Cell Diff test 3

Lesson 15

The actin cytoskeleton - Actin cytoskeleton focusing on non muscle cells - Actin filaments have certain basic similarities to microtubules but their structure is unique and allows them to function in a number of critical cell processes in nearly all types of eukaryotic cells

Lesson 11

communication basics/ ion channels - concepts related to how cells talk/communicate with other cells. Also referred to as cell signaling - how nerve and muscle cells communicate via changes in membrane potential

Lesson 13

communication: RTK signaling - The third major class of cell surface receptors, the enzyme coupled receptors, in some cases these are coupled to a separate protein that functions as an enzyme, hence the name enzyme coupled receptors, but more often the receptor has its own enzymatic domain that can become activated and carry out reactions to transduce a cellular signal into an intracellular signal - How are hydrophobic molecules detected

11. Neurons are polarized cells

- "antennae" where signals are detected / sensed - where signals are released to other cells - Since ion channeled coupled receptors are associated with nerve cell signaling, now is a good time to look at the morphology of a nerve cell or neuron - Neurons are polar cells, this does not mean polar in the sense that it is able to form hydrogen bonds with water. When we talk about cells and use the term polar, we are talking about cells that are asymmetric, one portion or one end is different from the other. In the case of neurons, there are several extensions of membrane referred to as dendrites at one end, these protrude off of a structure known as a cell body, where the nucleus, ER, and Golgi are located. The dendrites are where signals are detected, next an electrical signal travels along an extended protrusion, known as the axon. The axon can vary from less than one millimetre in length to more than a meter in length. Each of us has a nerve cell where the cell body is located near the base of our spine and the axon extends down the length of our legs toward the big toe. At the end of any axon is a nerve terminus or terminal where electrical signals are converted back to chemical signals and those signals are released into the synapse to trigger other cells to respond, those other cells could be nerve cells or muscle cells or sometimes various types of glandular cells

14. Microtubules are assembled from globular ab-tubulin dimers

- 13 protofilaments form MT wall - Both bind GTP but only beta can hydrolyze GTP - MTs are polar due to head-to-tail assembly of dimers - Unlike intermediate filaments, which have a number of different types like keratins and lamins, there is only one type of microtubule. - The fundamental subunit of a microtubule is a heterodimer composed of alpha and beta tubulin - In the upper left of this fig, a cartoon tubulin and heterodimer is shown in which alpha is a light green sphere, and beta is a dark green sphere - Alpha and beta tubulin are globular proteins, unlike the fibrous intermediate proteins. We never find alpha or beta individually, they are always found as a dimer - Both alpha and beta bind GTP, but only the beta tubulin can hydrolyze GTP, and this ability of the beta to hydrolyze GTP is critical for the control of assembly and disassembly of microtubules - Microtubules are tubular structures. A wall of a microtubule is formed from 13 structures referred to a protofilaments. Each protofilament shown in panel A is an arrangement of head to toe assembly of tubulin dimers - Microtubules are polar structures because head to tail assembly into a protofilament creates one end in which beta tubulin is exposed and another end in which alpha tubulin is exposed. The end at which beta tubulin is exposed is termed the plus end, and end where alpha tubulin is exposed is termed the minus end. Plus and minus are arbitrary terms, they do not imply anything about electrical charge ad there is not an electrical charge distance between the two ends of the microtubule. - The 13 protofilaments assemble laterally to form a wall of microtubule as shown in panels B and C. Panel B is the end on view of a microtubule, so in this case, given that we are looking into the dark green end, we are looking at the plus end - Notice the open structure referred to as the lumen shown in gray, that is the inside of the hollow tube. Given both the head to tail assembly and a lateral association of protofilaments, any tubulin dimer, except those at the very end will have another subunit in front of it, behind it, and on either side of it - The cartoon views in panel B and C are based on electron microscope views shown in panels D and E

15. Actin Binding Proteins (ABPs) control actin assembly, organization, movement 2. Organizers

- Actin filaments more _________ than MTs => actin usually found as__________ - The second group of ABPs act as organizers - Actin filaments are much more flexible than microtubules, they are much thinner and lack a hollow core structure that actually provides an amount of rigidity to microtubules. We typically find actin filaments in bundles, which have greater structural integrity and more strength to allow the actin filaments to preform their function. - 3 types of organizers are shown in this Fig. starting at the right most red outline and moving clockwise: (1) bundling proteins help to bundle actin filaments together. The bundling protein here is shown as the green oval. (2) side binding proteins help to stabilize and organize the actin filaments. These are very critical for actin filaments in muscle cells. (3) cross linking proteins are found in the cell cortex. These green Y shaped proteins help to create networks of actin filaments that help to support the plasma membrane. The actin filaments in each example are the same fundamental actin filament, it is the specific actin binding protein that determines what array the actin filaments will form which determines the function of the actin filament in a particular cell or particular part of the cell

12. Signal amplification in rod cells

- Amplification occurs in all G protein-coupled pathways - Transducin = Gt - - Rod cells are classic examples of signal amplification. This process is thought to occur in all signal pathways, but is relatively easy to quantify in rod cells. In this case for each photo absorbed, each rhodopsin in turn is capable of activating 500 transducin molecules. Transducin is just another name for the Gt protein shown in the previous slide. Each transducing molecule in turn activates one cGMP phosphodiesterase so there is no amplification at this step, but those 500 cGMP phosphodiesterase molecules can hydrolyze 10^5 cGMP molecules. In turn this reduction in cGMP levels cause approximately 250 sodium channels to close, because the sodium channels close, roughly 1- 10 million sodium ions per second are preventing from entering the cell for a period of roughly one second, this causes the rod cell membrane to be hyperpolarized by one millivolt. This is one example, but amplification is believed to occur in all GPCR pathways, and probably most all types of signal pathways

15. Actin Binding Proteins (ABPs) control actin assembly, organization, movement 1. Assembly regulators

- Another similarity between actin filaments and microtubules is that actin filaments also have a number of associated proteins that control their assembly organization and movement - The proteins that carry out these actions or actin filaments are referred to as actin binding proteins (ABPs) and there are a variety of different classes of these ABPs. The first group are proteins that regulate actin assembly. The major examples in the Fig are outlined in red. Starting at the upper left and going clockwise: Nucleating protein serve to stimulation the formation of actin filaments from actin monomers. The next example of assembly regulators are the monomer sequestering proteins, these proteins bind to actin monomers and keep them from assembling into filaments, if a monomer sequestering protein was active we tend to find the actin in the G actin form rather than in filaments. At the bottom, capping or plus end blocking proteins help to stabilize actin filaments and are ways to adjust the normal treadmilling behavior described previously. Severing proteins bind to and sever actin filaments, actin filamnets are then converted from long polymers into very short polymers changing the functional properties of those filaments - Different actin binding proteins can operate in different regions of the same cell at the same time - Actin filaments depending on which actin binding proteins they tend to be interacting with can carry out different roles in the same cell at the same time

13. Monomeric G proteins like Ras are turned off by GTPase activating proteins (GAPs)

- triggers G protein to hydrolyze GTP - In the case of monomeric G proteins, like Ras, these proteins are stimulated to undergo GTP hydrolysis by a group of proteins called GAPs (GTPase activating proteins) - GAPs work in opposition to GEFs (Guanine Exchange Factors) which activate monomeric G-proteins, so if the cell wants to switch from the active Ras GTP state at the bottom of the figure to inactive Ras GDP state, a GAP will come along and interact with Ras GTP and stimulate the Ras protein to carry out GTP hydrolysis and release the inorganic phosphate - The G protein, not the GAP, carries out the hydrolysis reaction, the GAP just triggers the G protein to carry out this reaction

12. Receptor activation of trimeric G proteins

- Both alpha and beta-gamma can alter activity of target protein but alpha is most common and best understood - Signaling via GPCRs involves activation of the couple G protein by the GPCR. In part A at the top, the receptor protein is floating in the plasma membrane, on the interface of the membrane is a trimeric G protein. Trimeric in the way that is has three subunits, alpha, beta, and gamma. In the absence of signal the G-protein is in its off state meaning it had GDP bound to it. - Note that the alpha subunit is the one that binds and hydrolyzes the guanine nucleotide. Notice also that the alpha subunit and the gamma subunit have lipid anchors, so these are integral membrane proteins associated with and able to diffuse around on the interface of the lipid bilayer. When a signal molecule is recognized by the GPCR as shown in part B, the receptor undergoes a conformational change so that the cytosolic domain is now able to interact with the G-protein. When a G-protein bumps into the receptor with a bound signal molecule, the receptor stimulates the G-protein to undergo nucleotide exchange, GDP is then released, and a new molecule of GTP is bound. This in turn activates the G-protein which separates away from the activated receptor as shown in part C, and then separates into two components, the alpha subunit with bound GTP and a complex of the beta and gamma subunits. Both parts are ow activated, they can diffuse away on the interface and interact with target proteins referred to as effectors and thereby alter the activity of these effector proteins - Although both the alpha and beta gamma proteins are capable of altering the activity of effectors, the alpha with bound GTP is the best understood of the two.

