Lab 4 Cellular Communication PDF

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Biology 212

Laboratory 4: Cellular Communication Background Cells have a need to communicate with each other. This happens for a bunch of reasons, which may include  determining of mating is possible,  sending or receiving signals about the environment or food,  determining if it is safe to reproduce, and  being told when to make or stop making certain chemicals. The process by which cells communicate is varied but boils down to a few fundamental ideas that are not actually that difficult to understand. In fact, you already know most of these concepts—all we need to do is add some simple reactions and the rest is easy. Basic mechanisms At its core, there are three steps to cell communication: 1. The signal must be received at some type of protein receptor, 2. There is an amplification of the signal along a set of protein-based mechanisms to eventually 3. Produce a cellular and/or genetic response.

Image 1: Cell signaling/communication or signal transduction Taken individually, none of this is difficult and it actually makes a lot of sense. This is how we make communicate with each other: we make noises that are detected by our ears, we process those noises in our brains, then we respond to those noises. For the sake of saying it, not every cell and organism needs to follow these steps directly. There are cells within your very body that can bypass these steps, as there are direct connections from cell to cell. They are given the names like gap junctions in heart muscle, or plasmodesmata in plants. These three steps are involved with external signals that are secreted from the cell. 1 Lab 4

Let’s look at how the cell does these steps…in a simplified way. Signal reception There are two different types of signals, in essence, that we will look at. (If you think about your senses, we can deal a lot with light, sound, touch, etc., but it’s going to make this conversation a bit more complicated.) Those are protein-based signals and lipid-based signals. One at a time!

Image 2: Different types of signal receptors Protein-based signals Because proteins are not capable of entering into cells without alternative means (bulk transport, a form of active transport), the receptor for protein-based signals are normally embedded inside of the cell membrane. The binding of the signal induces a change in the shape of the receptor, which will trigger one of several types of outcomes, depending on the type of receptor this is. These could include  G-protein coupled receptor, where the binding causes the replacement of GDP with GTP, activating the G-protein that then activates another enzyme through phosphorylation;  Ligand-gated channel, where the binding opens a chemical gate to allow movement of ions, typically Na+, K+, Ca2+ or Cl–; and  Enzyme-linked receptor, where the addition of the enzyme forms a dimer out of the receptors, which activate them to become kinases. Lipid-based signals These are typically referred to as steroid signals, which are lipids and are based upon the basic sterol structure of cholesterol, shown below. Very commonly steroid ligands bind to receptors that are present within the cell. These signals, once bound to their receptors, are often imported into the nucleus and begin the next stage of cell signaling. Amplification or transduction During this step, we take the one signal/receptor model and attempt to “blow it up” to having multiple signals. This allows for multiple outcomes to happen as a result of the signal or allows for there to be enough machinery within the cell to make the response actually occur. Protein-based signals For the most part, this requires the use of what are known as protein kinases. These are enzymes that 2 Lab 4

add phosphates (so they phosphorylate) to other proteins. Although this is not 100% true always, the addition of phosphates tends to make a protein go from inactivity to activity. Typically we steal phosphates from ATP or some other energy molecule. (Remember those? Can you name more?) The catch is, we can’t keep proteins in a constant state of activity, so we need an off switch. “Off” switching the proteins is conducted by protein phosphatases, which remove phosphate groups. Thus, we get a battle between kinases and phosphatases:

Image 3: Relationship between kinases and phosphatases Which one wins? Depends if we are activating more kinases due to a signal or not. It’s a remarkably smart system that doesn’t require an off-switch signal! But what you can get is from one starting signal, perhaps 100 molecules are activated; each of those 100 molecules can activate 100 molecules…and you can see how this will amplify. Lipid-based signals That sounds well and good, but what about for the steroid ligands? How do they do this step? Well, some of them do activate kinases as above, but these serve mainly as transcription factors. As you’ll learn later, these aide in the transcription of DNA into RNA, which is part of the gene-expression pipeline. Some of the gene products that are expressed first are used to make more genes becoming expressed…which, in a sense, is like the kinase cascade. One gene can turn on two genes, which turn on five genes, etc.

