Cell Bio Mcb 2210 Class Notes PDF

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1/20/16 Intro lecture Cell Theory - All living things are made of cells - The cell is the basic structural unit of living things - Cells can only arise by division from preexisting cells o Core concepts that are still believed to be true today Basic prosperities of cells - List is common in most cells but not all cells have all these properties - There are many exceptions within our bodies Central Dogma - How the cells exhibit all their properties - Information that allows cells to do and express things are encoded in their genome - Activation  transcription  Processing  Translation - More complex than what diagram shows - Vast number and types of proteins encoded in the genomes of different eukaryotes Cells are dynamic - Not all the info in a cell is encoded directly in DNA in a form we can read - Cells are either methylated or acetylated 1/22/16 - All cells have a common ancestry - First eukaryotic ells are more related to archea than bacteria - Prokaryotic cells arose first therefore they’re simpler cells but that doesn’t mean they’re inferior - Single celled organisms are generalists therefore complicated and do many functions Core Tools of Cell Biology Type of cells cell biologists study - Primary cells: cells that are isolate straight from a living organism that can be cultured to study cell specific functions. o Many are differentiated so they aren’t going to divide anymore/hang around in the dish longer o Can bring limited values bc we can’t understand their growth factors that keep them around longer. o Cells right out of the organ. Not many are differentiated so they don’t divide anymore - Transformed cells: cells that have become cancerous because they divide continuously therefore easy to maintain in lab - As technology increases, we can study cells in vivo/in their real environment instead of pulling them out in a dish bc you don’t know if how they act in a dish is relevant in their lives. - Light microscopy: one of the biggest tools o Limited (around 200 nm) in contrast, magnification and resolving power, limiting what can be detected with them o The quality of microscopes have been improving but again, there are hard limitations o Cells are mostly water therefore they’re very transparent. Light passes through them bc they have poor contrast. Need to enhance their contrast (poor contrast due to water) - Transmitted light microscopy: magnify a specimen using one or more lenses o White light passes thru the specimen before being collected o Cells are neither reflected or absorb much light so contrast is poor and little details can be made o Contrast is the difference in intensity between an object and its background o Bring out detail, we need to exploit changes in the phase of light or stain the object to make it darker o Phase contrast or differential interference contrast  techniques for enhancing contrast - Magnification vs. resolution

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Magnification is how much you blow up on image. Depends on lens. Resolution= how far apart two objects have to be to be seen as 2 different objects Depends on wavelength. Shorter wavelength = better resolution as well as how good ur lens are. Numerical aperture (how well lens can gather cone of light coming out of the specimen) Resolution of conventional light microscopes is ½ the wavelength of light being used

1/25/16 - Ability to resolve doesn’t mean ability to detect. - Just means you won’t be able to see if it’s smaller than a certain nm - Anything smaller will look the default nm - Epifluorescence microscopy improves contrast and allows specific structures to be labeled via staining. - Smaller wavelength = higher energy - Illuminates molecules w/ fluorophores via higher energy light - Antibodies: immune proteins that bind to specific proteins. o To make, obtain protein, pure of interest and inject it to animal. o Monoclonal vs. polyclonal antibodies - Immunocytochemistry only works for nonliving cells - Western Blotting- used to detect proteins with antibody detection. o Smallest proteins can elude through SDS PAGE gel faster o Gel separates protein by size of molecule o Use electric field to separate protein - Fluorescent proteins can keep the cell in tack so we never kill the cell - To get plasmid across membrane is called transfection o Infect cells with foreign DNA to cause a desired expression o Transfection can be transient or stable (expression from plasmid or DNA integrates into genome/heritable) o Transgenic lines of animals can be generated by stable transfection of germ cells o We can transfect mutant molecules to be active all the time by activating a protein to turn it on o Mutant molecules that are dominant negative don’t function right and block the function of the cell’s own version of the molecule  Mutant form of enzyme can’t catalyze enzymatic reaction aka doesn’t work/nonfunctional therefore causes the mutant form of the cell to not work as well. Has ability to even block function of normal enzymes 1/27/16 - A multi-color fluorescent image uses different proteins and antibodies as well as DNA dyes to add color to a black and white microscopic image - 200 nm is the lowest resolution of light microscopy you can see so any object smaller than 200 nm will always appear 200 nm, regardless of it’s size - siRNA help transfect things into cells using new technologies that exploit RNAi mechanisms to allow us to knock out specific proteins of interest in cells by targeting mRNAs o **Don’t memorize image on this slide. Not a genetics class. Just understand generally what is going on. - Two methods to deblur images (generally remove out of focus light from the focal plane) o Laser scanning confocal microscopy uses pinholes to deblur  Pinholes can focus length  Gives laser to stimulate fluorescent of image causing it to focus on one specific point when it passes through keyhole  When light comes back out, it passes through detector and another pinhole that focuses it as well o Digital deconvolution uses computational methods to deblur