12. calmodulin is activated upon binding calcium

- Ca++ binds to and activates calmodulin - CaM kinase is most common target - CaM kinase phosphorylates Target poteins on S/T - Ca++ / calmodulin binds to and activates target protein(s) - Calcium release from the ER into the cytosol leads to the activation of several proteins. One important target is the protein calmodulin. Calmodulin is an extended protein with calcium binding globular regions separated by an extended alpha helix. When calcium binds to the calmodulin the protein becomes active and the calcium calmodulin complex can bind to and activate specific target proteins. In the example shown, calcium calmodulin binds to and wraps around a portion of a target protein labeled CaM kinase, cam stands for calcium calmodulin and the binding of calcium calmodulin to the CaM kinase activates this enzyme to phosphorylate its target proteins thereby changing the activity of those target proteins

11. Channel proteins and nerve → muscle signaling

- Ca++ is common trigger for regulated secretion in various cells - Not all NT receptors are ion channels - When the action potential reaches the nerve terminus, the electrical signal containing the action potential is converted into a chemical signal in the form of a neurotransmitter - The left portion of this figure shows a resting nerve terminal, notice here there is a voltage gated calcium channel in the nerve cell membrane and the channel is closed. Also note there is a vesicle termed the "synaptic vesicle" that contains the neurotransmitter. In synaptic signaling, the signaling cell is referred to as a presynaptic cell and the target cell is referred to as a post synaptic cell. The post synaptic cell could be another nerve cell, it could be a muscle cell, but note it contains the neurotransmitter receptor on its plasma membrane, this is an ion channeled coupled receptor that is extracellular and ligand gated. The extracellular ligand in this case is the neurotransmitter. When the action potential reaches the nerve terminal as shown in the middle panel, the electrical signal that is the wave of membrane depolarization triggers the voltage gated calcium channel to open and calcium enters the cell. Recall that calcium influx is one of the more common ways to trigger regulated exocytosis or secretion at a defined time. There are a wide variety of known transmitters involved in nerve cell and muscle cell signaling. In the ion channeled coupled receptors found on the post synaptic cell, allow different types of ions into that target cell. If the ions depolarize the post synaptic cell and action potential will be initiated and propagate around that cell, if the ions hyperpolarize the postsynaptic cell, that is make its membrane potential more positive, that will suppress the formation of an action potential in the post synaptic cell. Although the vast majority of neurotransmitter receptors are ion channels, there are other types of surface proteins that also detect neurotransmitters and can initiate changes in membrane potential in neurons and muscle cells

14. MTs are nucleated and organized by the centrosome

- Centrioles are MT bundles but gamma-tubulin rings nucleate MTs - interphase cell: radial array - dividing cell: bipolar array - When we look at microtubules in cells they are organized in particular patterns or arrays. Such organization emanates from a central structure referred to as a centrosome, which is a relatively amorphous complex of a large number of proteins as indicated in the cartoon centrosome shown in the figure at left. An animal centrosome contains two small microtubule based structures called centrioles. The function of these microtubule bundles is still unclear, however it is clear that they do not serve as nucleation points or templates to seed assembly of microtubules, that role is instead carried out by ring structures that are created from gamma tubulin, a relative of alpha tubulin and beta tubulin. These ring structures serve as nucleating sites and tubulin heterodimers can assemble onto these rings to form the 13 protofilament microtubule structure. Additional tubulin dimers then add onto the plus ends so the microtubules grow away from the centrosome, with the minus end attached at the centrosome and the plus end extending distally towards the cell periphery - In the top right Fig we see the typical radial array of microtubules. The centrosome is shown as a light green oval with two black centrioles in the center. The microtubule minus ends are associated with the centrosome and the microtubule plus ends approach the plasma membrane. Most of the action in terms of assembly and disassembly in a normal cell occurs at plus ends, since the minus ends are attached to the centrosome - When a cell in interphase moves into M phase the centrosome duplicates and we end up with two radial arrays that are attached to each other. This bipolar array, shown at bottom is actually what forms the mitotic spindle

11. Signal transmission along axons

- Change in membrane potential travels from cell body to nerve terminus - Moving wave of depolarization is termed action potential - Channel receptors play critical roles in the signal transmission from dendrites to the nerve terminus. If we were to place different voltmeters in different regions of the axon, as describe in the Fig., we can measure the voltage change over time as a signal propagates along the axon. In this particular situation at time 0, there is a positive voltage change detected on voltmeter 1, this region has been what is referred to as depolarized. The 0 voltage baseline in the fig represents the resting membrane potential. At a later time, one millisecond site, the voltmeter at V2 now detects the depolarization event. Notice that when time is 1 millisecond, the voltmeter at V1 has returned back to resting potential. The movement continues as a wave of membrane depolarization moving from the dendrites at left toward the nerve terminus at right, this change in membrane potential as a function of time and location is referred to as an action potential

11. The action potential depends on voltage-gated Na+ channels

- Channels exist in three states: open, inactive, and closed - Transition from open to inactive state controlled by "timer" --> produces directional depolarization (prevents back-flow)

14. The axoneme forms the core of cilia and flagella

- Dynein-powered sliding of outer doublets is converted to bending by various crosslinking MAPs - The microtubule bundle present in cilia and flagella is referred to as an axoneme, it is a highly ordered and complicated bundle of microtubules and associated proteins - In panel A we see an electron microscope view of the cross section of an axoneme and panel B shows a cartoon view with several identifiable components - Microtubules are shown in green and are somewhat special microtubules in that they have a normal 13 protofilament structure referred to as an A microtubule and then piggy backed on that they also have an incomplete microtubule referred to as a B microtubule. Together the A and B microtubules are referred to as an outer doublet, and within an axoneme there are 9 such outer doublets organized around a central pair of normal microtubules. This organization is set up by a variety of MAPs, these maps are actually cross linking maps that help to form the microtubule bundle examples in this diagram include nexin shown in blue and the radial spoke proteins shown in brown. Each doublet, specifically the A microtubule has two types of dynein motors associated with it, one called outer arm dynein and one called inner arm dynein. The microtubules in this structure are very stable which is important for the action and the structure of the axoneme, so dynamic instability has to be suppressed by various MAPs not shown in this Fig. The dynein's are related to the dynein's we have already discussed. The motor proteins reach out and try to walk along the adjacent outer doublet microtubules with the dynein's in a sense trying to slide the one outer doublet past the other. The sliding action is tempered by the various crosslinking MAPs which prevent sliding, but sense the force generated has to be released somehow that sliding force is converted to bending force due to the constraints provided by the crosslinking MAPs

14. Nerve cell morphology

- ER & Golgi - It turns out that neurons, also represent a good Segway into the topic of IFs and MTs - An important issue in terms of synaptic signaling is that many of the proteins and peptides that function as neurotransmitters are actually products of the secretory pathway - Extracellular signals in the form of neurotransmitters are detected by the dendrites on the left, which initiates an action potential that travels along the axon and arrives at the nerve terminus on the right where neurotransmitter release is triggered to pass the signal on to another cell - If we look for the membrane bound organelles within the neuron, the nucleus, ER, and Golgi are all located in a structure called the cell body. Both dendrites and the axon can be considered membrane protrusions from the cell body. Any vesicle that buds from the Golgi will start off in the cell body. Many of these vesicles, especially those that contain neurotransmitters and plasma membrane proteins like a voltage gated calcium channel, will have to travel through the cytosol in the axon and eventually be delivered at the nerve terminus - Unlike the action potential, which occurs on the order of milliseconds, move into the vesicle if it was just to occur through the process of simple diffusion within an axon will take hundreds to even thousands of years. This tells us that the cell has to have a mechanism to more quickly move vesicles down from the cell body to the terminus. In humans, the longest nerve cell is roughly a meter in length with the cell body at the base of the spine and the nerve terminus near the big toe. Calculations here indicate that if a vesicle was just to diffuse within the axon from the cell body of the nerve terminus it would take more than 3,000 years for that vesicle to arrive, so simple diffusion cannot account for long distance vesicle transport on a reasonable timescale. In addition that neuron is under a great deal of physical stress as we move around. The axon is a relatively narrow structure and is twisted, pulled, bent, and stretched in various directions as our legs move so the cell has to have some mechanism to resist those physical stresses - Both the directed movement of vesicles within the axon and the resistance of physical stress result from the actions of cytosolic proteins that make up the cytoskeleton

13. Regulation of G proteins in the enzyme-coupled receptor pathway

- Example: The Ras (rat sarcoma) G protein - Guanine Exchange Factor stimulates nucleotide exchange - GEF is an intracellular signaling protein - Here is a more specific example involving The Ras G protein (ras stands for rat sarcoma) - In this Fig. the receptor has already been activated and undergone transphosphorylation following interaction with a signal molecule, which would be a growth factor - An adaptor protein shown in dark blue contains an SH2 domain and is able to interact with phosphorylated tyrosine. In doing so another protein, called The Ras activating protein is brought to the intracellular signaling complex and becomes activated - The Ras activating protein is a specific name for a general class of proteins called guanine exchange factors. - Guanine exchange factors stimulate nucleotide exchange by G-proteins, in this case, Ras, which is the G-protein is shown in green. Ras, unlike the trimeric G-proteins covered in the previous lecture, is a monomeric, single sub-unit protein - In the resting state Ras is inactive in that it has GTP bound to it. Ras also has lipid anchors attached to the interphase of the plasma membrane. Ras can diffuse across that surface until it comes into contact with an activated guanine exchange factor or GEF, then that guanine exchange factor can stimulate nucleotide exchange. Ras lets go of GDP and binds to GTP and the Ras protein is now in the active form. GEF in this case is an example of an intracellular signaling protein, it was brought to the complex indirectly through interaction with an adaptor protein, became activated, and then activated another component that allowed for the onward propagation of the signal. Much is known about the Ras protein because it is mutated in about 30% of all human cancers. In these cancers Ras cannot hydrolyze GTP, so it can be activated, but not inactivated, and therefore stuck in the active state, which continues to propagate the signal and response. RTK pathways are commonly used to detect growth factors, so the end result of this Ras mutation is that the cell thinks it is constantly being told to reproduce

