BIO 201 With Todd Hennessey Fifth Week of Notes
BIO 201 With Todd Hennessey Fifth Week of Notes BIO 201
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This 25 page Class Notes was uploaded by ChiWai Fan on Saturday February 27, 2016. The Class Notes belongs to BIO 201 at University at Buffalo taught by TODD HENNESSEY in Spring2015. Since its upload, it has received 114 views. For similar materials see CELL BIOLOGY in Biology at University at Buffalo.
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Date Created: 02/27/16
Cell Biology with Todd Hennessey Feb 24, 2016 (All images are taken from professor’s slides and information edited by ChiWai Fan) Biological Membranes: Early experiments to determine what membranes are: In the 1890s, E. Overton used hypertonic shrinkage of plant cells to estimate the composition of plasma membranes. He wants to know what plasma membranes are made of. What he knew already: Permeable compounds prevent hypertonic shrinkage. Hypertonic shrinkage happens when water leaves the cell causing the cell to shrink. Permeable compounds can prevent this from happening His Hypothesis: Like dissolves like, so if the membrane is hydrophobic then hydrophobic compounds should be permeable and prevent hypertonic shrinking— this suggests if the membrane is hydrophobic, a hydrophobic substance can dissolve into the membrane, come back out and dissolve into the membrane again. If membrane is hydrophobic then compound that prevent hypertonic shrinkage should be hydrophobi. (This is wrong but believed in 1890s) Hypertonic Shrinkage When you add solute, you decrease the water concentration. (High solute=low water) Water moves from a high concentration of water to a low concentration of water (High salt in ocean causes your skin to shrink) Cell shrinks in hypertonic solution. Because water is being removed Hypertonic solution: Adding any solute to water lowers the water concentration In a hypertonic solution, water moves down its concentration gradient, from high water concentration to low Since the water concentration is lower outside, water moves out and the cell shrinks Water moves toward the highest solute concentration Overton says if the hydrophobic membrane shrinks then the substance added is hydrophobic How can you stop this shrinking? -Add solutes to the inside to balance the solute concentrations What do we know so far? What did Overton know? 1. Adding any solute to water lowers the water concentration 2. In a hypertonic solution, water moves down its concentration gradient, from high water concentration to low 3. If the water concentration is lower outside the cell, water moves out and the cell shrinks 4. Permeable compounds can stop hypertonic shrinking Isotonic solution: Mo visible swelling or shrinking in an isotonic solution When everything is right. No shrinking or swelling. Hypotonic solution: Solute concentration is higher inside the cell Water concentration is lower inside Water moves down its concentration gradient into the cell Hypotonic swelling/ popping Hypertonic: Cell shrinks because water moves out. The outer layer is not plasma membrane. The plasma membrane is surrounding the round part. Hypotonic: Water moves in but the cell can’t expand because of the cell wall. How could Overton tell if a solution was hypotonic? –He couldn’t, the cell wall was too strong so he couldn’t tell if the cell was swelling. Why did Overton choose plant cell instead of animal cell? Because plant cells have cell wall so it is easier to tell if the cell has shrunk. And cell wall protects the plant cell from popping due to hypotonic swelling. Overton’s Conclusions: 1. The compounds that prevented hypertonic shrinking of plant cells were much like fatty oils in their solubility. Fatty oils are triglycerides. But Overton meant lipids! 2. The plasma membrane of plants must be fatty oil (hydrophobic lipid). It is a lipid membrane because the only thing that can get across it was lipids. Overton guessed it is a pure lipid membrane. But we know that it’s not. He probably looked at only amphipathic ones. Later experiments showed lipid bilayers We put lipids, proteins and carbohydrates in lipid bilayers now. The nonpolar, hydrophobic fatty acids “interact” with one another in the interior of the bilayer The charged or polar hydrophobic head portions interact with polar water. Does this look more solid or fluid? It looks very solid. Is eukaryotic membranes fluid in live cells? In the 1970s the Fluid Mosaic model proposed: 1. Membrane proteins for transport of non-permeable compounds 2. Fluid membranes with rapid lateral diffusion of lipids and proteins. They move like water in the pool. Membrane Proteins Two main types of proteins in the cell A. Soluble B. Membrane bound Two main types of membrane bound proteins A. Peripheral B. Integral—really hard to rip out An integral membrane spanning protein Not all