Image 4: Steroid signal activity Secondary messengers Sometimes transcription factors and kinases aren’t enough. Cells can also recruit the use of secondary messengers, which are small molecules that can trigger a variety of effects independently of the kinase cascade or gene expression mechanisms. These include calcium ions (Ca2+), cyclic AMP (cAMP), inositol triphosphate (IP3) and diacylglycerol (DAG). We need not focus on their mechanisms of study – although I’m sure some of you know what Ca2+ can do in skeletal muscle and neurons if you’ve taken a physiology course. 3 Lab 4

Image 5: Examples of various secondary messengers Response to the signal This particular step of cell signaling can happen in a multitude of ways. Cells can release chemicals as a response. They can divide. They can start to move. They can change their shape. These are rapid changes that are at the ready to occur. These are all cellular responses. Both steroid and protein signals can trigger these, although they tend to be protein signals. With steroid signals, they tend to produce genetic responses. Gene products start to be produced, altering the cell’s biochemistry and yield a much slower response. Gene products can show up in waves, so a steroid signal can have long-lasting effects, far beyond that of a protein. Recall that everything results in an amplification of the initial signaling. Below is one such example.

Image 6: Example of amplification pathway So, how do you know what a signal will do? The easiest way to find out is to either look it up or add it and find out yourself. We are going to examine how this all occurs through the lens of a neuron and examine how it does its job. For that, we need a brief refresher about neuron anatomy. It won’t be tedious, as your lab will do more than enough to fill in the gaps. 4 Lab 4

Neuron anatomy The basic structure of the animal neuron is relatively conserved throughout evolution. The soma, or cell body, contains most of the organelles we have come to know and love in eukaryotic animal cells. Off of the soma are branch-like extensions known as dendrites (“branches”), that are surrounded sometimes in a layer of fatty membranes that are not produced by the neuron. Neurons vary in the distribution, size and number of these dendrites. That fatty membrane layer is called myelin, and aid in the function of the neuron. On one side of the soma is a long extension, called the axon, which is attached to the soma by a swelling known as the axon hillock. The axon, much like the dendrites, is covered in myelin. The ending of the axon has a series of terminal boutons that resemble dendrites in a sense. Neuron function The language of the neuron is called an action potential, which is a temporary alteration of the membrane potential. (We’ve dealt with the membrane potential briefly with how ATP is produced.) It is akin to binary, where we alternate between 0s and 1s, or to a light switch, where lights are on or off. The role of the membrane pumps and gates are to control whether or not an action potential occurs, and, if so, to have it move from the dendrites, where the signals to “fire” are received primarily, to the hillock, where the final decision to fire or not occurs, and down the axon to the terminal boutons. The synapse and neurotransmitters The gap between the terminal boutons and the next membrane is called the synapse or synaptic cleft; we refer to the membranes as either presynaptic or postsynaptic. In the terminal boutons, vesicles that contain chemicals known as neurotransmitters are signaled to bind to the membrane and release their contents into the synapse. These neurotransmitters bind to receptors on the postsynaptic membrane, where their receptors are either ligand-controlled Na+ gates or ligand-controlled K+ gates. Certain neurotransmitters bind to only Na+ gates, while others only bind to K+ gates. A few bind to both, but it depends on location in the nervous system.

Image 7: The synapse showing the pre and post synaptic membranes Each neuron will have, on average, 7000 synapses with other neurons—either to send neurotransmitters or to receive neurotransmitters. This amount of interaction leads to the complexity of determining the next step in the cell signaling cascade of neurons.

In Class We are going to walk through Action Potentials Explored, which you will find on SimBio. This is the same 5 Lab 4

application as previously used. There are two sections of simulation we will work with; you are encouraged to read the text and attempt the problems on your own, although they are not required for this lab exercise. Section 1: Neurons Communicate, focusing on pages 2, 4-5, 8-9, 12, 14, 15. Page 2: you are told the number of neurons the average brain contains. Recalling each neuron makes 7000 connections, how many connections exist within your brain? (If the math perplexes you, let’s look at a simpler example. If one neuron makes 5 connections, there are a total of 5´1 = 5; if there are two neurons, then there are a total of 5´2=10. We are not looking at the combinations, which is a more complex calculation.)

Page 4: What is the path a pain neuron takes when you hit your finger with a hammer? How long is this distance on you? These signals tend to travel at around 100 m/s; how long should this take?

Page 5: Show the basic anatomy of neurons by including a screen capture of various neurons. Figure 1. Neuron examples. Pages 8-9: Describe what happens in terms of membrane potentials during a hammer strike to the finger. What part of cell communication would this be?

How does the position of the electrode affect the resulting graph?

