Unit 2 Outline
Unit 2 Outline BIOL 30603
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This 8 page Class Notes was uploaded by TCU2461 on Saturday April 30, 2016. The Class Notes belongs to BIOL 30603 at Texas Christian University taught by Dr. Akkaraju, Dr. Misamore, Dr. Chumley, Dr. McGillvray in Spring 2016. Since its upload, it has received 75 views. For similar materials see Molecular, Cellular, and Developmental Biology in Biology at Texas Christian University.
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Date Created: 04/30/16
Dr. Chumley - The Membrane: • Provides a barrier from outside to inside • There must be a transport system which are proteins imbedded in membrane • Membranes are 50-100Aand consist mainly of lipids and proteins. The proteins have important functions allows cells to adhere to each other or send signals from one side of the cell to the other side. • Lipids: - 1.) Phospholipids • They have a fatty acid tail (long carbon chain are hydrophobic) • Platform in which the fatty acid tail is attached to (typically glycerol) • Phosphate group • Head group, typically an alcohol (the head group is polar and hydrophilic) • Phosphatidyl-serine is IMPORTANT TO KNOW! It has a hydrophobic tail and hydrophilic head. • Free fatty acids only have one carbon chain tail and tend to take on a structure where hydrophobic tails point toward each other forming a MICELLE. • Aphospholipid is more in the shape of a cylinder, and forms a sphere called a a LIPOSOME. This form is energetically favorable. - 2.) Glycolipids • Alipid where the alcohol has been changed to a sugar • Made in the ER and in Golgi • G M1Ganglioside is a ver important glycolipid that makes up majority of plasma membrane of neurons. - 3.) Cholesterol • Contains a steroid ring structure, a non polar hydrocarbon tail, and a polar head group • They sit right between phospholipids • Cholesterol does not change fluidity of membrane (unless you add a LOT) • Functions: - Increase degree of lipid packing (lipids will come closer together when there is a cholesterol in the membranes because the affinity of the cholesterol to the phospholipids. It tightens up interactions between lipids.) - Decrease membrane permeability (if membrane is permeable to a gas, then the membrane becomes less permeable as you increase the cholesterol percentage.) - Decrease membrane flexibility (note difference between fluidity and flexibility) - Enhances formation of lipid rafts Rafts are areas of unequal distribution of membrane lipids • If you have a 1:1:1 ratio of sphingomyelin, phosphatidylcholine (PTC) and cholesterol, • then you form PTC domains. Phosphatidyl-serine is typically on the inner side of the plasma membrane. When cell undergoes • stress, the PTS will flip to the outer side of the membrane. Proteins are made to flip it back inside the cell. When cell death starts to occur, the PTS will flip on the outside. Macrophage recognizes this as a dying cell and degrades it. • -RAP—Fluorescence RecoverAfter Photobleaching You fluorescently tag a protein in the membrane then bleach an area so it is no longer fluorescent. Then you can see how the bleached area will fill back in with fluorescents which indicates the membrane is move around and fills in the bleached area. - The Lipid Bilayer: • Membrane assembly begins in the ER • Phospholipids are added to the cytosolic half of the bilayer and SCRAMBLASES mix the added phospholipids in the ER membrane. It mixes them up and distributes phospholipids. • Some proteins are specific to one side of the membrane (PTS) and FLIPPASES will transfer those proteins to their respective membrane side. - As mentioned before, if cell is stressed, flippases stop working and PTS is on the outer layer of membrane. Dr. Chumley Carbohydrates are added inside the lumen of the ER and the golgi—they are not added in the • cytosol. When the vesicle fuses the carb residues will always be on the outside of the cell. - Certain Phospholipids are confined to one side of the membrane: • Glycolipids are located mainly in the plasma membrane and ONLY in the noncytosolic half of the bilayer. • Glycolipids acquire sugar groups in the Golgi and are added on the lumen side of the Golgi membrane. Once the vesicle fuses with the cell membrane, the sugar will be flipped out to the noncytosolic side of the cell. - Membrane Proteins • Proteins serve functions: - Transporting ions across bilayer - Anchor the membrane to macromolecules on either side - Receptors that detect chemical signals in