Superresolution microscopy techniques allows people to see below 200 nm If resolution limit is 200nm, how did we come up with all the textbook figures of organelles and cell structures that are smaller than that? o Electron microscopy Electron Microscopy - Another means of getting around the resolution limits of light - Electrons are used to illuminating specimen of interest - TEM and SEM are two popular electron microscopy microscopes Sample Prep for TEM - EM must be done in a vacuum for electron gun to work - Sample must be dry so it’s low density to scatter electrons - Need to stain the metal to bind to structures of the cell and stick there Immuno-electron microscopy - Label structures in EM - You can’t see antibodies in the EM, but you can attach dense particles to antibodies to make them visible in the EM (gold beads) - Allows you to visualize the beads in your image/the fine details in structures Scanning electron microscope - Coat sample with heavy metals - Electron beams bounce off metals, collect rays with detector to give you a different type of image (see pictures on the slides) Differential centrifugation - Take samples of cells and break them up in solution - Put solution into ultracentrifuge tube and spin sample at different speeds and time - Based on size or density of what is in the solution, more dense things go to bottom of tube in lower speeds and in less time compared to things that are less dense Gradient centrifugation - Put sample on sucrose gradient - Organelle moves through gradient until it reaches a sucrose level as the same density as itself - Finer separation of organelles Membranes and Proteins - Proteins specialize one organelle from another - Each organelle contains its own subsets of proteins therefore differentiating their functions and structure - Membranes are barrier between inside and outside worlds, and the barriers are selectively permeable - Membranes are important for cell-cell interactions and provide scaffold for biochemical activities  glycolysis, electron transport chain, etc. -

1/29/16 - Lipid rafts: specialize regions w/ specialize proteins - Three major lipid components of membrane: - Amphipathic molecules (hydrophilic and hydrophobic ends) - Level of saturations in fatty acid tails is important to consider. o Unsaturated creates spatial difference therefore kink in the tail. o Overall height dimensions of kinks are shorter o Taller vs. shorter phospholipids contribute to thicker or thinner membranes o Straight saturated chains = packed more and less fluid membrane compared to unsaturated chains o Double bonds creates a kink - 1. Phospholipids o Cholesterol o Proteins that can be associated w/ or inserted in membrane to give it variety of functions o Phosphoglycerides: made up of hydrophilic head group and hydrophobic tail