Page 12: This is a bit more muscle physiology than cell communication, although we can connect the dots if we need to. (We should.) If we view an action potential, or a signal from the neuron, as the first step of cell communication with a skeletal muscle, try the following pattern. Make graphs that show the following trends and paste these into the boxes. o One pulse in 1 s. Wait 1 s. One pulse in 1 s. Figure 2(a) o Ten pulses in 1 s. Wait 1 s. Ten pulses in 1 s. Figure 2(b) o Twenty pulses in 1 s. Wait 1 s. Twenty pulses in 1 s. Figure 2(c) What patterns do you observe between pulses, time and tension? Provide support from above.

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Page 14. Repeat above if you add botulinum toxin, an excretion from the bacterium Clostridium botulinum: trends and paste these into the boxes. o One pulse in 1 s. Wait 1 s. One pulse in 1 s. Figure 3(a) o Ten pulses in 1 s. Wait 1 s. Ten pulses in 1 s. Figure 3(b) o Twenty pulses in 1 s. Wait 1 s. Twenty pulses in 1 s. Figure 3(c) What do you suppose this toxin is doing to cell communication? Justify your response.

Page 15: Lidocaine is a chemical derivative of cocaine and is meant to diminish the sensation of pain as it is used when you go visit a dentist. Describe how it affects action potentials and the delivery of those signals after striking a finger with a hammer.

Section 2: Ion Movement, focusing on pages 2-3, 5, 7, 12-17. Page 2: Ions are the true drivers of what occurs during action potentials. Follow the directions found on the page, then answer the question presented, Q2.1. Place a screen capture of your response that is correct below. Figure 4. Page 3: Now get to watch the membrane potential in real time. Notice the initial concentrations of Na+ and K : more Na+ outside the cell and more K+ inside the cell. Please note that they incorrectly label the ion gates as ion channels. Recall we cannot close or open channels. Describe the resulting membrane potential and ion movements based upon the configurations of Na+ and K+ gates below. Remember to reset between tests! Na+ Gate K+ Gate Observations Closed Closed Open Closed Closed Open Open Open +

Page 5: Repeat the above test, but rather than describing your observations, paste the graph that is produced. Na+ Gate K+ Gate Observations Closed Closed Open Closed 7 Lab 4

Closed Open Figure 5.

Open Open

Page 7: There are five key points to recall regarding membrane potentials and action potentials. List them below.

Page 12: Shift the membrane potential. How does the voltage gate change? Would you consider this a K+ or Na voltage gate? Justify your response. +

Page 13-14: We see that the action potential continues to propagate under one of the two conditions. What part of the cell communication requirements are you observing? How do you know? What condition allowed for the propagation?

Page 15-16: What must occur after gate activation in order to ensure the membrane potential can be restored? Explain how this makes sense.

Page 17: Assemble the correct picture and paste it below. Figure 6. Section 3: Action Potentials Challenge, focusing on pages 2-3. Page 2: We get to play with manipulation of the transduction of the action potential. Your goals are to determine how   

gate density affects the propagation and restoration of the membrane potential, how K+ gate opening speed affects propagation and restoration of the membrane potential, and how K+ gate closing speed affects propagation and restoration of the membrane potential.

Describe your “experimental” approach to answer these three bullet points. Give enough detail so that I could replicate your approach.

What type of data did you examine to address these three goals?

Give your three claims that address/answer these goals.

Page 3: We get to manipulate two major factors that are rather important in membrane potentials:  conductance of the ions – how easily they may move through the membrane, and 8 Lab 4



intra- / extra- cellular concentrations of Na+ and K+.

Determine the balance of conductance and ions to allow for a resting membrane potential that is yields all of the following. You may report the numbers or use screen captures, whichever is easier for you. Membrane Potential (mV) Conditions + 70 + 55 + 30 – 30 – 55 – 70

Follow-up questions Complete the following questions regarding your experiences from this lab. 1. Look back at part 2, page 12. Explain, in terms of protein structure, how this opening and closing occurs.

2. Taken in total, we saw all three portions of cell communication if we look at sections 1 and 2, especially if we focus in on muscle contraction. Describe those three stages of cell communication by using sections 1 and 2.

3. The last portion of section 1 involved myelin, which is a substance that alters the rate of the action potential. How is the presence or absence of myelin akin to the use of kinases and phosphatases?

4. Explain, based upon your previous knowledge, how the initial ion distributions and concentrations could be formed. You may wish to look at previous lab activities.

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