15. Actin filaments exhibit treadmilling in the cell

- Filament length is constant (assembly at one end = disassembly at other end) - Actin filaments are polar - ATP hydrolysis occurs after assembly => weakens bonds between subunits - Similar to microtubules actin filaments undergo a distinctive assembly behavior that depends on a bound nucleotide - Actin filament assembly occurs via treadmilling rather than dynamic instability and the nucleotide is ATP rather than GTP - During treadmilling shown in the left, Actin filament length remains constant as assembly at one end leads to disassembly at the other end. Actin filaments are polar, one end is different from the other. This is most easily observed as the differences in assembly and disassembly at the two ends - Although the subunits at the two ends appear the same by electron microscopy, there structure is subtly different, and this is related to which nucleotide, ATP or ADP is associated with the subunit. If we look at the right end of the figure at right, then actin with ATP bound to it, AKA ATP actin, is able to assemble on to the end of an actin filament, specifically the plus end of the actin filament - Similar to microtubule assembly hydrolysis of the nucleotide, ATP in this case will occur after assembly and this weakens the bonds between subunits. Like microtubules assembly and disassembly are limited to the ends of actin filaments, so that even though the subunits have weakened bonds with their neighbors, they still stay within the filament, until they get to the end of the minus end of the actin filament. When they get here they fall off as ADP actin. That ADP can later be regenerated through nucleotide exchange. The actin subunit lets go of ADP and can bind to a new molecule of ATP to be ready to add to the plus end of another actin filament. If we look at a particular subunit undergoing assembly in this manner, as shown in the left Fig, it would add to the plus end and overtime it would move toward the middle of the actin filament as additional subunits were added at the plus end, and subunits were lost simultaneously at the minus end, eventually the subunit will pop out on the minus end and be released to have its nucleotide regenerated. This movement of subunits within the filaments is what meant by treadmilling - Energy from hydrolysis is not needed to assemble the filament. Hydrolysis actually sets up the disassembly of the filament and if we were able to block ATP hydrolysis for actin, filaments would just continue to grow. The same is true of microtubules, if we block GTP hydrolysis from microtubules, these filaments would also continue to grow and would not be able to disassemble

14. The cytoskeleton consists of three dynamic protein filament networks

- Filaments are assembled from protein subunits held together by noncovalent bonds - Subunits: keratins, neurofilaments, lamins cytoplasmic or nuclear arrays resist mechanical stress, support nuclear membrane - Subunit: ab-tubulin dimer radial array vesicle transport, organelle positioning, mitosis, cell swimming - Subunit: G-actin Cell cortex cell shape, cell contraction, cell crawling - Previous slide describes just one of the roles of the cytoskeleton in a particular type of cell - The cytoskeleton in some form is found in all types of eukaryotic cells - In animal cells the cytoskeleton consists of three different protein filament networks, each network is a dynamic system meaning that protein subunits are held together by noncovalent bonds so those subunits can rapidly assemble and disassemble as required to form or eliminate the filaments - Each of the filament systems has certain functions and processes in which they play critical roles. - In this diagram all three filament systems are highlighted in intestinal epithelial cells. In addition we cell an electron microscope view and a cartoon view of the subunit structure that forms each of the three filaments ; intermediate filaments, microtubules, and actin filaments - There are a variety of types of intermediate filaments and their subunits. Keratins, which are found in our epithelial cells, hair, and nails. Neurofilaments which are found in nerve cells. Lamins which help support the nuclear envelope. MOst of these filament systems are found in cytoplasmic erase, which is found in the cytosol. Lamins are found in the nucleus and form a nuclear array. The blue filaments in the intestinal epithelial cell cartoon, represent an example of a cytoplasmic array, but if you look closely, you can see that the brown nucleus actually has a blue line around its rim and that is supposed to represent the nuclear lamins. The job of the intermediate filaments is to resist mechanical stress on cells. In the previous slide when the nerve cell resisting mechanical stress was discussed, that was the job of neurofilaments. The nuclear lamins support the nuclear membrane. So the job of intermediate filaments is to provide structural support in animal cells - Microtubules have other functions and arrangements within eukaryotic cells. The subunit that makes up a microtubule is referred to an alpha-beta tubulin dimer. If you look at the cartoon view of the microtubule, note that there are alternating light green and dark green dots, those dots represent the alpha and beta tubulin, two related proteins that together form a dimer. It is the assembly of that dimer that generates the microtubule structure. In most types of cells, microtubules form a radial array, then emanate from a central structure and are referred to as a centrosome or a microtubule organizing center and they extend toward the cell periphery in a radial pattern. Microtubules are involved in vesicle transport and axons are chalk full of microtubules because they serve as roadways for the movement of vesicles traveling from the cell body to the nerve terminus. Microtubules also play a role in organelle positioning, that is where the cell locates its ER, Golgi, Mitochondria and even its nucleus. Microtubules play a critical role in mitosis where they form the framework for the mitotic spindle and they preform a role in cell swimming, which is exhibited by sperm cells or unicellular eukaryotes - The actin filaments (F-actin) are formed from actin monomers or actin subunits called G or globular-actin. In most types of eukaryotic cells, we find actin filaments at the cell cortex, a region just underneath the plasma membrane, these actin filaments play a role in cell shape, so you also notice the vili that protrude at the top of the intestinal epithelial cells have actin filaments with in. Actin filaments play a role in cell contraction, most commonly muscle cell contraction, but they actually form the contractile ring which divides one cell into two. Although we often think about actin having a major role in muscle cells, those are specialized cells. In reality all types of eukaryotic cells have actin filaments, and they use actin filaments occasionally for contraction, but also for other functions like cell crawling. This is the ability of our white blood cells to crawl around our body to sites of infection and it depends on actin filaments

11. Signal transduction .

- For target cell to respond to signal - extracellular signal must be transduced into intracellular signal - A critical aspect of cell signaling is the ability of a target cell to recognize information in the form of an extracellular signal and can convert that information into an intracellular signal that will lead to an appropriate response. This conversion is referred to as signal transduction since the information represented by the extracellular signal is transduced across the plasma membrane to a different representation in the form of an intracellular signal

14. Dynamic instability : MTs alternate between phases of growth and shortening

- If we watch individual microtubules in a cell we see that the filaments undergo an interesting form of assembly in which the ends alternate between phases of growth and shortening in the cell because myosin's are attached to the centrosome. This behavior called dynamic instability occurs predominately at the plus ends - If we watch a single microtubule over time, it will grow for some period of time and then will undergo a transition to shortening. After a while it may return back to the growing state, or it may shorten all the way back to the centrosome and then a new microtubule will grow out - This behavior allows microtubules to probe the cytoplasm and this constant growing and shortening turns out be a critical part of microtubule functions in most types of cells

12. Molecular switches in signal pathways

- GDP bound = inactive - GTP bound = active - There are a number of so called molecular switches involved in cell signaling - For GPCRs the most important switch is the G-protein to which the receptor is coupled (interacts with). G-proteins bind GTP and can hydrolyze the GTP to GDP. Whether bound to GTP or GDP determines the active state of the G-protein - In the right panel, a G-protein at top is in the off or inactive state when GDP is bound to it. When a signal is detected the g-protein is triggered to release the molecule of GDP, which flows away into the cytosol. In its place a new molecule of GTP binds, this process is referred to as nucleotide exchange. One nucleotide molecule is being exchanged for another. The G-protein is now in the on/active state and the signal can be passed onward. - Notice that many of these signal figures have radiating red lines that indicate when a protein is active. Sometime later when the signal goes away the G-protein will hydrolyze the GTP to GDP and an inorganic phosphate will be released. The G-protein is then switched off - In sum, a G -protein that is GTP is active, but a G-protein that is GDP bound is inactive and they can switch back and forth between these states depending on input signals

12. GPCRs are a large family of 7-pass transmembrane proteins

- GPCRs are involved in responding to • some neurotransmitters • odors and tastes • photons • various endocrine hormones - Roughly half of known drugs bind to GPCRs (most inhibitors, some potentiators) - GCPRs represent a large family of related 7-pass transmembrane proteins, meaning each protein passes through the membrane 7 times, as shown in the fig - Cells utilize GPCRs to detect a variety of signal molecules including some neurotransmitters, odors and tastes, photons and a variety of endocrine hormones - Each member of the family shares a conserved transmembrane domain and is somewhat similar to the cytosolic domain, that is the region of the protein exposed on the cytosolic side of the plasma membrane, but they differ in the extracellular domain, which allows each family member to detect a different extracellular signal molecule. - The relative importance of GPCRs in cell signaling and in life in general can be recognized when one considers that approximately ½ of all known drugs target GPCRs. Most of these drugs represent inhibitors, which shut down responses dependent on GPCRs while some represent potentiators, drugs that activate the receptor and induce the response

13. The trimeric G protein shuts itself off via GTP hydrolysis

- GTP binding or interaction of G protein with target protein activates a "timer " that eventually triggers GTP hydrolysis and inactivation of G protein - In the case of trimeric G proteins, the G protein will shut itself off via spontaneous GTP hydrolysis - In this Fig we start with an activated G protein at the top, the binding of GTP to the Alpha subunit, or the interaction of the G protein with its target effector enzyme, typically activates a timer, which is really just a tension or stress in the protein structure, such that eventually GTP hydrolysis will be triggered and the G protein will become inactivated. Once the alpha unit hydrolyzes to its bound GTP, then it will interact with the Beta-gamma subunits and reform the inactive trimeric protein

14. Dynamic instability depends on a GTP cap

- GTP-Tb adds to plus end - Hydrolysis after assembly weakens bonds w/ neighbors - Shortening MTs have lost the GTP cap - Growing MTs have a GTP cap - The ability of microtubules to undergo dynamic instability depends on the ability of beta tubulin to both bind and hydrolyze GTP, and it is the presence of GTP vs GDP bound to this protein that is the basis for dynamic instability - Although alpha tubulin can bind GTP, because it can't hydrolyze GTP, it does not appear to play as critical a role in microtubule assembly - When talking about GTP tubulin or GDP tubulin, the identity of the nucleotide on a beta subunit is what is being talked about - In the Fig GTP is indicated as the red dots. If we start at the top of panel A, GTP tubulin is adding on to the plus end and as it adds on the microtubule grows. However, sometime after assembly the beta tubulin will hydrolyze GTP to GDP. This creates conformational stress in the protein and so the subunits that have undergone hydrolysis under GDP have a weaker affinity for their neighbors. As long as there are GTP tubulin subunits at the end of the microtubule, it will continue to grow - These GTP tubulin subunits form a GTP cap. All growing microtubules have a GTP cap, but occasionally either do to dissociation of GTP tubulin from the end or to random hydrolysis that catches up with the very end of the microtubule, GTP tubulin will be exposed at the microtubule end. Since these subunits do not associate as tightly to their neighbors, they begin to peel away from the neighbors, and subunits begin to fall off the plus end of the microtubule. The microtubule then begins to shorten and the subunits fall off. Once the GTP cap is lost, the microtubule will be in the shortening phase - The growing microtubule has a GTP cap, but a shortening microtubule has lost that cap There is some small chance the cap can be regained, in which case a shortening microtubule transitions back to the growth phase - Panel B refers to a shrinking microtubule but n reality the microtubule is not really shrinking, rather its subunits are falling off the end, so that is why a better term is shortening - An analogy that is used to commonly describe the GTP cap is a cork and champagne bottle, as long as the cork is present everything stays in the bottle, like when the cap is present the microtubule stays in the growth phase, once the cork or the cap is lost, the contents will come spewing out like in the case of the microtubule shortening