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 Glycerol group links chemical compounds of head to the tail  Most abundant in membranes o Sphingomyelin: still a phospholipid but instead of glycerol, it has a sphingosine group 2. Glycolipids: head group linked by sphingosine group o head group is sugar group linked to FA hydrophobic tails 3. Sterols: made up of 4 fused rings o Cholesterol can alter quality of phospholipid membranes Lipids behave badly in membranes o Form either micelles or liposomes if you put them in water o Micelles: hydrophilic heads face aqueous solution and tails facing each other so they don’t face water  Single phospholipids layers  Form w/ lipids that have one phospholipid chain o Liposomes: formed in test tube with lipids  Usually bilayers Bilayers can self assemble if there’s ever a tare Biological membranes are mixture of different types and amounts of different mixtures with proteins embedded and associated with them (different functions because of that) ** Don’t memorize the major lipid components of selected biomembranes table*** Composition can affect curvature of a membrane Variety of head groups on phospholipids can all be different sizes Phospholipids are not fixed in space. They float around in sea of phospholipids at different rates. Very dynamic o Lateral diffusion happens rapidly o Transverse diffusion/flip flop- occurs rarely about 1 time a day because the phobic tail would be interacting with water and that’s not thermodynamically favorable. o Rotation o Flexion is also very rapid Method to measure mobility of phospholipids in membrane: o Generate liposomes in lab made up of phospholipid of interest. Conjugate fluorescence to head group of phospholipids to treat cells with that. o FRAP- use photo bleaching technique. Irreversible killing of the fluorescence While things are mobile in membranes, things are not completely uniformed. o Two leaflets of membranes can have different compositions o Flippases are enzymes that can flip flop lipids together or bring leaflets back to their proper side Proteins are basic machinery of cells and important part of membranes because proteins allow things to come in and out of membrane if cells want that Side chains give amino acids different characteristics Hydrophilic = polar hydrophobic = non polar Peptide bond forms when amino and carboxyl sides interact with each other N terminus comes out of ribosome first aka made first Protein folding occurs co-translationally (as protein undergoes translation) Protein needs to be folded in it’s native form for it to function properly o Chaperones and chaperonins help proteins fold properly o Heat shock family of proteins are proteins that act like chaperones o Regions on chaperones (hydrophobic regions) interact with hydrophobic interactions on unfolded protein to help them fold by hydrolyzing ATP. o Hydrolyzing energy allows chaperone to fold up protein as it undergoes conformational change Most membrane proteins almost always glycosylated

2/1/16 - Proteins fold co-translationally. Some can fold into proper shape on their own, some cannot. - Disulfide bonds = covalent bonds. Occurs in oxidized environment to stabilize the protein - Most membrane proteins are glycosylated  sugar chains added onto proteins in ER as they’re being synthesized - Many proteins have multiple functional domains which allows catalysis to occur better - Many different proteins can share functional domains therefore causing similar protein structures - Protein complexes can be stable, or can be triggered by a signal to assemble - Protein-protein interactions occur due to non-covalent interactions - Quat structure: interactions of multiple subunits. 2 same subs = homodimer, 2 different subs = heterodimer - More non-covalent interactions = stronger interactions therefore bind together tightly and stay bounded together longer before falling apart again. Weaker interactions bind and release rather quickly. - Strength of interactions between proteins = affinity o Higher affinity interaction will have relatively small koff (kd) and off rate, complexes will stay bound longer, so there will be more of them at equilibrium o **Don’t go deeply into the equations o On rate depends on the concentrations of the reactants (high concentration = high interactions) and Kon (association) rate constant, which depends on size, rate of diffusion, and whether there is a favored orientation required for binding, etc. o Koff rate depends on the sum of the forces that will hold reactants together o Affinity controls what fraction of a molecule will be bound as the concentration of its binding partner is varied o Glutamate has higher binding affinity than glu-azo  lower kd = stronger affinity interaction  If concentration is below kd value then low interactions occurs - At equilibrium, there are more complexes - High protein concentration than kd than you’ll see them complex - Nano lower than micromole therefore A-C will have a higher affinity interaction bc higher affinity has lower kd - A-CD complex decrease binding affinity bc kd increased Classes of Membrane Proteins - Integral: AA sequence is tightly associated into the lipid bilayer o Transmembrane proteins span the bilayer - Lipid anchored: AA sequence has undergone lipid modification which sticks it to hydrophobic region on the membrane o Different fatty acids used to attach to inner leaflet o GPI can be added to protein and insert themselves and become outer leaflet - Peripheral: have protein-protein interaction with either lipid anchored or integral membrane protein - Immunofluorescence or immune-EM are two techniques used to show a protein associated w/ the membrane - Proteases cleaves or digest accessible protein regions and can be used to deduce topology of a protein in the membrane o Topology = what kind of domain does the protein possess o Treat the cell with trypsin in culture medium to digest extracellular domains of protein while keeping intracellular domains in tact 1/3/16 - One way to deduce topology of protein is to do an assay with proteases such as trypsin. - Transmembrane region of proteins are amphipathic o If alpha helix based, it can be a single or multipass Transmembrane protein - Neg = hydrophilic pos = hydrophobic on graphs - Beta sheets can also interact w/ membranes