cell’s environment and relay them to cell interior - Work as enzymes to catalyze specific reactions at the membrane Functional Class Protein Examples Speciﬁc Funcitons + + Transporters Na Pump actively pumps Na out of the cells and Na Pump + + Ion Channels K Leak channel allows K ions to leave cells, thereby having a major inﬂuence on cell excitability Anchors integrins link intracellular actin ﬁlaments to extracellular matrix proteins Receptors platelet-derived growth factor binds extracellular PDGF and, as (PDGF) receptor a consequence, generates intracellular signals that cause the cell to grow and divide Enzymes adenylyl cyclase catalyzes the production of the small intracellular signaling molecule cyclic AMP in response to extracellular signals - Membrane proteins associate with the lipid bilayer in different ways: • Some proteins pass all the way through the membrane and called transmembrane/integral proteins and they areAMPHIPATHIC—meaning it has both hydrophobic and hydrophilic regions • Some are located in cytosol and associate with cytosolic half of lipid bilayer by an amphipathic alpha-helix exposed on the surface of the protein • Some lie entirely outside the bilayer (on either sides) attached to the membrane by covalently attached lipid groups • The primary mechanism used to imbed proteins into the membrane, is the alpha-helical region. The helix forms for two reasons: - 1.) Side groups are hydrophobic and like to be impeded in hydrophobic region of membrane - 2.) One side of peptide bond that holds peptide together, one side is positively charged and the other side is negatively charged. This causes the twisting of the alpha-helix. • It takes about 30-40 amino acids to pass through the membrane. • Multipass membranes—aquaporins • Membrane proteins ca nee solubilized in detergents - Detergents have a polar and non polar regions. The hydrophobic part of detergent interacts with the hydrophobic parts of protein (the same with the lipids). You can isolate the proteins this way. Dr. Chumley • The cell membrane is reinforced by underlying cell cortex - Spectrin and Actin are two proteins embedded in membrane and give it rigidity. This happens on inside and outside of cell. When two cells interact and stick there are proteins that hold them there —an example is the synapse in neurons. - Cytoskeletal “Corral” • Lattice work can also affect fluidity of membrane with some proteins. IF you put fluorescent marker on integral protein inside corral, you would see how it bounces around in one region then bounce around in another region. The corral will open and close all the time allowing the protein to move around. - Membrane Interactions: • Tight junctions - If two cells interact with each other, no proteins can move in or out between the two cells. The apical plasma membrane and basal plasma membrane are completely separate - The cell surface is coated with carbohydrates and forms the GLYCOCALYX. • The main function of the glycocalyx is to protect the cell. - LECTINS are imbedded in the membrane that can bind to carb residues of other cells. These are able to reach out farther than the glycocalyx. - Dissolved gases and steroid hormones can move across synthetic, protein-free bilayer. There are NO movement of ions across. - All steroid hormones are derived from CHOLESTEROL. They have receptors inside cell since it can diffuse through the membrane. Component [Intracellular] [extracellular] Na + Low High K + High Low Ca 2+ Low High H + Slightly Higher Slightly Lower Cl- Low High - Membrane Potential: • The very edge of cell is where the potential lies. • In general, the pH is lower on the inside than on the outside. • The resting potential is (on average) -70mV across a membrane which is why things tend to want to move across. Since there is a difference in potential across the membrane it has the potential to do work—this is • called the Membrane Electrical Potential. - Membrane Transport Proteins: • Transporter - Bind solute and undergo conformational change to transfer solute • Channel - Much weaker interactions with solute and faster solute transfer such as through an aqueous pore • Two types of transporters: - 1.) Passive Transport • Solute passes through membrane simply due to concentration gradient (also due to chemical gradient) - 2.)Active Transport Require energy for conformational change to move solute across membrane • Dr. Chumley • Water moves through aquaporins, a multi-pass protein, down it’s concentration gradient—osmosis • Active transport - Ways to move solute uphill: • 1.) Coupled transport—movement of one solute uphill with another solute downhill • 2.)ATP-driven pumps—use hydrolysis ofATP to pump solute uphill • 3.) Light-driven pumps—use radiant energy (typically found in photosynthesis) - EX. You can use the Na /glucose gradients to transport across membrane. You build up so much glucose inside the cell that a gradient for glucose forms and it wants to leave. Sodium is used as a coupled read to bring glucose back into the cell since sodium’s gradient wants to move into the cell.Acell in intestine takes glucose in from intestinal lumen by a coupled reaction with sodium then moves it out to where the other cells are by passive transport. Sodium must be actively transported outside the cell (via sodium/potassium pump) since the concentration is always greater outside. SGLT1 (Sodium-Glucose Transporter 1) is the transporter that brings sodium/ glucose into the cell. GLUT1 (Glucose Transporter 1) is the passive transporter that moves glucose out of the cell. Sodium/Potassium Pumps • - Pumps 3 sodium for every 2 potassiums (“Too much salt on my banana”) - The pump must hydrolyzeATP in order for this to happen. 2+ • -a Pumps: 2+ Calcium pumps keep the cytosolic [Ca ] low. • These concentrations are low since Ca is used as a second messenger. - This transport usesATP hydrolysis to move calcium across membrane - PMCA—Plasma Membrane CalciumATPase pump…removes calcium to the outside of the cell - SERCAPump—Serca and Endoplasmic Reticulum CalciumATPase pump…moves calcium back into the sarcoplasmic reticulum by hydrolyzingATP • Both of these pumps are active transport! • Pumps: - Uniport • Transport a single solute. IT does not couple the movement of solute with anything else. PMCAis uniport. - Symport • Two solutes move in the same direction. They can use electrochemical gradient orATP.An example is the SGLT1. - Antiport • The solutes move in opposite directions across the membrane. They can use electrochemical gradient orATP. Typically when one solute moves down it’s gradient, it brings another molecule against its gradient. - Ion Channels: • Aselectivity filter allows only certain ions to go through. It is lined with carbonyl oxygens and is just large enough for potassium (for example) to pass through and the carbonyl interaction is stronger than the interactions with water that are solvating the potassium ions. This allows only the potassium ion to pass through the channel. • Membrane potential is governed by the permeability of a membrane to specific ions • Ion channels randomly snap between open and closed states but spend more time in the closed than open position. • Patch clamping • Different kind of stimuli influence opening and closing of ion channels - Voltage-gated - Ligand-gated (extracellular/intracellular) - Mechanically-gated When the stereo cilia vibrate, the hair cells rub back and forth. Liquid on outside of the cell has • higher potassium concentration on the inside (THIS IS DIFFERENT THAN NORMAL CELLS). So when stereocilia move, they open a channel allowing potassium to rush in causing the electrical potential to change. Dr. Chumley • If you remove all the potassium from the outside of cells then the potassium cannot rush in and change the potential difference and will not allow calcium to enter the cell and release neurotransmitters. • Action potentials are mediated by voltage-gated cation channels - All cells have the electrochemical gradient and it isALWYAS negative on the inside and positive on the outside. • Voltage-gated Sodium channel - If a sodium channel is initially closed, an action potential must come about causing it to open allowing sodium to INTO the cell making the membrane DEPOLARIZED.After the channel opens briefly, it goes into an inactive state where it is open, but inactive because protein plugs channel—this is the REFRACTORY PERIOD allowing REPOLARIZATION to occur. - When the cell depolarizes, the change in voltage can be ~15-30mV causing an action potential. - Depolarization is the loss of potassium from inside the cell. - (Chumley 5-pg. 4) - The propagation goes in only one direction because when sodium rushes in and goes in their direction along the negatively charged membrane, when it goes to the left, the channels are inactive and cannot recognize the change in voltage. But when the go to the right, the channels are closed and can detect the voltage difference and open. There are sodium-potassiumATPase pumps that pump the sodium back out and brings