11. The action potential is a wave of membrane depolarization

- Here are two sequential snap shots of an action potential, the upper fig is the instantaneous view at time zero that was shown in the previous slide, the lower panel is the instantaneous view at time = 1mm, so 1 mm after the top panel snap shot. Notice the blue region of depolarization has moved toward the right during the intervening time, and as indicated in the previous slide, new sodium channels have opened up due to that depolarization. On the backside of the wave of depolarization, the channels have become inactivated and will eventually close. The inactivation is critical to prevent back flow by preventing further sodium from entering the cell at the back end of the wave of depolarization. Notice if we go even further back then the channels have switched from the inactivated status to the closed status, this region of the cell is being repolarized in preparation for the action potential. - To summarize, opening of the voltage gated sodium channels is critical for the propagation of the action potential for the dendrite towards the nerve terminus. The timed inactivation of the channel, even though the local membrane is still depolarized is equally important to prevent back flow of the wave of depolarization and ensure directional propagation of the electrical signal

11. Ion channel-coupled receptors are gated.

- Here we see four different types of ion channeled coupled receptors: the voltage channel gated receptor, the ligand gated extracellular signal receptor, the ligand gated intracellular receptor and the mechanically gated receptor. Each type is associated with certain nerve or muscle functions. In the case of voltage gated channels, these open when the membrane potential or voltage changes, these are critical for forming action potentials, the mechanism by which cells travel electrically along nerve cells - Ligand gated extracellular signal receptors are commonly associated with muscle contraction - Ligand gated internal channels are involved in the sense of smell and mechanically gated channels are involved in the sense of touch, so there are different ways that different channel proteins may be induced to open, but because we are talking about ion channels, opening one will lead to a change in charge distribution across the plasma membrane and hence a change in membrane potential

14. Several chemotherapy drugs target microtubules

- Inhibition of either assembly or disassembly will block MT function Either will prevent mitosis and hence cell reproduction - We know that dynamic instability is the critical for the functional role of microtubules and cells. - Evidence of this is that several chemotherapy drugs target microtubules, and in fact it is possible to disrupt the function of microtubules either by preventing their assembly or by preventing their disassembly - If assembly is prevented, then the cell will have no microtubules, so it makes sense that any cellular process dependent on microtubules would not work - If disassembly is prevented then microtubule turnover is prevented and tubulin is locked into fixed filaments. It turns out the drugs that prevent this assembly are equally If not more effective against cancer than the drugs that block assembly - In either case the reason why these drugs work is that they prevent mitosis, which depends on highly dynamic tubules by preventing mitosis cell reproduction is blocked, which makes cancer so dangerous

13. SH2 domains allow other proteins to bind to active RTKs

- Intracellular signaling proteins are recruited to P-Tyr on receptors - Many intracellular signaling proteins activate G-proteins in response to GFs - Contain SH2 domain that binds to P-Tyr =direct recruitment - Bind to adaptor proteins w/ SH2 domain = indirect recruitment -The phosphorylated tyrosine's on the cytosolic domain of a receptor tyrosine kinase represent binding sites for proteins called intracellular signaling proteins that specifically interact with phosphorylated tyrosine, or P-Tyr - Most of these intracellular signaling proteins contain a specific protein domain called an SH2 domain - Proteins that have an SH2 domain can bind directly to the phosphorylated tyrosine on another protein and are therefore directly recruited to bind to activated RTKs and form an intracellular signaling complex - Other intracellular signaling proteins may not have an SH2 domain, but can still be indirectly rooted to the complex by being able to interact with adaptor proteins that contain an SH2 domain - In this particular Fig. the intracellular signaling protein shown have been directly recruited and presumably have an SH2 domain - intracellular signaling proteins labeled in blue, orange, and purple are activated by recruitment to the intracellular signaling complex and then act to propagate the signal into the cells interior for the appropriate response - The idea that these intracellular signaling proteins are able to pass the signal on was reinforced by the discovery that many intracellular signaling proteins active G-proteins in response to growth factors.

13. Ras-GTP activates the MAP-kinase cascade

- MAP Kinase phosphorylates and activates transcription factors - Transcription factors stimulate gene expression - Protein products induce cell reproduction - Coming back to the normal situation, the previous slide described how Ras was normally activated - How then does Ras GTP, Ras with bound GTP or the active Ras, propagate the signal onward? The typical target for the Ras protein is an enzyme called Raf, AKA MAP-kinase kinase kinase. The pathway described here is commonly termed the Ras pathway or the MAP kinase pathway - By activating Raf, Ras-GTP kicks off the MAP kinase cascade, which one kinase phosphorylates and activates another. MAP in this case stands for Mitogen Active Protein, a mitogen is similar to a growth factor and often the terms are used synonymously for any signal that stimulates mitosis or cell reproduction - Ras activates Raf (Map Kinase kinase kinase) which is activated and phosphorylates its target protein which is MAP kinase kinase (MEK). MAP kinase kinase then phosphorylates and activates MAP kinase (ERK), which is extracellular signal kinase. The end result of the MAP kinase cascade is that target proteins will be phosphorylated by MAP kinase. These targets of MAP kinase are mostly transcription factors or regulators, so the primary consequence of activating this pathway is a change in gene expression - In this example if the original signal molecule was a growth factor then the expressed proteins will act to induce cell reproduction

12. GPCRs detect odors to provide our sense of smell

- Mammals code for roughly 1000 odorant receptors - Each olfactory neuron expresses one or two types - Golf activates adenylyl cyclase = [cAMP] increases => opens cation channels => action potential - Here is an example that ties together GPCRs, a second messenger in the form of cAMP, and ion channel proteins. - GPCRs are involved in detecting odors and therefor are critical for the sense of olfaction, otherwise known as smell - Olfactory neurons are specialized cells that recognize odors, these cells form part of the olfactory epithelium which lines our nasal passages - Fig shows olfactory neurons embedded in olfactory epithelium in a cartoon view and a scanning electron microscope view. The protruding structures at the top are referred to as modifies cilia, this is where the odor receptors are located. That region up there, when it detects an odor molecule will activate an action potential, which travels along the axon until the olfactory bulb is reached, and it is there that we begin to process different odors. - When we look at the GPCRs involved in the detection of odors, it is interesting to note that mammals code for 1000 different odorant receptors, that is 1000 different GPCRs just for the sense of smell. Each type recognizes a specific odor molecule, or class of odor molecules, when we look at a given olfactory neuron, each neuron expresses only one or two types of these various odorant receptors, thus each specific neuron is really only effective at detecting 1-2 types of odors - The GPCR on the surface of modified cilia recognizes the odor molecule and then turns around and activates the G-protein, and the alpha subunit of the G-protein, which is referred to as G-olf activates the enzyme adenylyl cyclase, which synthesizes cAMP - When the -protein is activated because there is an odorant molecule outside the cell, intracellular cAMP levels increase, because this is a neuron that intracellular chemical signal will lead to a change in membrane potential and in fact there are intracellular ligand gated cation channels that let sodium into the cell. As cAMP levels go up, these channel proteins open up and sodium flows into the cell which generates an action potential in the olfactory neuron. Which is then sent forward to the olfactory bulb and allows us to detect odors.

14. cilia and flagella are MT-based appendages for cell swimming and flow generation

- Microtubule motor proteins also play critical roles in certain specialized cells to allow those cells to swim or allow them to generate flow of liquid across the surface. These motor proteins and microtubules are found as part of microtubule containing appendages called cilia and flagella - In panel A we see a paramecium which contains 100s-1000s of short little appendages called cilia. Each of which is formed by a bundle of microtubules and associated motor proteins - In panel C we see a sperm with a single flagellum, that appendage is very similar to the cilia of paramecium, it just happens to be a single longer bundle - In panel B we see cilia on the surface of epithelial cell sheet that lines out lung passages. The cells don't move, rather they are fixed in places as part of the epithelial cell sheet, but the bending of the cilia creates flow across their surface which helps move gunk and other material out of our lungs

14. MT motor proteins are MAPs that transport cargo in the cell

- Motors hydrolyze ATP to walk toward specific end of MT - Not all MAPs regulate microtubule assembly or disassembly, some instead function as microtubule motor proteins which transport cellular cargo inside the cell - There are two major classes of microtubule motor proteins (1) kinesins and (2) dyneins - Most kinases move cargo along microtubules from the minus end to the plus end - Dyneins transport cargo from the plus end to the minus end - The motor protein has the ability to bind tubulin subunits, similar to a series of bricks in the road and can step from one to the next carrying their cargo - Microtubule motor proteins carry a variety of cargo along microtubules, including vesicles, organelles, and even other microtubules. This movement is directed and requires energy input - All microtubule motor proteins are ATPases that can bind ATP and hydrolyze it to power the structural changes necessary to step along the microtubule