o Amphipathic character o These sheets can role up to form a barrel Fluid Mosaic Model of Membranes - The lipid bilayer is a flexible 2D fluid sheet - Helps us picture the membrane of cells - Membrane proteins float in this sheet - Many proteins can and do move laterally in membrane but once a protein is inserted, it can’t easily leave the membrane - Topology of protein is set once it’s inserted and synthesized - Conformation can change, however. - Not all proteins are mobile, but many are - Figure 10-43 shows what ways can prevent movement of proteins in the membrane Method 1 for measuring protein mobility: cell fusion - Label proteins of one cell w/ red dye and second with green dye - Fuse membrane of two cells to form heterokaryon with force bc usually membranes don’t like to fuse - Watch what happens to the two dyes - Result: overtime they become mixed Method 2: FRAP of fluorescently labeled protein - CD2 is a membrane protein that links to yellow fluorescent proteins - Take laser, photobleach a spot. Light is so intense it alters covalent bond of molecule so it can’t fluoresce again. - Track over time does the fluorescence recover - Recovery = mobile in membrane Method 3: single particle tracking - Quantum dots are crystals that can fluoresce - Can link to an antibody - Track the dot in the membrane Lipid rafts - Contain cholesterol, sphingolipids, and proteins - Tend to accumulate diff proteins than non-raft areas Membrane Transport - Lipid bilayer is impermeable to most things - Small, nonpolar typically can diffuse right across membrane - There are transporters in the membrane to control entry for those molecules that cannot easily cross the membrane Membrane transport proteins - Passive Transport: allows net movement down a chemical concentration, electrical or electrochemical gradient; no energy beyond thermal motion needed (high areas of concentrations to low) o Facilitated diffusion by uniporter carrier proteins o Ion channels - Active transport: move molecules against a chemical, electrical or electrochemical gradient; they require extra energy (low to high) o ATP-dependent pumps o Symporters o Antiporters 1/8/15 Membrane Transport Make up lecture - Membrane lipids and proteins associated with them create a selective permeability barrier - Transporters decide what gets across the cell membrane o Required because not many molecules can diffuse across a lipid bilayer

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o This is due to hydrophobic tail thus creating hydrophobic barrier on either side of membrane and therefore only smallest hydrophobic molecules can pass across the membrane o Charged, polar molecules can’t really get across Small ions with charges cannot move through bilayer bc of that charge If the cell wants to transport the biomolecules that can’t get across via passive diffusion, a solution are membrane transport proteins Transport proteins act enzymatically to move substances across membrane o Passive transport proteins allow net movement down an electrochemical gradient  No extra energy besides thermal energy  Carrier proteins or ion channels are the proteins that move molecules passively  Facilitated diffusion = done by carrier proteins o Active transport when molecules move against a gradient therefore needs extra input of energy to make that happen  Pumps directly use ATP to do this  Symporters and Antiporters are carrier proteins that indirectly use ATP to move things against gradient Concentration + chemical = electrochemical gradient Carrier proteins vs. channels (passive) o Carrier proteins have binding site in 3 structure that interacts with solute (molecule being transported)  Binds one side of membrane, conformational change in carrier protein tht opens it up on opposite side and releases molecule o Channel have little interaction w/ solute  Either is in open or closed confo. When open, molecules move directly through an aq. Pore in center of channel. Diffusion doesn’t peak, no saturation. Transport peaks when binding sites on transporters are saturated. Due to binding of solute to carrier. Produces max rate of transport (vmax) Cells use channels to set unique ionic environment inside and outside the cell Osmolality refers to concentration of solute in solution o Water floats to higher solute concentration Cells use transport proteins to keep [solutes] on both side membrane are equal Hypertonic = cell shrinks bc water moves out to high solute outside the cell Charged molecules add another force o If the inside of the cell is net neg, an anion will be repelled and a cation will be attracted o Vise versa if inside cell is positively charged o The combo of the chemical concentration gradient and electrical gradient determines the rate and direction of transport of a charged molecule.  Known as electrochemical gradient o Need to consider which way a charged molecule will move across a membrane Diffusion of ions gives rise to electrical potentials Lipid bilayer can separate charges (when you typically cannot) Only a small number of ions need to move across membrane to create...