potassium back in (3Na/2K). - If you have a resting neuron and create an action potential in the middle, propagation will go in both directions. - At the terminal of the cell, there are voltage-gated calcium channels and once the propagation reaches these channels, calcium rushes in and causes the release of neurotransmitters into the synapse. Sodium rushes in to the adjacent ligand-gated sodium pump on the dendrite causing a voltage change. - The postsynaptic cell has receptors that when activated, open and cause a change in membrane potential allowing the chemical signal to be turned into an electrical signal. - Neurotransmitters can be inhibitory by making the membrane more polar and making it harder to depolarize. Excitatory is opposite making the membrane easier to polarize. • Optogenetics uses light-gated ion channels to activate or inactivate neurons - Force expression of a protein CHANNELRHODOPSIN in specific neurons then insert a photooptic cable. The light turns on and off which causes neurons to work. By changing orientation of the channel causes the neurons to fire an action potation or not fire an action potential. - The inside of the lumen of the ER is the same as the outside of the cell. If a vesicle bids off of ER and fuses the cell, the membrane will be the same. - Transport: Signal sequences direct proteins to the correct compartment • - Import into ER - Retention in lumen of ER - Import into mitochondria - Import into nucleus - - Export form nucleus Import into peroxisomes • There is a tough mesh of cytoskeleton (nuclear lamina) and proteins need a vehicle to transport them through the pore. - Nuclear Localization Signal (NLS) sequence on the cargo protein allows the nuclear importer to transport the protein through the pore and into the nucleus. - Hormone receptors float around in the cytosol that contain a nuclear localization sequence, but is hidden in the tertiary sequence. The transporter cannot recognize the sequence. Once the conformation is altered of the protein then the import protein can take it inside. • Ex. - Inside nucleus, GTP is attached to protein RAN. RAN+GTP binds to import protein which shuttles back out to cytosol. Once in the cytosol, GTP is hydrolyzed to RAN-GTP + Pi and Dr. Chumley dissociates from import protein. The import protein is not able to attach to another NLS and transport another protein into the nucleus. • Ex. - NF-AT (Nuclear Factor ofActivated T-Cells) is a transcription factor which is found in the cytosol. Phosphate groups attached to NF-AT hide the import sequence to take it into the nucleus. Calcineurin is a phosphatase that removes the phosphates and binds to NF-AT bringing it into the nucleus where activation of gene transcription can occur. Kinases phosphorylate NF-AT which expose its export signal to be moves back out into the cytosol. - Protein Sorting: Proteins unfold to enter mitochondria • • Proteins may need to: - Imbed in outer membrane - Remain in inter membrane space - Imbed in inner membrane - Remain in the matrix • Asignal sequence on a protein will bind to the protein translocated in outer membrane (TOM) of mitochondria then through the transporter of inner mitochondria membrane (TIM). - Hsp70- Heat Shock Protein helps unfold and stabilize the protein as it goes through TOM.ATP is needed to remove Hsp70 form the protein. • Peroxisome - Proteins enter the peroxisome from both the cytosol and the ER - Peroxisomes produce phospholipid myelin. Myelin wraps themselves around axon and push all of the channels and pumps creating nodes of pumps and channels. - What else on peroxisome • When a cell begins translation, it may or may not have a signaling sequence that tells the ribosomal complex to connect to the ER so co-translational transport can occur. - Asignal-recognition particle (SRP) binds to the signaling sequence on the growing peptide and binds to the SRP receptor on the ER. SRP is displaced and moves the peptide signal into the protein translator (Sec61) which moves peptide through the membrane as it is being synthesized. - BIPproteins reside inside ER lumen and bind to peptide coming in the ER and pull it through. They are powered byATP. - Vesicular Transport: • ER—>Cis side of Golgi—>leave via trans side of Golgi in vesicles—>endosome or membrane • COPproteins: - COP-1 and COP-2 work similarly to clathrin that coats vesicles. Vesicles that bad from ER have coating of COP-2. They form by creating a sphere-shape and pull the membrane up and ultimately buds it off. COP-2 is also used to