13. NO Signaling

- NO = dissolved gas (hydrophobic - Very reactive => short lifetime => local signaling - GPCR Ca++ pathway activates NO synthase => NO production - Target = guanylyl cyclase => GTP to cGMP - cGMP activates Ca++ ATPase pumps in ER - cytosolic Ca++ decreases => relaxation - Like steroids, the dissolved gasses are hydrophobic and can therefore diffuse through cell membranes. Unlike steroids, dissolved gases like nitric oxide are very reactive and have a short lifetime. Dissolved gases are more involved with local signaling events as opposed to endocrine signaling - The fig shows the series of signaling events that lead to vasodilation. TO start the process, acetylcholine is released by nerve cells that terminate around the blood vessel. The acetylcholine diffuses across the short distance, but instead of directly effecting the muscle cells, it is recognized by a GPCR on the endothelial cells, and this activates a GPCR calcium pathway within the endothelial cell, which leads to the activation of an enzyme called nitric oxide synthase. Nitric oxide synthase converts the amino acid arginine into nitric oxide. As a dissolved gas nitric oxide can diffuse out of the cell and across into adjacent smooth muscle cells. With in the smooth muscle cells nitric oxide is recognize by the enzyme guanylyl cyclase, which is essentially the receptor for the nitrous oxide signal - Guanylyl cyclase is activated by the nitric oxide and convert GTP into cGMP. cGMP then activates calcium pumps in the ER of the smooth muscle cells, and these pumps work to decrease the cytosolic calcium concentration within the smooth muscle cells which promotes relaxation and vasodilation

14. Intermediate filament are assembled from fibrous, tetrameric subunits

- Overlapping subunits = strong filament - Phosphorylation of Ser in head causes disassembly - Dephosphorylation promotes assembly - Least understood of the three filament systems because they do not have a role in motile processes and just seem to preform structural roles - In terms of intermediate filament subunits, they are fibrous or elongated in shape. They have a globular head and a globular tail as shown in panel A of the fig. In between they have an extended alpha-helix, this structure is the monomer form and the N terminus at one end forms the head and the C terminus at the other end forms the tail. If we look at this subunit we can see it is polar, one end is different from the other end. This is not the chemical definition of polarity, but rather a structural definition of polarity. We never find monomers just floating in the cell, because one monomer will quickly associate with another to form a dimer. Two monomers associated in a parallel way so that both heads are at one end and both tails are at the other end, and the alpha-helical regions coil around each other. This still creates the structure that is polar (one end diff from the other). Also like monomers, we never find individual dimers in cells, instead one dimer will associate with another dimer relatively quickly, but now the arrangement is antiparallel in terms of the tetramer, one dimer is pointing in one direction, and the other dimer is pointing in the other direction. The heads of one dimer and the tails of the other dimer are near each other at one end, and the same arrangement is seen that the other end. The tetramer is nonpolar, we cannot tell one end from the other. The two dimers are slightly staggered in that that coil coiled regions have substantial overlap but are shifted. This nonpolar tetramer turns out to be the fundamental subunit for intermediate filaments and is lowest common denominator we find in cells. Teo tetramers can then associate with each other to generate a longer structure and start to form a filament which will be nonpolar. In the actual intermediate filament itself there are tetramers associated side by side and twisted into a rope like filament. Those tetramers then assemble into end and generate a much longer filament that we would call the actual intermediate filament as shown in panel E. Because the fundamental subunit is nonpolar, the intermediate filament itself is nonpolar, we can't tell one end of the filament from the other end. Given that the initial subunit monomer is fibrous, all subsequent arrangements are fibrous and there is overlap between these fibrous extended subunits. Intermediate filaments are very strong and resist the physical stress. Cells can control the assembly and disassembly of intermediate filaments by phosphorylation. Phosphorylation of a serine in the head domain will cause the subunits to disassemble from the filament to form the lowest common denominator, the tetramer. If that phosphate group is removed by the action of the phosphatase, tetramers will assemble to form an intermediate filament

12. Many cells use GPCR cAMP pathway to control gene expression

- PKA phosphorylates transcription factors - Signals may lead to cell reproduction, differentiation or metabolic changes - Going back to our example the second messenger cAMP can be used to control gene expression. cAMP binds to and activates an enzyme called Protein Kinase A or PKA. - PKA in the inactive form is normally found in the cytosol, but upon activation it moves through nuclear pores into the nucleus and is able to phosphorylate specific target proteins - When we talk about controlling gene expression the target protein will typically be a transcription factor, shown in green in the diagram. PKA phosphorylates and activates the transcription factor so that the transcription factor binds to the appropriate gene sequences and activates the transcription of one or more genes - Although not shown in this diagram, the mRNA produced by transcription will be translated to produce a protein and this it is the protein products expressed by this pathway that make the target cell respond as appropriate. By using this pathway various signals could promote cell reproduction, cell differentiation, or initiate metabolic changes within the cell. The GPCR cAMP pathway is a very common pathway, but how it is used by a cell depends on the type of signal molecule, and ultimately the type or transcription factors that are activated

12. The GPCR Ca++ pathway

- PKC phosphorylates target proteins on S/T - DAG activates PKC - Ca++ also causes Protein Kinase C to moves to inner face of PM - Calcium also cause another enzyme, protein kinase C, to move to the interphase of the plasma membrane, when it arrives there protein kinase C or PKC, binds to and is activated by diacylglycerol. PKC will then phosphorylate target proteins on serine and threonine and alter the activity of those target proteins. Taken together, this means that two kinases have been activated by the GPCR calcium pathway. Those kinases are cam kinase and protein kinase C. The two kinases are able to phosphorylate and change the activity of multiple target proteins.

13. Steroid hormones often induce changes in gene expression

- Receptors are typically transcription factors - Some receptors are always bound to DNA - There are a variety of steroid hormones - Steroid hormones are involved in endocrine signaling but they can be involved in other signaling modes as well - In the right Fig. we see a typical signaling mechanism for a steroid signaling molecule, in this case cortisol. The steroid is in the extracellular environment and diffuses across the plasma membrane into the cytosol, there it is recognized by an intracellular receptor protein. That protein undergoes a conformational change upon binding cortisol and becomes activated. The active receptor with bound signal then migrates into the nucleus through the nuclear pore and interacts with specific DNA sequences. In most cases the intracellular receptor for a steroid is in fact a transcription factor. Once in the nucleus the activated receptor functions as does any transcription factor, it binds to the regulatory region of a target gene to activate transcription. Steroid hormones commonly control gene expression in the target cell. Some steroid receptors are always bound to DNA and are already waiting in the nucleus for the steroid molecule to diffuse across the nuclear membrane as well as the plasma membrane in order to activate the receptor

12. Example uses of the GPCR Ca++ pathway

- Sperm contact => block to polyspermy - Neurotransmitters => smooth muscle contraction - Neurotransmitters => secretion of digestive enzymes - Growth factors => genes expressed => cell reproduction - The g protein coupled calcium pathway is used in a number of cell signaling examples - In this figure we see the fact that fertilization, and in particular the blocked polyspermy depends on calcium release. The sperm contacts the egg at the arrowhead, and calcium shown in red spreads from that initial point of contact across the cell. As it does do it raises the eggs defenses to prevent additional sperm from fertilizing the egg. The sperm in this case represents the signal molecule and it is recognized by GPCRs on the egg surface. The GPCR calcium pathway is also involved in the detection of a variety of neurotransmitters. For example smooth muscle cells use this pathway to initiate contraction and pancreatic cells use this pathway to trigger secretion of digestive enzymes other cells use this pathway to recognize growth factors leading to changes of gene expression and initiation of cell preproduction

13. Receptor tyrosine kinases(RTKs) are best understood enzyme-coupled receptors

- The best understood enzyme coupled receptors are receptor tyrosine kinases (RTKS) - These will be focused on as the primary example of the enzyme coupled receptors - While may different cells use many types of enzyme receptors for a variety of signaling activities, a significant reason why we know so much about RTKs is that animal cells often use these detectors to detect growth factors, the signal molecules that trigger cells to reproduce. This role puts RTK and the signal pathways they activate front and center when it comes to research on cancer, the disease of uncontrolled cell reproduction that often exhibits defects in RTK pathway components - Unlike GPCR, which in passing through the membrane 7 times can change their confirmation, enzyme coupled receptors are single pass proteins, therefore they have to activated by a different mechanism in order to transduce the presence of an extracellular signal into an intracellular signal. - For enzyme coupled receptors, including RTKs, the individual receptor proteins are inactive in the absence of appropriate signaling molecule. When the signal appears, that signal molecule is bound by two separate receptor proteins, this brings the two receptor proteins together in the membrane, which by necessity brings the intracellular enzymatic domains of the two receptors together. As the name implies, the enzymatic activity of this receptor is a tyrosine kinase, so the receptors are dimerized by the signal and then each enzymatic domain is brought in contact with this target protein which is the other tyrosine kinase molecule, the two kinases then phosphorylate each other by attaching phosphate groups to tyrosine side chains in a process known as transphosphorylation or crossphosphorylation. The only way to get target groups on tyrosines is on the intracellular side of the receptor, as if there is a signal molecule on the extracellular side that brought two receptors and tyrosine kinase domains together

11. signaling events may be classified in a variety of ways including the type of signal molecule, type of origin (signaling cell), chemical nature of the signal and response of that particular signaling event.

- This is only a small subset of known specific signaling examples - The primary focus in this course will be the general principles of cell signaling and a few detailed examples of the most common signal pathways

15. Actin Binding Proteins (ABPs) Control Actin Filaments Assembly and Organization 3. Motors

- Unlike microtubules which have 2 different families (kinesins and dynines), all actin dependent motor proteins fall within the myosin family - Myosin is involved in muscle contraction, but many non muscle cells, including unicellular eukaryotes also contain myosin's for processes such as cell crawling or the formation of a contractile ring.

