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CELLBIO2382 - MIDTERM I Cumming's Lectures (Lectures 1 - 8).docx

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Biology 2382B
Robert Cumming

Biology 2382 – Cell Biology Cumming’s Lectures Lecture 1 – Introduction to Cell Biology What Is Cell Biology? • Anything that’s alive has a cell / needs a cell • Cell Biology: academic discipline that studies cells, basic structural, functional and biological unit of all known organisms • Symbiotic relationship between cells and genes Why Study Cells? • In order to treat something that’s abnormal, you must know what is normal What Is A Cell? • Ultimate goal is to understand how macromolecular systems and organelles work and cooperate to enable cells to function autonomously and in tissues How Do We Study Cells? • Need a way to isolate and maintain cells in vitro (artificial situations, outside of the body) where we can manipulate them • Know how to view cells, separate them and identify proteins that drive processes Cell Culture • Cell Culture: technique used to grow cells / tissues outside organism under strictly controlled conditions • Cells are isolated from any tissue by breaking down cell-cell and cell- matrix interactions • Proteins stuck outside the cell make them sticky  need to break apart • Trypsin cleaves all the proteins off outside the cell, allow the cells to not stick together • You can add a chelating agent EDTA • Cells are supplied with proper nutrients, serum and grown at 37°C in a CO 2ncubator (pan of water of body for more moisture to evaporate) • When you culture cells outside of the body, there is higher oxygen concentrations, by infusing more CO 2t maintains the normal gas ratio that is found inside the body • Take cells and put into vessel (dish / flask), supply with proper nutrients (medium – amino acids, minerals, vitamins, salts) so it can properly grow • Recreating the environment of the body – adding the cells to a medium, mimicking with artificial concoction • Add serum to vessels (liquid within blood) that will contain insulin, growth factors that helps the cell to divide • Red dye is added to observe pH of the cells • Put inside spin bottles or roller bottles for suspension cell cultures compared to adherent (dish / flask) • Primary Cell Culture: cells taken directly from an organism • Contact Inhibitions: cell density is high, only divides a limited number of times before it stops dividing • You can add trypsin to dissociate cells and put them in fresh media to repeat it over and over again (passaging) • Hayflick Limit: primary cells can only maintain ~50 generations • Cell in cultures can do this indefinitely are called a cell line • Cell Line: transformed cells that are able to grow indefinitely • In cell line, they lose contact inhibition and start to grow on top of each other • Cancer is uncontrolled cell division, lose regulatory mechanisms that control how often they divide • Making cell from tumours  you can promote transformation by introducing an oncogene, and overexpress to disrupt the normal cell cycle • First human cell line called HeLa cells (1952) from cervical carcinoma • Fibroblasts is one of the major cell types in skin (grown most readily in culture) o Normal – elongated, aligned, orderly packed, grow in parallel arrays, contact inhibitions o Transformed – rounded, hairlike processes, disorganized, grow on top of each other, loss of contact inhibition Stem Cells • Stem cell has two major properties: o Self renewal of stem cell can make more identical copies of itself (cell lineage) o External cue to differentiate, take cell and instruct it to be a different cell that has a function (programming event) • Embryonic stem cells (ES cells) can be maintained in culture and form differentiated cell types • Inner cell mass is the accumulation of the ES cells • ES cells are pluripotent – can be grown indefinitely in culture and differentiate to three germ layers • Can be induced to differentiate into precursors for various cell types • In cell culture, you interfere with the gene and take genetically-altered line and introduce into a fertilized egg • ES cells incorporate into developing embryo Adult Stem Cells • Found in most tissues, required to maintain and repair the tissue • Intestinal epithelium are replaced every 5 days • Cells that are in close proximity to the stem cells (helper cells) help the stem-cell niche (provide signals to either self renew or differentiate) • Intestinal stem cells are not pluripotent, can only give rise to one or two cells (help replenish destroyed tissue) • As we get older, stem cells don’t work as well (not as good to