proteins are spanning proteins Spanning proteins mean they’re on the outside to the inside Inside of the cell, we have a lot of hydrophobic R groups that are nonpolar Outside of the cell, we have a lot of hydrophilic R groups that are polar uncharged All integral membrane proteins are spanning proteins. –FALSE!!!!!! Some examples of integral membrane proteins that are not spanning proteins The part that is in the membrane is hydrophobic. Are these all amphipathic proteins? NO. The one inside the membrane is not. How can you tell if these are integral or peripheral? By doing experiments. Centrifugation. In pellet= no soluble. When you centrifuge all the membranes down the pellet at high speed, if they do not come off the membrane with high salt, they’re integral. Peripheral membranes are held together by ionic bonds If they do not come off of the membrane with high salt, they are integral How can you get an integral membrane protein out of the membrane and solubilized in water? Use detergent to rip it out of their membrane Insolubleadd detergentnow it is soluble positive charge protein at pH 7 Peripheral proteins can be on either side of the membrane. They can be attached by ionic bonds to proteins, lipids or sugars. If they come off at high salt, it is peripheral There are no peripheral proteins on the outside of RBCs. Why? Because blood has high salt!! How can you tell if it is peripheral? -Centrifugation Two possibilities: 1. Everything in pellet (protein didn’t come off) so it is integral membrane protein 2. If the protein is in supernatant, it came off. So it is peripheral Would peripheral proteins be in the pellet or supernatant after centrifugation with no salt added? They will all be in pellet. If added high salt, peripheral will be in supernatant. Would integral proteins be in the pellet or supernatant after centrifugation in detergent? If you add high salt and centrifuge an integral membrane protein, it will be in pellet. If you centrifuge in detergent, it will be in supernatant Feb 24, 2016 Overton: Membrane only has lipids (WRONG) Testing the Fluid Mosaic model 1. Label two different kinds of plasma membranes so that the proteins of one kind are green and the others are red 2. Fuse the membranes together. A. If half stays green and the other half stays red, then the membranes aren’t fluid because the proteins didn’t mix B. If the proteins mix and form mosaics, then the membranes are fluid The Frye-Edidin experiment (1970) 1-3. Fuse two cells together to make one. Half is human and half is mouse. A. Label human cell membrane proteins red with red antibodies. B. Label mouse cell membrane proteins green with green antibodies. If it is half and half it isn’t a mosaic (mix=mosaic) 4. Wait 40 minutes. If mosaics form, then the membranes are fluid Fused cells at the start of the Frye-Edidin Experiment Is this a mosaic? NO The percent mosaics increase at higher temperatures. Why? Because that is the phase transition temperature (from solid to fluid) Cell grows in different temperatures so phase transition temperature isn’t always the same. It Is a problem if the membrane becomes too solid. Membrane fluidity is very important. What do you think would happen to this curve if you increased the degree of unsaturation of fatty acid side chains on the membrane phospholipids? It would shift to the left because you lower the melting temperature when you increase unsaturation Current model of a typical animal membrane (Note that the description tells you about the structure) A way when the frye-edidin experiment can be fooled: when a protein is stuck. Membrane Carbohydrates I. Glycoproteins. Proteins with covalently attached sugars A. The sugars face the outside of the cell when they are on the plasma membrane. B. Not all membrane proteins are glycoproteins but most receptors are if they face the outside of the cell. II. Glycolipids. Lipids with covalently attached sugars A. Like glycoproteins, the sugars face the outside of the cell when they are on the plasma membrane. You must have their sugar receptor in order for them to attached onto the membrane Sugar facing outside of cell=receptors Glycoprotein=target to binding Viruses bind to different types of receptors, depending on what type of glycoproteins they have Can a glycolipid be a receptor? Yes. But most are glycoproteins FYI Glycoproteins outside of HIV (not a cell, no DNA until it has a host to do reverse transcription to make its own DNA to infect cell). It helps HIV to target to the right cell. Amounts of Lipid, Protein and Carbohydrate in different biological membranes (in general) When red blood cell matures, it is just a plasma membrane Myelin: lipid wrapped around nerve cell as an electrical insulator NOT ALL MEMBRANE ARE THE SAME THUS THEY DO NOT HAVE THE SAME PERCENTAGE. What do you think are the most important things to know from this slide? ONE SIZE DOES