14. Vesicle transport uses the directional tracks established by microtubules

- Vesicle transport along microtubules is critical for the movement of vesicles from the cell body to the nerve terminus and backwards from the nerve terminus to the cell body during endocytosis - Left Fig shows microtubules in the axon arranged in a parallel array so that all plus ends point to the nerve terminus and all minus ends point towards the cell body creating directional roadways. These microtubules are stabilized roadways due to the action of other MAPs - If we look at vesicle transport from the cell body to the axon, that represents movement towards the plus end of the microtubule. This outward transport is powered by kinesin and is indicated by the red colored vesicles. In comparison, some material has to travel back, this could be recycling of the plasma membrane as a result of endocytosis - Inward transport, or transport towards the minus end is powered by the motor protein dynein - Vesicle transport doesn't just occur innerve cells, it occurs in all types of eukaryotic cells - The right Fig shows a typical radial array in any generic animal cell. The radial array is set up so the plus ends are near the cell periphery and the minus ends are attached to the centrosome. The centrosome is usually somewhere near the nucleus. Outward flow of vesicles will be carried on microtubules by kinesin and inward flow will depend on dynein

15. Myosin powers vesicle transport and filament sliding along plasma membrane

- While the job of myosin II is to slide actin filaments past each other generating contraction, other types of myosin's are involved in other types of movement. - Here for example is myosin I, as shown in panel B, myosin I can bind to a vesicle and power the movement of that vesicle along an actin filament toward the plus end of that filament. Alternatively, as shown in Panel C, myosin I can bind to the plasma membrane and slide an actin filament along the plasma membrane. In both cases myosis I walks toward the plus end, it is just moving a different cargo - There Is good evidence that a number of different types of vesicles can contain myosin motors as well as kinesin and or dyneine motors, and thus such a vesicle would be able to move along an actin filament or along a microtubule depending on the vesicles location in the cell - Myosin and actin are not limited to muscle cells, all types of eukaryotic cells use actin in a variety of different ways. Some of that may involve contraction, but it may involve other types of movements as well

12. Many cells use GPCR cAMP to control gene expression

- cAMP activates Protein Kinase A (S/T kinase) - The examples shown previously still dealt with nerve cells and changes in membrane potential, it is important to realize that many cells use GPCRs, and particular cAMP levels to trigger other responses. - For example it is very common for cells to use this pathway to control gene expression. In this Fig. we see the signal molecule, adrenalin, recognized by a receptor which activates the G-protein and the alpha subunit then binds to and activates adenylyl cyclase which then converts ATP to cAMP, cAMP then binds to ans activates an enzyme called protein Kinase A (PKA), PKA is a serine/threonine (S/T) kinase that can regulate the activity of specific target proteins

12. Most effectors are enzymes that create second messengers

- cAMP or Ca++ are most common - Most effectors downstream of trimeric G-proteins are enzymes that produce intracellular signal molecules called second messengers. Usually the alpha subunit with bound GTP interacts with and activates the effector enzyme as show in this Fig. - In this example the active enzyme converts a substrate, shown as the blue squares, into a product, shown as the blue circles, which represents a second messenger - Second messengers are typically soluble molecules that can diffuse throughout the cytosol and interact with other proteins to change the activity of those proteins. The most common and best understood examples of second messengers are cAMP and Ca++ ions. - The term second messenger highlights the concept that these molecules only appear at significant levels in the cell. after an extracellular signal molecule, the primary or first messenger has been detected by a receptor and signal transduction across the plasma membrane has occurred

12. GPCRs enable our sense of vision

- cGMP is second messenger - Receptor (rhodopsin) has retinal chromophore - Retinal isomerization activates Gt - Gt activates cGMP phosphodiesterase converts cGMP to GMP - At rest : [cGMP] high = Na+ channels open - In light : [cGMP] drops = Na+ channels close - Lack of signaling is interpreted as light -GPCRs also play a critical role in vision, like olfactory neurons this involves a second messenger as well as ion channels, however here the second messenger is cGMP instead of cAMP - The GPCR, specifically called rhodopsin has a chemical associated with it that serves as a chromophore, this chemical retinal allows for the absorption of light, and the retinal can change its structure through the process of isomerization as part of the activation process - In this slide we are looking at rod cells, on the left in the dark and on the right in the light. In the dark rhodopsin is inactive because no light is being detected, under these conditions sodium channels are open, meaning the cell is depolarized and the nerve terminus is undergoing a high rate of neurotransmitter release. When light is detected rhodopsin becomes activated, which in turn activates the G-protein called GT which then activates the enzyme cGMP phosphodiesterase, this can cGMP to GMP. At rest cGMP levels are relatively high and sodium channels are open because cGMP serves as an intracellular ligand to open the sodium channels. In light cGMP levels drop because cGMP phosphodiesterase has been activated by the Gt, when cGMP levels drop, the sodium channels begin to close and the cell actually becomes hyperpolarized which reduces the rate of neurotransmitter release. The interesting thing here is that lack of signaling (lack of passing the signal onto the next cell in the sequence) is interpreted as light, whereas constant release of neurotransmitter and activation of the next cell is interpreted as dark. Here there are a couple of different things going on, we have a different second messenger, and we have a different way of regulating the level of the second messenger. Instead of creating more second messenger when the signal that is light is detected, the second messenger is destroyed and the channels close. Any change in any component whether it increases or decreases can be used by a particular type of cell to propagate or to stop the signal process

11. Gap junctions

- cytoplasms are continuous - fastest and most direct - variable duration - are formed when protein channels present in the plasma membrane of adjacent cells align - they allow small molecules (1000 molecular weight or less) to pass from the cytoplasm of one cell directly into the cytoplasm of another - they represent the fastest and most direct mode of communication between cells since the signal is never released to the extracellular environment - the duration here is variable depending on the variation of the signal in one cell and whether the gap junction is open or closed - gap junctions are found only in certain tissues that require rapid and extensive communication, such as the cardiac muscle

12. Level of cAMP depends on relative activities of adenylyl cyclase and cAMP phosphodiesterase

- important for shutting off pathway - As previously described by signal control or phosphorylation of G-proteins, every component of a signal pathway that is activated has to have some means to be inactivated. In the case of second messengers, if activation of a signal pathway leads to an increase in second messenger levels, then when the extracellular signal goes away there also has to be a mechanism to reduce the concentration of second messenger. - In the case of intracellular cAMP, the level of this second messenger depends on relative activities of adenylyl cyclase, an enzyme that converts ATP to cAMP, it also depends on the activity of the enzyme necessary for converting cAMP into a nonfunctional form, AMP, this enzyme is called cAMP phosphodiesterase and it is important for shutting off pathways that involve cAMP. - cGMP phosphodiesterase is a similar protein that is responsible for degrading cGMP in rod cells. In terms of controlling detection of light. cAMP phosphodiesterase typically works at some background level in the cell and cells tend to control the relative activities of adenylyl cyclase in order to regulate cyclic AMP levels

11. the same signal may trigger different responses in different target cells

- in reality cell signaling is one of the most complicated activities that a cell carries out - there are plenty of examples where different pairs of cells use the same signal molecule to communicate, but produce quite different responses in three different cell types - while acetylcholine triggers skeletal muscle contraction, it essentially has the opposite effect in a heart pacemaker cell, in that it reduces the frequency of contractions in this case. - The salivary gland example is really just regulated secretion which is one of the main types of exocytosis covered in the previous lecture - if you look closely at the Fig. you will notice the acetylcholine receptors on the heart and salivary gland cells are the same, but differ from the receptor on the skeletal muscle cells. - there are in fact different types of receptor proteins that have evolved to bind to the same signal, but transmit the signal on by different mechanisms and lead to very different responses

11. Signal molecule chemistry and location of receptor

- intracellular receptors • steroids • dissolved gases - cell surface receptors • peptides • proteins • neurotransmitters - Cell signaling can also be classified based on the chemistry of the signal molecule and its receptor location - On the right we have a cell trying to detect a hydrophilic signal molecule. This could be a peptide, or protein, or some type of neurotransmitter that by itself is unable to pass through the plasma membrane, thus in order to recognize a signal molecule and transduce the information that the signal molecule is present, the cell must have a cell surface receptor capable of recognizing the signal. What is implicit is the binding of the signal molecule by the cell surface receptor which is typically a transmembrane protein somehow changes something on the inside of the cell as part of the signal transduction event. Many cells also detect hydrophobic signals including steroids or dissolved gases, because these signal molecules are hydrophobic, they are able to diffuse across the plasma membrane. In most cases the cell still needs a receptor protein, but now the receptor is found inside the cell and these receptors are therefor termed intracellular receptors.