replenish – part of the reason why we age) Normal Cells  Stem Cells • The path from stem cells  differentiated cells is not necessarily a one-way trip (can reverse back to earlier state) • Mature cells can be reprogrammed to become pluripotent • Pluripotent stem cells can be obtained from differentiated normal cells • Reprogramming of fibroblasts can be accomplished by introducing into differentiated cells three genes that are characteristics of ES cells and one in cancer cells • These cells are called induced pluripotent stem cells (iPS cells) – believed to be the future of transplantation medicine • ES cells is creating life in order to destroy it (ethical issues) • Notion of personal medicine  custom tailor a therapy for patient Origins Of Cancer • Genetic mutations can arise that result in uncontrolled cell division / prevent programmed cell death • Mutations can occur in stem or normal cells • Cancer stem cells differentiate into different cells, harder to control • Reprogramming normal cells to stem cells could potentially promote transformation Lecture 2 – Imaging in Cell Biology Anton Van Leeuwenhoek • First practical microscope about 300 years ago • First to observe living protozoa and bacteria (animalcules) and went on to visualize human RBCs and sperm Modern Compound Microscope • Bright-field Microscopy: o White light as the light source o Condenser lens to focus light on specimen o Objective lens to collect light after it has passed through specimen o Ocular / eyepiece lens to focus image onto eye o Typical light microscope magnification is 40 to 1000x o Only structures with high refractive index (ability to bend light) are observable Resolution Of Microscopes • Resolution: ability to distinguish between two very closely positioned objects as separate entities • Conventional microscope can never resolve objects / cellular features that are less than ~0.2 μM apart • Smaller resolution = better • Resolution = D = 0.61λ / Nsinα (distance resolved between 2 points) o λ = wavelength of light o Nsinα = numerical aperture (higher = better) o N = refractive index of medium between specimen and objective lens o α = ½ angle of light entering objective o Limit of resolution is 0.2 μM = 200 nm Wavelength Spectrum Used In Microscopy • Obtain contrast in light microscopy by exploiting changes in phase of light • Certain parts of the cell refract light more than other parts • Cellular constituents with high refractive properties can slow the passage of a light beam by a quarter wavelength (~ ¼ λ) • When you have interference (causes area of the cell to look darker), amplitude is lower = dim light • To cause maximal wave interference, we use phase contrast microscopy • Phase Contrast Microscopy: used to examine live “unstained” cells • Small differences in refractive index and thickness within the cell are further exploited and converted into contrast visible to the eye • Differential Interference Contrast Microscopy: used to examine live “unstained” cells o Similar to phase contrast, allows for conversion of small differences in refractive index and thickness within the cell into contrast visible to the eye o Based on interference between polarized light and equipped with polarizers o Defines outline of large organelles and provides better detail of cell edge • Interference between polarized light generates contrast Fluorescence Microscopy • If we want to see precise details about proteins or cellular structures in better resolution, we can use fluorescence • Fluorescence Microscopy: uses a property of certain molecules to fluoresce, emit visible light when they absorb light at a specific wavelength • Location of fluorescent dyes or fluorescent protein molecules can be imaged, can visualize more than one structure • Dyes (fluorophores) absorb energy kicking electrons into a higher orbital (unstable) – this instability causes electron to drop into its normal orbital releasing energy as visible light (fluorescence) • Excitation light is different from emission light, an optimal heat for excitation and optimal heat for emission • Fluorescence images are obtained by using a more powerful light source and filtering out the wavelength of light that is optimal • Signals of fluorophores are bright on a black background • Variety of fluorophores exist with different excitation and emission wavelengths that allow labeling of more than one organelle at the same time • Fluorescent dyes are available to stain cell structures and organelles • Dye can be conjugated with antibodies to localize any molecules of your interest in cells Monoclonal Antibodies • HAT medium is toxic for myeloma cells, which have mutation