NOT FIT ALL. DIFFERENT CELLS HAVE DIFFERENT PHOSPHOLIPIDS Membrane Lipids Exoplasmic=outside. Cytosolic= Inside|This is different for every cell. You know what PC, PS, PE and PI are. SM is sphingomyelin, It is more on the outside than inside, its head group faces outside of bilayer (net charge =0 at pH=7.0) Asymmetry of phospholipid outside and inside of cell, Why? Because Cl here is cholesterol Red blood cell will not have peripheral proteins because it has high salt. (You want charged polar head group to bind to peripheral proteins so you put it outside) Phospholipid: Charged head group facing inside because that’s where there’s peripheral proteins This is from red blood cells. On which side would you expect to see more peripheral proteins at pH=7.0? Why? INSIDE because you don’t have any charged peripheral protein on the outside and it has too high salt! PS PE are negatively charged and they are needed inside Membrane Transport Two main types of solute movement across membranes: Passive diffusion: No extra energy (-∆G) coupling needed. Concentration driven if it is uncharged (move from high concentration to low) Electrochemical if it is charged (protein moved to opposite charge) Active transport: Needs energy to make it go. Energy does not have to be in ATP form If movement is predicted as +∆G, couple it with a sufficiently - ∆G to make it go. Example of active transport: ∆G for influx = +5.3 kcal/mol ∆G for ATP hydrolysis = -7.3 kcal/mol Add them together by coupling ATP hydrolysis with transport in: +5.3 kcal/mol + -7.3 kcal/mol = overall ∆G of -2.0 kcal/mol Passive Diffusion Energetics of solute movement of an uncharged compound ∆G is depended on concentration of inside and outside ∆G is the free energy change R is the gas constant T is the absolute temperature At 25 C, this equation become: [Cin is the concentration inside the cell [C ] is the concentration outside of the cell out This is only for passive diffusion of an uncharged, permeable compound If ∆G is negative, then net influx is thermodynamically favored so it will move in. What if the ∆G is positive? Then it will move out. No net flux at equilibrium G=0. Dead cell What if it is charged? Electrochemical driving forces 1. Chemical force (Wc): Wc = work to oppose a chemical force Wc = 1.4 log [in]/[out] = ∆G Concentration dependent movement 2. Electrical force (We): (only if it carries a full net charge) We = work to oppose an electrical force We = FzVm F is Faraday constant z = valence (valence is zero when it is uncharged) Vm = voltage across the membrane What is membrane potential? Vm is the voltage across a membrane It is the membrane potential Depolarization is a positive-going change in a cell's membrane potential, making it more positive or less negative inside. Hyperpolarization is a change in a cell's membrane potential that makes it more negative. If it is positively charged, it can be brought inside. How to Make a Membrane Potential Asymmetric charges inside and outside generates a membrane potential Different ion concentrations on different sides of a permeable membrane No net flux at equilibrium. What If We Apply a Voltage? + What do the K ions do? The inside is hyperpolarized relative to the outside K+ flows in, against its concentration gradient. The electrical force overcomes the chemical force Hyperpolarized: forces it to go where its againist its concentration gradient if it is charged How do we know the voltage necessary to do this? By electrochemical Electrochemical Energy ∆G influx Wc – We ∆G influx2.3RT log [in]/[out] - FzVm (chemical force minus electrical force) At equilibrium, Wc = We (because ∆G = 0 at equilibrium) What happens to this equation if it is uncharged? Then the valence is zero and has no electrical force (no Fzvm) What’s the point? Chemical energy for the movement of uncharged molecules across the membrane is provided by the concentration gradient For charged molecules, it is electrochemical energy instead of simply chemical energy True or false? If the external concentration of K+ is 100mM and the internal concentration is 10mM, K+ will go into the cell. Answer: False. It can but not always. Only if the electrical force allows it. If the inside is too positive (depolarized), K+ can be kept out by electrical force. Like charges repel preventing it from going in. Feb 26, 2016 Ligand-gated ion channel This diffusion is not just concentration-driven. Why? Because it is charged Ligand binding causes a conformational change to open the channel and the polar substance can move across the membrane Ligand is anything that binds to a receptor What is its charged? Then you want to think about electrochemical Other kinds of ion channels Voltage-gated ion channels. If you change the membrane potential, then to you open or close a channel Voltage across the membrane causes a conformational change to open or close a channel