13. Signal molecule chemistry and location of receptor

- intracellular receptors: steroids, dissolved gases - cell surface receptors: peptides, proteins, neurotransmitters - Signaling via hydrophobic signal molecules - These signals can diffuse across the plasma membrane and are recognized by intracellular receptors as shown in the figure at left. There are different types of hydrophobic signaling molecules, but both represent either steroids or dissolved gasses

11. contact dependent signaling

- local - fast - variable duration - the signaling cell and the target cell are in direct contact - the signal molecule is attached to the surface of the signaling cell, rather than being a soluble molecule released to the extracellular environment - the duration here is dependent on the duration of the interaction of the two cells. - This type of signaling is often seen in situations where a cell influences its neighbors to differentiate into a particular cell type

11. endocrine signaling

- long distance - relatively slow - relatively long duration - the signaling cell and the target cell are relatively far apart from each other, so this is considered long distance signaling. - the signal molecule, which is often referred to as a hormone will travel throughout the body via the bloodstream before being detected by the target cell. This means that in comparison to other modes, endocrine signaling is relatively slow, which makes it relatively long lasting because it will take longer to remove the signal molecules and thus terminate the target cell response

12. The GPCR Ca++ pathway

- phospholipase C- β splits PIP2 into DAG and IP3 - PIP2 (phosphatidylinositol 4,5- bisphosphate - Ca++ increases in cytosol - Although many GPCRs lead to changes in cAMP levels, some instead control levels of calcium ions in the cytosol. This pathway referred to as the GPCR calcium pathway is shown in this diagram. Similar to the cAMP pathway, when a cell using this pathway recognizes a signal molecule, the GPCR will become activated, it triggers the trimeric gene protein to undergo nucleotide exchange. The activated gene protein then diffuses across the interface of the plasma membrane, where in this case the gene protein recognizes an enzyme different from adenylyl cyclase. The enzyme in this case is referred to as phospholipase C. Phospholipase C converts a phospholipid called PIP2 into two products., diacylglycerol which represents the fatty acid hydrocarbon tail and glycerol component and inositol 1,4,5 trisphosphate, IP3 and represents the hydrophilic head group of this phospholipid. DAG remains associated with the interface of the plasma membrane, but IP3 is free to diffuse throughout the cytosol and can interact with other target proteins within the cell. At this point the production of diacylglycerol and IP3 means the pathway has begun to branch and these branches have different targets. IP3 will interact with the calcium channel and the ER membrane, that calcium channel is gated by IP3 and so it is referred to as an IP3 gated calcium channel, when the channel protein binds IP3, the gate opens and calcium runs down its concentration gradient. Calcium is higher in the lumen of the ER then it is in the cytosol so calcium begins to increase in the cytosol in response to detection of the extracellular signal

11. Autocrine signaling

- short distance = local - fast - variable duration - the signaling cell is the target cell, so a very short distance, and as local as it gets - very fast type of communication - come cells can control their own reproduction by secreting autocrine signals, but mutations that lead to inappropriate secretions of such signals has been identified as a contributing factor for certain cancers

11. synaptic signaling

- short distance = local - fast - brief - may be considered a specialized form of paracrine signaling this involves a neuron as the signaling cell and most commonly another neuron or muscle cell as a target cell - the distance between the cells is spanned by a very short gap termed the synapse, hence the term synaptic signaling. Although the distance between the cell is very short, synaptic signaling is really a component of a much longer distance, nervous system signaling that involves electrical signals traveling down the length of neurons to allow rapid signal propagation over relatively long distances

11. paracrine signaling

- short distance = local - fast - brief - occurs over a short distance and thus is local and relatively fast - here the communication duration is relatively brief because the signal molecule can be moved fairly quickly since it does not have a chance to diffuse very far

13. Nitric Oxide (NO) signaling in vasodilation and regulation of blood pressure

- smooth muscle concentration = vasoconstriction - smooth muscle relaxation = vasodilation - Signaling via dissolved gasses was not a recognized mechanism of cell communication until about 20 years ago - The now standard example of signaling via a dissolved gas involves nitric oxide control of vasodilation and regulation of blood pressure - Fig shows a cross sectional view of a blood vessel. The vessel is formed form endothelial cells and surrounding these cells are smooth muscle cells that are innervated by nerve cells, which are not shown in the fig. The nerve cells control the contraction and relaxation state of the muscle cells indirectly via the endothelial cells - If the smooth muscle cells contract, vasoconstriction occurs as blood vessel diameter decreases - If the smooth muscle cells are told to relax, the blood vessel diameter increases and vasodilation results

15. Cell crawling depends on all three actin arrays

- step 1: (parallel bundles) - step 2: Focal contact formation at front ( stress fibers ) - Step 3: 1. Focal contact release at rear 2. Contraction of stress fibers and/or release of cortical tension -The ability of animal cell to crawl depends on all three actin arrays described in the previous slide - Cell crawling can be broken down into 3 steps - Step 1 is the protrusion or extension of the plasma membrane at the leading edge of the cell, this is shown as the red region as the cell moves toward the right in the direction of the green arrow. This step depends on the parallel bundle in the filopodium and lamellipodium, specifically the polymerization of actin subunits on to the plus ends of those actin filaments. Actin assembly or polymerization actually provides the energy to push the membrane forward. Step 1 is similar to when you are walking and you extend your foot forward, but have not yet contacted the ground - Step 2: the membrane that has just been extended forms an attachment to the surface. This attachment is known as a focal contact. The focal contact is the region where the actin cytoskeleton inside the cell is indirectly connected through the plasma membrane to the surface the cell is crawling across. A cell contains many focal contacts on its underside. Focal contacts contain proteins call integrins, which are cell surface receptors that bind to extracellular substrates or the extracellular matrix. Contractile bundles called stress fibers terminate or embed into the interface of a focal contact and typically stress fibers extend from one focal contact to another as indicated in blue. Focal contacts and stress fibers are a way for a cell to use the process of contraction to move relative to a surface - Step 3: in order for a cell to continue to crawl, the back end has to come forward, this process described in the figure is contraction, but also known as retraction. It involves the detachment of the rear most focal contact, which allows the plasma membrane in that region to be brought forward. The force for bringing the backend forward is thought to depend on contraction of stress fibers. Another way for the bac of the cell to come forward is through a mechanism known as release of cortical tension. If we look at the top portion of this Fig. the cell cortex is shown in orange. When the front end of the cell extends forward and new focal contacts are formed, that cortex is put under tension (like stretching a rubber band forward) and then attaching it to the surface. When the attachment point at the rear of the cell is released or removed the rubber band then springs back to its original dimensions and that will pull the back of the cell forward. There is evidence that some cells use stress fibers to power contraction, while others use release of cortical tension, and some may use a combination of both. - This is a repeating process. Cycling through steps 1-3, extension, attachment, and then contraction in order for a cell to crawl across a surface

11. cells typically detect and integrate multiple signals

- the second complexity is that a target cell is rarely exposed to just one signal molecule at a time, thus target cells function in some ways like computers in that they must integrate information from a variety of signals and calculate how to respond - in this example the cell may respond in different ways depending on which signal or signals are detected or not detected

11. The three classes of cell surface receptors

1. Ion channel coupled receptors 2. G protein coupled receptors 3. Enzyme coupled receptors - There are three main groups of cell surface receptors. The one we will talk about in this lecture is ion channel coupled receptors. These are most commonly found in nerve cells and muscle cells, which depend on changes in electrical gradience or membrane potential as well as synaptic signaling to communicate. As the name implies these receptor proteins recognize signals of various types, and those signals in turn control the ability of ions to move across the plasma membrane through a channel protein

15. Actin arrays in non-muscle cells

1. Parallel / close ABP = fimbrin. extends plasma membrane 2. Anti-parallel/loose ABPs = α-actin and Myosin II Contraction 3. Random/crossed ABP = filamin Supports PM (cell cortex) - This slide and the next highlight several important concepts related to actin and actin arrays in non muscle cells. - The first, which is indicated in the fig, is that actin can be arranged in a number of different arrays in different regions of the same cell. In panel A we structures referred to as filopodium and lamellipodium as well as contractile bundles. This particular cell could be crawling across the surface and the region shown to the right of the cell represents the leading edge, which is the front of the cell during the process of cell crawling - At the leading edge, structures are present including filopodium and lamellipodium - Filopodium are spike like protrusions of the plasma membrane and lamellipodium are very flattened extensions of the plasma membrane, both can be seen in the upper right scanning electron microscope image of a crawling cell which is moving in the direction of the arrow - The internal actin arrangement in both filopodium and lamellipodium is somewhat similar. They are often referred to as parallel or close arrays. Parallel is certainly more descriptive of the filopodium - A specific actin binding protein called fimbrin bundles the actin filaments in these organized arrays. The job of both of these structures is believed to be to extend the plasma membrane in the direction the cell is crawling - Notice the arrow heads in the cartoon images for both filopodium and lamellipodium. The actin filaments are shown as the red lines and the arrows point to the plus end of each actin filament - In both filopodium and lamellipodium the actin plus ends are right underneath the plasma membrane - The second type of array in non muscle cells is the contractile bundle. Even non muscle cells have antiparallel bundles of actin filaments. These filaments as indicated in the cartoon in panel B are loosely arranged in comparison to the close arrangement of filopodium and lamellipodium and thus the actin filaments are farther apart from each other in contractile bundles. The arrows indicate the antiparallel arrangement of the actin filaments. Given this arrangement and the contractile function of these bundles, it should not be surprising that myosin II is a key actin binding protein in these structures. - We also find a bundling protein called alpha actinin, it is the job of alpha actinin to set up the antiparallel arrangement of the bundle as well as the loose spacing of the actin filaments. That spacing allows myosin II to get into the contractile bundle and carryout its function - The third array which is not shown in the diagram, but was alluded to in the earlier description of actin binding proteins as organizers is the random or crossed array of actin filaments. Here the actin binding protein is filamin. It is the green Y shaped protein in the cartoon at lower right. The job of the random or crossed array is to form the cell cortex, which supports the plasma membrane and helps determine cell shape

13. Signal pathways are reversible

1. Signal production by the signaling cell stops and the signal is destroyed or absorbed from the system 2. As signal concentration decreases the Signal molecules will dissociate from the receptor and the receptor will become inactivated 3. This begins a step-by-step process of component inactivation - the actual mechanism of deactivation depends on the specific depends on - the specific component - If we are talking about G proteins, they evolved in signal pathways and will hydrolyze GTP to GDP & Pi (GAP or timer) - How this occurs depends on some extent on whether we are talking about trimeric or monomeric G proteins. - All signal pathways must be reversible. There will be a series of events that occur, just like the series of events that occurred to lead to the activation pathway - The reverse of the pathway must start at the signal molecule, rather than at the end of the pathway