of specific gene • Hybrid cells survive in HAT medium because they obtain a missing gene product from spleen cells • Hybrid cells are immortal like myeloma cells and produce antibody Immunofluorescence Microscopy • Antibodies are made to specific proteins and added to cells fixed on a slide which bind specific protein they were design to recognize • How are you going to find where the specific antibody bound? • You add secondary antibodies with attached flurophores and bind the primary antibody • Each flurophore has a unique excitation and emission wavelength that can be detected with appropriate filters in the microscope • Performed on fixed (dead) cells Dual Fluorescence Microscopy • Uses appropriate microscope filter set for each flurochrome then digitally overlay images • Fluorescent imaging in live cells uses green fluorescent protein (GFP) – derived from a naturally occurring protein found in a jellyfish capable of bioluminescence • Protein contains a short sequence of amino acids (chromophore) that are capable of fluorescing when excited with blue light • Gene was isolated and heavily modified so it would encode a protein with properties ideally suited for live cell fluorescent imaging • GFP-fusion proteins allow fluorescent imaging in live cells o Use recombinant technology to put GFP gene inside plasmid with gene of interest  o Transflect into a cell and translate the synthetic protein inside the cells (GFP-fusion protein) • Fluorescent proteins come in many different flavours • By mutating various amino acids in GFP, new types of fluorescent proteins were created with different excitation and emission profiles • Two or more different fluorescent fusion proteins can now be visualized in live cells Laser Scanning Confocal Microscope • Uses a laser to shine through a pinhole and to the dichroic mirror • Fluorescent specimen is illuminated with a focused point of light from a pinhole • Emitted fluorescent light from in-focus point is focused at pinhole and reaches detector • Emitted light from out-of-focus point is out of focus at pinhole and is largely excluded from detector • Detcting fluorescence only from focal plane produces a sharp image (thin optical section) Deconvolution Microscopy • Computationally intensive math procedure to remove fluorescence contributed from out-of-focus parts of the stained sample • Considers so-called point spread function which determines degree of blurriness by comparison to a reference set of tiny fluorescent beads • Images are taken at different focal planes (called a Z-stack) and restored by deconvolution display impressive details without any blurring • Fluorescence resonance energy transfer (FRET) measures protein interactions in live cells • If no protein interaction occurs then excitation of cyan fluorescent protein (CP) will only result in cyan fluorescence (480 nm) • If protein interaction occurs then excitation of CFP with result in yellow fluorescence (535 nm) • In picture, protein interaction detected at front of migrating cell Fluorescence  Electron Microscopy • Electron microscopy provides better resolution than fluorescence microscopy • EM needs fixed and sectioned samples or metal-coated samples (living cells cannot be imaged) • Gives highly magnified images o Wire filament is an electron source and when heated, electrons accelerate towards anode o Magnetic (not glass) condenser focuses electrons on specimen o Specimen is stained with electron-dense heavy metals • Electrons are excited and go towards the anode, focused through condenser lens and pass through and focus the electron path onto a detector screen • If it hits it, it will make a bright white signature • Images are formed from electrons that pass through a specimen (TEM) or scattered (SEM) from a metal coated specimen • SEM looks at the outside and TEM looks the inside • Resolution of TEM = D = 0.61λ / α o No N as light is replaced by electrons in a vacuum o Sin α is now α since electron scatter is almost 0 o Theoretical resolution is 0.005 nm, the effective resolution is 0.1 nm (2000x greater than light microscopy) o Very fine “D” • To prepare sample for TEM, tissue / cells must be chemically fixed (aldehyde) and dehydrated • How is the image formed? o Electrons hit specimen but deflect due to metals deposited on organelles o Unobstructed electrons are focused by lenses onto a phosphorescent screen o Crystals in the screen, excited by the electrons, give off energy as visible light o B&W image is made up of shadows where electrons failed to penetrate • Detection of specific proteins using immunoelectron microscopy • Degradation of fatty acids in peroxisomes is accompanied