Mechanosensory channels. Mechanical stretch can open channels in a stretch receptor cell Think of a rubber band, pin a hole in it, stretch the rubber band and you can’t see the hole. Membrane lipid bilayer which is impermeable to ions If you want to get ion across, put in ion channel and use it to give you information about things, Information such as ligand in an external environment the voltage in a cell and etc. Voltage-dependent change in conformation Facilitated Diffusion (ex. Door from NSC to Talbert when there are too many people trying to get pass) Facilitated diffusion is saturable Active Transport Example of energy coupling—requires energy but not necessarily ATP What if there is a +∆G for influx for something? Can it still go in? Sure ∆G = +5.3 kcal/mol You can get a -∆G from ATP hydrolysis ∆G = -7.3 kcal/mol Add them together by coupling ATP hydrolysis with transport: +5.3 kcal/mol -7.3 kcal/mol ∆G of -2.0 kcal/mol Even if there is a +∆G for a reaction, it could still go if coupled to a sufficiently -∆G reaction Types of Proteins for Active Transport Active transport is necessary to overcome a positive ΔG for transport Active transport is necessary to overcome a positive ∆G for transport Primary Active Transport: The Sodium–Potassium Pump When 3 Na+ go out for every 2 K+ that go in, we have hydrolyzed enough ATP to overcome any positive ∆G so the overall ∆G is negative How can Na+ move from low Na+ inside to high Na+ outside? We pump it out. The energy for this is provided by ATP This can be used to control cell size. How? Hypertonic or hypotonic situation across the cell depending on how active it is. Secondary Active Transport (requires primary to set up energy that it can use.) Glucose influx has a +ΔG When you hydrolyze ATP in primary, you make the sodium outside higher than inside so the ∆G is negative. But glucose ∆G is positive. But as long as sodium is negative you can run this as a symport What provides the energy for glucose influx? What provided the energy to establish the Na+ gradient? Primary active transport by sodium potassium ATPases As long as you have high sodium outside, you can run it as primary. Negative ∆G does not always have to be provided by ATP. You can have active transport without ATP, you can use ion gradient. Symport of glucose from the intestine into the gut cells <inside of intestine. Primary active transport This is active transport because the ΔG for glucose influx is positive What provides the energy for glucose uptake here? The electrochemical energy of Na+ influx provides a negative ΔG We’re using energy to fight against equilibrium! Summary: Extracellular Matrix—a bunch of stuff on the outside of cell Proteoglycans are just excessively glycosylated glycoproteins Many types of animal cells are surrounded by an extracellular matrix (ECM). The ECM is composed of a network of several kinds of fibrous proteins and sugars on the outside of the plasma membrane. The ECM proteins are secreted by the cells into the extracellular space where they assemble into an interconnected 3-D network and serve diverse functions such as scaffolds, girders, wire, glue and trail markers. The ECM plays important roles in: 1. Holding together cells in tissues in their proper positions 2. Mediating cell-cell interactions within the tissue. 3. Regulating the shapes and activities of cells. 4. Cell adhesion, migration and differentiation during development 5. Cell growth 6. Cell adhesion to a substrate or to other cells 7. Signal transduction Examples of two types of proteins in the ECM: Cadherins and Integrins Integrins and the Extracellular Matrix Integrins are anchored both inside and out The Integrins bind to extracellular matrix proteins by reversible non-covalent interactions Cadherins (calcium-dependent adhesion proteins) 1. Cadherins are transmembrane glycoproteins of the plasma membrane that mediate Ca++ dependent cell-cell adhesion. 2. Cadherin-cadherin interactions form adherents’ junctions between cells like Velcro (reversible) or rivets holding cells together. 3. Because Cadherins are transmembrane proteins, they are also involved in cell signaling from the ECM to inside the cell. Cell communications Signal transduction Intercellular: Between cells Intracellular: Inside cells General types of chemical communication between cells Also signal transduction from the outside world (smell, taste, etc.) Syanatpic: small distance Paracrine: smell and taste, longer distance Endocrine: in blood General types of ligand-activated receptors A. Plasma membrane receptors 1. GPCRs (G protein coupled receptors) a. Metabotropic: second messenger involved b. Ionotropic: 2. Ligand-gated ion channels 3. Others (RTKs, etc) B. Intracellular receptors Lipids and proteins can be receptors You don’t only have receptors on plasma membrane! Some are inside the cell.
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