13. Signal Pathways are Reversible

1. Signal production stops and signal is destroyed or absorbed 2. Signal falls off receptor 3. This begins a step-by-step process of component inactivation - G proteins will hydrolyze GTP to GDP & Pi (GAP or timer) - Second messengers will be destroyed or pumped out of the cytosol cAMP phosphodiesterase or Ca++ATPase - Phosphatases will remove phosphate groups added by kinases - Failure to turn off signal pathway when the signal disappears may cause as many or more problems then failure to turn on the pathway when the signal is present - If a signal pathway uses second messengers, the second messenger like cAMP or calcium have to be destroyed or pumped out of the cytosol -This mechanism involves a phosphodiesterase enzyme which will degrade the cyclic form of the nucleotide and convert it into the monophosphate form - If a signal pathway uses calcium, and the calcium increases in the cytosol, then in order to reverse this part of the pathway, calcium ATPase enzymes in the ER or plasma membrane will pump calcium out of the cytosol, either into the lumen of the ER or to the extracellular environment, thus returning cytosolic calcium levels to the resting state. - For any activation event that involves a kinase, the action of the kinase must be reversed by the activity of the phosphatase and so the phosphatase must remove phosphate groups from target proteins that were added as part of the activation process - Failure to turn off signal pathway when the signal disappears may cause as many or more problems then failure to turn on the pathway when the signal is present. In fact cancer largely results from failure to turn off one or more growth factor signal pathways when the growth factor/factors are no longer present

11. the spatial and temporal relationship, the distance and timing of communication between a signaling cell and a target cell can be classified into various modes

1. endocrine signaling 2. paracrine signaling 3. synaptic signaling 4. contact dependent signaling 5. autocrine signaling 6. gap junctions

15. Myosin motor proteins hydrolyze ATP to move along actin filaments

> 15 types of myosins - Most walk to plus end - Myosin II powers actin filament sliding that produces contraction - Since myosin's provide directed movement along actin filaments they require an energy source - Myosin's like kinesins and dyneins for microtubules combine to and hydrolyze ATP to allow the myosin to walk along actin filaments - There are more than 15 different types of myosin. Most types walk toward the actin filament plus end - The best understood member of the myosin family is myosin II, its action is depicted in the Fig. Note that there are important structural considerations for contractile movement. First myosin II itself is able to assemble to form a filament, the simplest version of which is shown in dark green. Here the Myosin II filament consists of two motor proteins assembled in an antiparallel arrangement. Each myosin protein consists of two head domains, the large green globular regions that walk along active filaments and an elongated stock domain that allows myosin II proteins to assemble into a filament. In this example, the head of one myosin II protein are pointing to the left and the head of the other is pointing towards the right, which is an antiparallel arrangement. Since myosin II walks towards the plus end of an actin filament, the heads of one end of the myosin II filament will walk to the plus end of the upper actin filament in this cartoon and the myosin heads at the other end will walk toward the plus end of the lower actin filament. Functionally the myosin II filament does not move in and of itself, instead the two actin filaments are moved in the direction indicated by the blue arrows, that shortens the entire length of the structure and that shortening is contraction. The second important structural consideration relates to the organization of the actin filaments. These must also be in an antiparallel arrangement in order for contraction to occur since myosin's only walk toward actin with plus ends. - Myosin II powers contraction, which is really the sliding of actin filaments past each other

Lesson 12

Communication: GPCR signaling - Another major class of cell surface receptors, the G-protein coupled receptors usually abbreviated as GCPRs, cells express many different types of these receptors and use these proteins to recognize a variety of signals

Lesson 14

Cytoskeleton - Intermediate filaments & microtubules - Intermediate filaments are found only in animal cells, but microtubules are found in all eukaryotic cells, actin is also found all throughout eukaryotic cells - IF is the abbreviation for intermediate filament, MT= microtubule

12. Molecular switches in signal pathways

Ser (S) Thr (T) Tyr (Y) - What is a kinase? And how do such enzymes control protein activity? - Like G-proteins kinases represent a kind of molecular switch commonly found in regional pathways. This switch is based on modification by a phosphate group and can regulate the activity of the modified target protein - In the left fig., we star with a blue target protein at top, the signal comes in and that activates a kinase which hydrolyzes ATP and covalently attaches a cleaved phosphate group to the target protein. In this Fig., that phosphate is the yellow circle with the yellow P in the middle. The presence of the phosphate group on the target protein activates the target protein and that can activate other proteins, this is labeled as signal out. The activity of a kinase is counterbalanced by the activity of enzymes termed phosphatases. The job of a phosphatase is to remove that phosphate group from the target protein and reverse the action of the Kinase. In this example, by removing the phosphate, the phosphatase converts the target protein from the on state to the off or inactive state. The process of adding a phosphate group to a target protein is termed phosphorylation, while the process of removing a phosphate group is termed dephosphorylation. Phosphate groups are added to the side chains of 3 amino acids, serine (ser(s)), threonine (Thr(T)), and Thyronine (Tyr(Y)). Some Kinases are able to phosphorylate target proteins on either Ser or Thr and they are referred to as S/T kinases, whereas other kinases only target Tyr and these are Tyr kinases. The same terminology can be applied to phosphatases

11. cell communication via signaling molecules

signal= ligand= stimulus - signaling cells produce and release signals - target cells use receptor proteins to detect signal and then responds - to terminate a response 1. signaling cell stops releasing signal molecule and 2. the signal molecule is destroyed or ingested - almost all cells, even single celled organisms communicate with other cells and they certainly respond to chemicals or other information in the environment - communication, or signaling between cells is generally controlled by chemical signal molecules which are often referred to as ligands and can be thought of as stimuli - we can describe the relationship between two communicating cells. One acts as a signaling cell which produces and releases the chemical signal and the other acts as the target cell, which detects the signal and responds in some appropriate way. We often focus on how a signal leads to a response, but keep in mind that in cell signaling. it is also very important that communication be terminated at some point, so that the target cell stops responding - there are 2 main events that must occur to terminate the response, first the signaling signal must stop releasing the signal molecule and second the signal that has been previously released, must be removed from the extracellular environment, either by destruction of the signal or by uptake of the signal by surrounding cells

13. NO signaling and health

• Angina is chest pain that often results from atherosclerosis - Nitroglycerin has been used for years to treat angina - Atherosclerosis now known to inhibit NO production - Nitroglycerin is converted to NO => blood flow increases • Sildenafil inhibits cGMP phosphodiesterase - cGMP remain high => vasodilation persists - There are a number of medically relevant issues involving nitric oxide signaling. The first is associated with angina, which is the pain that often results from atherosclerosis or hurting of the arteries - Nitroglycerin has been used for years to treat angina and to relieve the chest pain that is associated with angina - It is now known that atherosclerosis inhibits nitric oxide production and it turns out that nitro glycerin works as a treatment for angina because the nitroglycerin is converted within the body to nitric oxide and by inducing vasodilation, you increase blood flow in the heart and relieve the chest pain caused by angina - Another medical implication of nitric oxide signaling involves one of the most popular drugs currently on the market, Slidenafil. Slidenafil inhibits cGMP phosphodiesterase and so cGMP levels remain high and vasodilation persists. This drug is more commonly known as Viagra

14. Microtubule-Associated Proteins (MAPs) modify dynamic instability

• MAPs bind along MT wall or at MT ends to regulate assembly • MTs become more dynamic when cells enter M phase - lifetime ~ 10 seconds - probe cytosol for chromosomes • MTs are more stable in axons of nerve cells - lifetime of hours-days - roadways for vesicles - Dynamic instability is an intrinsic property of microtubules. If we purify tubulin and provide the appropriate conditions of a test tube, microtubules will assemble from that purified tube and will undergo dynamic instability - Within cells dynamic instability can also be regulated depending on the appropriate function of the microtubules within that cell, such regulation is controlled by microtubule Associated Proteins (MAPs) - Cells can use these MAPs to modify dynamic instability. These proteins may bind to microtubule walls or to microtubule ends to regulate assembly - Although tubulin itself forms dynamic microtubules, there are situations in which a cell may need to make the microtubules even more dynamic or may wish to suppress dynamics and make the microtubules more stable - One example in which microtubules become more dynamic is when a cell enters M phase, the time in the cell cycle when a cell carries out mitosis and then divides to produce two daughter cells. The average lifetime of a microtubule in interphase is 10 minutes, but when the cell moves into M phase the average lifetime of a microtubule is reduced to 10 seconds. SO microtubules convert between growing and shortening much more frequently during M phase. This increase in dynamics is believed to be associated with the formation of the mitotic spindle and allows for microtubules to probe a greater area of the cytoplasm in search of chromosomes so that the chromosomes can be quickly attached and moved to the appropriate location on a spindle. The analogy here is that someone going fishing makes more casts in as many directions as possible in order to sample a greater number of areas and increase their odds of catching fish - There are also situations where microtubules need to be relatively stable. One example is the axons of nerve cells. In nerve cells the average lifetime of microtubules is hours or even days, this makes since given that the role of microtubules and axons can serve as roadways for vesicle transport. If the microtubules here exhibited normal dynamics, that would be equivalent to the roadway disappearing as the vesicles tried to move. In order to ensure vesicles can travel along the length of the axon, these cell stabilize the microtubules and hence they are roadways.

11. Nerve cell signal propagation also depends on:

• Voltage-gated K+ channels to re-polarize membrane - delayed activation so open "behind" Na+ wave - K+ flows out of cell • Na+K+ATPase re-establishes Na+ and K+ gradients for next action potential • Other transport proteins play critical roles in signal propagation along neurons. For example voltage gated potassium channels are necessary to repolarize the membrane behind the wave of depolarization. This returns the membrane potential to the resting state. Voltage gate potassium channels experience delayed activation following depolarization, so they open behind the wave of sodium entry. When these channels open, potassium, which is higher inside the cell, flows from the inside to the outside and helps repolarize the membrane back to a more negative state. Note that following the action of voltage gated potassium channels, this particular region of the cell has a membrane potential closer to the resting level, but the sodium and potassium gradience are backwards, sodium is higher in the cell than it should be and potassium is lower in the cell than it should be. To correct the distribution of these ions, nerve cells contain a large number of sodium potassium ATPases who work to reestablish the sodium and potassium gradience to prepare the axon for the next action potential. Recall that nerve and muscle cells expend a lot of ATP to power the sodium and potassium ATPase. This need to reestablish the gradience to the potassium and sodium ions is precisely why