by forming H O 2 2 which is inactivated by enzyme catalase Lecture 3 – Isolation & Analysis of Cell Organelles and Molecules Labeling Live Cells With Fluorescent Antibodies / Stains • Antibodies made against specific cell surface proteins can be linked to fluorophores • Membrane permeable fluorescent dyes can be used to label intracellular structures • Cells with bound antibodies or that have taken up the dyes can now be sorted and counted Fluorescent Activated Cell Sorting (FACS) • If we want to quantify something…FACS o Cells pass single file through laser light beam o Both fluorescent light emitted and scattered are measured by detectors o Individual cells are forced through a nozzle and given a charge proportional to the degree of fluorescence detected o Cells with different electric charges are separated by an electric field and collected • Quantification of cells expressing two different cell surface markers by FACS o As cells pass through FACS, intensity of green and red fluorescence emitted by each cells is recorded o Each dot represents a single cell o Proportion of each cell population can be calculated • We can use certain dyes that can permeate the cell membrane • Cells that have replicated their DNA but not fully divided (G2) will have twice the Hoechst stain fluorescence intensity of non-dividing cells (G1) • Much more cells in G1 phase compared to S and G2 due to length of time spent in G1 • At any point of time, most cells are in G1 (longer phase in cell cycle) How To Isolate Cell Organelles? • Step 1: disruption of cell plasma membrane o Mechanical homogenization o Sonication (ultrasound) o Pressure (cells forced through narrow valve) o Non-ion detergents (triton x-100) o Placing cells in hypotonic solution • Step 2: centrifugation of cell homogenate o Differential o Equilibrium density-gradient • Differential Centifugation: spinning homogenate yields pellet and supernatant, increasing centrifugal force (gravity) to isolate organelles based on mass • Filter homogenate to remove clumps of broken cells, connective tissue, etc. • g = relative centrifuge force (RCF) • Using sequential increase of g for different pellets, increasing spinning will lead you to isolate smaller and smaller things • Equilibrium Density-Gradient Centrifugation: separation based on density, homogenate is applied to a gradient of sucrose • At high speed / several hours, organelles migrate to sucrose layer equal their own density and remain there How Are Proteins Separated From The Organelle? • We can use different detergents to punch holes in membranes • All detergents have two properties (amiphipathic molecules) – have hydrophilic and hydrophobic domains • This allows the detergents to disrupt the lipid bilayer in organelles • Nonionic detergents disrupts lipid bilayer without interrupting the proteins folding / function • Ionic detergents denature by disrupting ionic and H-bonds, also disrupts lipid bilayer (good way to extract proteins but loses all function) SDS-Page • How do you find one particular protein? Using SDS-page • Electrophoretic separation of proteins is most commonly performed in polyacrylamide gels (PAGE) o Carried out in presence of negatively-charged detergent SDS (denatures and binds / destabilizes hydrophobic side chains within protein core) • All polypeptide chains are forced into extended negatively-charged conformations with similar charge-mass ratio • Mobility of SDS protein complexes are influenced by molecular size • How do you see and quantify actual proteins? o Apply some sort of chemical stain / dye to show bands • There is a lineal relationship between log molecular weight and electrophoretic mobility How To Detect A Specific Protein? • Western blotting (immunoblotting) • Electrophoresis & transfer  antibody detection  chromogenic detection o Take SDS-page and transfer onto membrane using electrical current o Proteins will leach out of gel onto the membrane (tougher) o Incubate with hybridized solution with antibodies and recognize desired protein o Enzyme-labeled second antibody recognizes the first antibody – enzyme reacts with a substrate (catalyze reaction causes substrate to glow only where it makes contact with enzyme) o Enhanced chemiluminescence substract reacts with enzyme of second antibody (glows) o Blot will glow where substrate is • X-ray film – dark bands represent specific proteins (positions of marker proteins are indicated by a pen while overlaying film on blots) Lecture 4 – Protein Synthesis & Transport Protein Sorting / Targeting • Typical mammalian cell has 10,000 cells that must be localized currently • Newly made peptides must be directed to correct