the receptor will then activate or induce the signal transduction pathway and ultimately lead to the cellular response. Slide 15 Specificity of the signal response is achieved at two levels. The first is the specificity of the ligand for binding to the receptor. Many cells may be exposed to a signal, but only cells with the corresponding  receptor will be able to respond. The second is the specificity of the intracellular response that is mediated by the signal transduction pathway. Different cell types may receive the same signal, but respond differently through the activation of different intracellular proteins. Here, two different cells respond to the same signal by activating different intracellular transcription factors. Alternatively, some cells may respond to the same signal by either moving or altering metabolic activity. Overall, we see that the activated signal transduction pathway is the collection of intracellular steps that are required to translate an extracellular signal into a cellular response. With that, specificity of the response will be determined by the internal signal transduction pathway. Slide 16 Here are two examples of cellular responses. At the top is a fast response. Here, an extracellular signal binds to a membrane-associated receptor. In response to the activation of the receptor, a cytosolic enzyme is activated, perhaps through a modification such as methylation, acetylation or phosphorylation. This is a fast response because the cell is able to quickly respond to the signal by simply changing the activity of a cellular protein that is already present in the cell.I n contrast at the bottom, we see a slow response. An example of a slow response is where the binding of a signal causes a change in protein levels within a cell. In this example, a soluble receptor is in the cytosol and the signal is able to pass through the cell membrane. In response to the activation of the receptor, the receptor itself is transported into the nucleus, where it acts directly or indirectly as a transcriptional activator producing messenger RNAs. Ultimately the mRNAs are translated to increase protein levels in the cell. This would be a slow response, because the response depends upon transcription, translation, protein folding, and protein modifications, and each step takes time before we see a change in cellular response. Note that in cells, the same signal may exert these different types of responses in different cell types and that either membrane-bound or soluble receptors can exert either type of response. Slide There are two ways in which signaling can be 17 assayed or measured. The affinity of the receptor for a signal can be measured in the same way that protein-ligand affinity is measured. We see here a plot where the pink line represents the reaction kinetics of receptor-signal binding. On the X-axis is ligand (or signal) concentration and on the Y-axis is the fraction of receptors bound by the signal. 100% would mean that all receptors are filled. 50% (or 0.5) would be half maximal binding (similar to ½ Vmax). Kd (dissociation constant) is the concentration of ligand required to have half of maximal binding and represents receptor-signal affinity. This can be compared to the concentration of signal required to achieve a physiological response. The blue line represents the physiological response of the cell. On the X-axis is the ligand (or signal) concentration and on the Y-axis is the fraction of cellular response that is the fraction of cells responding. A maximal physiological response can be measured (where all cells respond) and a half maximal response can be calculated. As you can see, the concentration of ligand to achieve half of the maximal physiological response is much lower than the concentration of ligand required to fill half of the receptors. This suggests that the even with a small amount of signal available to bind to a target cell, the signal must be amplified inside the cell , and because of this, very little signal is required to exert a response in the target cell! Let’s take a closer look at types of intercellular signaling, or signaling between cells. Slide There are different types of intercellular signaling. 18 In endocrine signaling, secreted signals are released into the circulatory system. In this way, cells throughout the body are exposed to the signal. While only cells that have the appropriate receptor can respond to the signal, this also means that many different cells in different tissues can respond to the same signal at the same time. This type of endocrine signal is commonly a secreted hormone. With endocrine signally, the signaling cell and the target cell are usually far away from one another in a multicellular system. We also see here, paracrine signaling. In this type of signaling, the secreted signals are released in the extracellular space where they can diffuse to neighbouring cells. In this case, the signaling cell and the target cell are near one another. Growth factors and neurotransmitters are common examples of paracrine signaling molecules. Slide Proximal signaling is also seen when the signaling cell 19 and the target cell are in direct contact with one another. Here, both the signal and the receptor proteins may be transmembrane proteins on different cells. Because of this, interaction between the signal and the receptor requires that the two cells are attached together through cell adhesion mechanisms. This is accomplished by integral membrane proteins. Neighbouring cells can also communicate by sharing cytosolic messengers. Examples of this are seen in plants and animals. In plants, plasmodesmata are junctions between two neighbouring cells. These junctions span the cell membrane and the cell wall between plant cells. The plasmodesmata effectively connect the cytoplasm of neighbouring cells, allowing messengers to move very quickly from one cell to the next. In fact, these connections form a vascular system within plants in which signals produced in the root can be transported from cell to cell up to the leaves of the plant. A similar structure is seen in some animal cells. Gap junctions are channels connecting the cytoplasm of neighbouring cells that allow the fast diffusion of small molecules from one cell to another. In this way, one cell may respond to a primary extracellular signal by producing an internal secondary messenger. This secondary messenger can then move through diffusion from one cell to another to exert the same response and in the end, coordinate the behavior of a series of cells. Slide 20 Finally, autocrine signaling is the process in which a cell communicates with itself. Here, the signaling cell and the target cell are the same. In this case, a cell produces a secreted signal and the same cell also carries receptors for that signal. Examples of this are growth factors that are produced to induce cell division or stop cell division, depending upon internal and external conditions. Slide 21 While we have just looked at different types of intercellular signaling, we can further classify cell signaling based on the types of receptors that are involved. In particular, there are seven major classes of cell surface receptors. While we cannot look at all of these in our course, we will focus on three types that highlight the major principles of cell signaling. These are the cytokine receptors, the receptor- tyrosine kinases (or RTKs), and G-protein coupled receptors (or GPCRs), Slide 22 In this module we will look at 1) the cytokine receptors of the JAK/STAT pathway that control the production of red blood cells 2) a receptor tyrosine kinase (or RTK) that is linked to phosphorylation cascadethrough a small G-protein called Ras to regulate gene expression and 3) a G-protein coupled receptor (or GPCR) that activates an effector protein inside the cell to produce a second messenger, cAMP, that ultimately regulates cell metabolism. Slide 23 And that’s all for today’s lecture. In summary, we have seen through this introduction, that signaling is the process by which a cell receives and responds to signal in its local environment. The steps that govern signaling includes not only signal production by a signaling cell, but also, signal reception by a target cell, along with the activation of internal signal transduction pathways that interpret the signal and lead to a cellular response. We will explore cell signaling further in our next 2 lectures.