moves between sister cisternae in Golgi. • Adaptin recognizes protein imbedded in membrane, then clathrin binds to adaptin. Clathrin interact with each other and pulls on the membrane. Dynamin squeeze the stalk which will fuse the membrane and bud off. - There are some mutations where dynamin is overreactive making too many vesicles, and some where it doesn't make enough. the Charcot-Marie-Tooth disease is a mutation in dynamin that prevents it from contracting in certain tissues. This destroys ability to release neurotransmitters in neurons for muscle contraction. Type of Coated Vesicle Coat Proteins Origin Destination Clathrin-coated Clathrin + adaptin 1 Golgi apparatus Lysosome (via endosome) Clathrin-coated Clathrin + adaptin 2 Plasma membrane Endosomes Dr. Chumley Type of Coated Vesicle Coat Proteins Origin Destination COP-coated COP proteins ER Golgi apparatus Golgi cisterna Golgi cisterna Golgi apparatus ER • Vesicle docking depends on tether and SNAREs - RAB proteins are responsible for vesicle docking by interacting with a specific tethering protein which holds vesicle away from target membrane. (Think of neurotransmitter release, they can be tethered to membrane waiting to be sent through. Vesicle tethering requires calcium.) SNARE protein on vesicle (v-SNARE) interact with SNARE on membrane (t-SNARE). In presence of calcium, SNAREs twist together and vesicle comes closer to membrane, fusing and releasing cargo. - Synaptogamin-1 is the calcium detecting protein, without this no fusion of vesicles occur. - The type of RAB protein determines where the vesicle goes. - Secretory Pathways: Most proteins are modified in the ER • - Glycosylation • Most proteins that are membrane bound have carbs attached to them. This requires Oligosaccharyl transferase which catalyzes transfer of precursor oligosaccharide to the peptide chain in the ER. - GPI-linked proteins also made in ER • GPI-Glycosylphosphatidyllinositol links an inositol to a protein binding it to the membrane. • Proteins are further modified in the Golgi apparatus and can be released from the cell by exocytosis - Endocytic Pathways: • Fluid and macromolecules are taken up by pinocytosis. • Receptor-mediated endocytosis provides a specific route into animal cells (Chumley 8-pg. 19) • Endocytose macromolecules are sorted in endosomes - Transport vesicles bring molecules to the endosome and from there can undergo transcytosis where it simply releases the molecule on the opposite side of the cell (required in cells with tight junctions for example), or go to the lysosome for degradation, or become recycled and go back to the same membrane the vesicle came from. • Lysosomes are principal site of intracellular digestion. They contain H pumps to make the lysosome more acidic than the cytosol. pH of the cytosol~7.2 while the pH of the lysosome is ~5.0. - Innate Immunity: • Two classes of immunity: - Innate Immunity—What happens FIRST. The very first thing that happens when pathogen enters body. You HAVE to have this. Phagocytosis is the main function of innate immunity. - Adaptive Immunity—activation of T-lymphocytes Innate immunity is an immunity against any type of agent, not specific for any one type of pathogen. • • Major sites of entry for pathogens include respiratory, gastroinestinal, and urogenital systems. 90% of energy is focus on these tissues because they are so thin and pathogens simply need to cross epithelial cells with tight junctions. • Note the duration of infection with the lack of innate immunity and adaptive immunity. • Phagocytes — cells that engulf and digest microbes and cell debris - Binding of bacteria to phagocytic receptors on macrophages induces their engulfment and degradation • Complement — set of proteins that form a nonspecific defense mechanism against many different microorganisms. They can coat a pathogen enhancing phagocytosis. - Binding of bacterial components to signaling receptors on macrophages induces the synthesis of inflammatory cytokines. • Bacterial cell surface induces activation of complement which then binds to the complement receptor on the effector cell. The effector cell engulfs the bacterium and breaks it down. Dr. Chumley • Methods of pathogen recognition by pattern-recognition receptors (PRRs): - LPS is a pattern certain kinds of glycolipids or sugars are also patterns. • Major Histocompatibility Complex (MHC) —proteins that tea short macrophage amino acid strands and presents it outside the cell.
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