destination • Targeting: direct proteins to right destinations (organelles), during or after synthesis • Sorting: direct proteins to secretory pathway (ER, golgi, lysosomes) • Many proteins are synthesized just by cytosolic ribosomes o Remain in cytosol o Targeted to intracellular organelles (specific signal sequence) • Other proteins are synthesized by ribosomes attached to rough ER o Reside in ER and proteins which are sorted to membrane, golgi, lysosomes • Two major protein-sorting pathways – nonsecretory and secretory • Basic mechanisms of protein targeting to the membranes of organelles are common: signal sequence  receptor for signal sequence  translocation channel  source of energy ER Structure • Endoplasmic Reticulum: uninterrupted membranous tubules and vesicles separated from cytoplasm o RER has ribosomes on tubules (cisterna) which are stacked o Extends from nuclear membrane • Secretory proteins have to enter the ER before they get out of the cell • Secreted and membrane proteins are sorted through RER • Sugars / carbohydrates are added to polypeptide, disulfides bonds are formed • Proteins are folded by chaperones (doesn’t happen spontaneously) Contranslation Translocation • Translocation and translation occur simultaneously • Cell-free experiments demonstrated that translocation of secretory proteins into microsomes is coupled to translation o Amino terminal signal sequence of newly initiated polypeptide (nascent proteins) o Signal-recognition particle (SRP) o SRP receptor embedded in ER membrane o Translocon: protein channel o Cleavage site where signal sequence is cut by a signal peptidase • mRNA will be recognized by a ribosome and will be translated • Signal sequence is going to be recognized by SRP who will bind to it and help direct ribosome polypeptide to ER and bind to SRP receptor • SRP and its receptor will also bond GTP (important for process) • Hydrolysis of GTP to GDP once it binds and helps promote movement of polypeptide to translocon • Newly made polypeptide will go through the now open translocon • GDP dissociates from the structure • Structure pushes it way into lumen, signal peptidase cleaves signal sequence off • Continue to have translation and will fold within the lumen • Translocon closes and it can start process over again Protein Modifications In ER • Specific proteolytic cleavage, glycosylation, formation of disulfide bonds, folding of polypeptide chains • Glycosylation: putting sugar groups onto a protein o Enzymatic transfer of 14- residue oligosaccharide precursor o From dolichol carrier (glycolipid) to an asparagine (Asn) residue of a nascent polypeptide by oligosaccharyl transferase o Dolichol carrier – mixture of 3 sugars (glucose, mannose, and N- acetlyglucosamine) o Several glycosidases work to subsequently modify N-glycan o Functions: protein folding, confer protein stability, cell adhesion • Formation of disulfide bonds in ER o Sulfhydryl / thiol groups (-SH) of two cysteine residues can form a disulfide bond (-S-S-) – oxidized o Disulfide bond formation is catalyzed by protein disulfide isomerase (PDI) o PDI regeneration requires a protein called Ero1 o When a protein is first synthesized, it has reduced sulfhydryl groups • Several proteins contribute to proper folding of proteins in ER: chaperone BiP, calnexin and calreticulin (lectins) and PDI • Binding of BiP and the lectins is believed to prevent misfolding / aggregation • Only properly folded proteins are transported from RER to Golgi Protein Import Into Mitochondrial Matrix • Proteins are targeted into mitochondrial matrix (two membranes) • This is achieved by: matrix targeting sequence o 20 – 50 amino acids at N-terminus rich in both hydrophobic, basic, and hydroxylated amino acids (amphipathic) • Chaperone proteins in the cytosol binds to hydrophobic regions to prevent misfolding and help guide towards mitochondria where it will be recognized by other proteins o TOM – translocon of outer membrane o TIM – translocon of inner membrane • HSC70 is found in cytosol and mitochondria, it will help pull protein through into mitochondria and the translocon will close (ATP required) Lecture 5 – Vesicular Traffic of Proteins: Golgi Apparatus Golgi Complex • Consists of flattened, disk-like cisternae with no ribosomes o Vesicles at cisterna tips fuse / pinch off o Three types of cisternae (cis, medial, trans) and two flanked networks of tubules (CGN faces RER and TGN Is opposite) o Processes and sorts proteins (secreted, membrane, lysosomal)  Transport Vesicles: Budding & Fusion • Soluble cargo protein attach to membrane cargo- receptor proteins
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