Chapter 9 Notes
Chapter 9 Notes Biol 3302
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Date Created: 01/25/16
Chapter 9 Culturing Visualizing and Perturbing Cells Culturing Plate coated with AA and proteins that help the cells stick to the plate Average animal cell takes approx 24 hours to divide Serum has all growth factors Visualization Techniques and Microscopy Fluorescence absorbing light at one wavelength and emitting light at a specific and long wavelength Major function To determine localization of specific cellular molecules ex proteins Major advantages 0 Sensitivity glow against dark background 0 Specificity immuno uorescence antibody specifically binds to one protein 0 Cells may be fixed or living Flurochromes uorescent dyes or proteins 0 Flurochromes may be indirectly or directly associate with the cellular molecule 0 Multiple urochromes may be used simultaneously Transformation process of introducing rDNA into prokaryotes Transfection process of introducing rDNA into eukaryotes Recombinant DNA technology living cells 0 Ex Hydra expressing GFP I Hydra transfected with plasmid DNA containing the GFP gene that is driven by the hydra betaactin promoter I Early experiment that showed the usefulness of uorescent proteins Purpose localization of protein X 1 Find gene X 0 2 Fuse gene X with GFP gene I aka chimeric fusion protein or recombinant DNA construct or plasmid construct O 3 Transfect eukaryotic cells I Only eukaryotic cells will have the machinery to recognize and translate 4 Wait 2448 hours for plasmid to be translated O 5 Use uorescence to find protein X I Couldn t do western blot because you don t know anything about protein X you don t have an antibody that recognizes protein X Immuno uorescence fixed cells 0 Process of making antibodies I Inject epitope into rabbitmouse I Extract antibody test specificity with ELISA develop specific ab I Add uorescent tag to primary not all because difficultexpensive or use uorescent secondary OO O 00 Direct immuno uorescence uorescentlytagged primary antibody Indirect immuno uorescence normal primary ab uorescentlytagged secondary Tagged proteins myc or ag be prepared to draw for exam I Scenario looking for protein C but don t have a primary antibody for it gene sequence is know can t use live cell rDNA method because transgene is too largewon t translate I Alternate method add myc or FLAG approx 12 AA to the Nterm or C term I Transfect cells time then use primary ab to mycFLAG Fluorescent dye fura2 is used to monitor Ca2 concentrations within a cell I Cannot be used for proteins only detects calcium ions Process of immuno uorescence Prepare sample on slide incubate with primary wash incubate with urochromeconjugated uorescentlytagged secondary antibody wash microscope I Doublelabel uorescence microscopy 0 O O O 0 Can be used with fixed or living cells Visualizes the relative distributions of two proteins Overlay 2 channels to visualize both Just because in overlay they re in the same area doesn t mean that they re interacting because they could be in different focal planes Ex visualizing microtubules indirect immuno uorescence and actin filaments with phalloidin drug that specifically binds to actin Fluorescent Microscopy I SPED stimulated emission depletion uorescent microscopy has a resolution of 20nm I Confocal deconvolution and superresolution microscopy overcome the limitations of uorescence microscopy blurred images thick specimens I Confocal O O O 0 Laser as the energy source Laser scans the specimen across and down to build an image Uses a pinhole in front of the detector to block light from other focal planes 2 types Laserscanning and spinning disk I Deconvolution O Takes image and uses a computer program to reconstruct a nicer image I TIRF microscopy Total internal re ection O O O Gives better resolution of the area that is closest to the cover slip Restricted focal plane Use regular TIRF to create a combined image I Superresolution Microscopy techniques I FRAP Fluorescence recovery after photobleaching O Gives information on the dynamics of a protein of interest I Want to know if proteinphospholipid moves around in the phospholipid bilayer Fluorescently tag protein or phospholipid 0 Bleach uorescence in a specific ROI region of interest I Bleaching quenching O Whenif region regains uorescence it shows that the protein is moving because the uorescent protein from other areas migrated into the ROI I Know it s moving and not just making more because not enough time 30 sec 0 Compare to another control region that is not bleached 0 FRET uorescence resonance energy transfer 0 CFP and YFP are used because emission wavelength of CFP excitation wl of YFP 0 Measures the distance between proteins I To find out if proteins are close enough to interact using chromophores I Fuse gene X with CFP cyan uorescent protein I Fuse gene Y with YFP yellow FP I Transfect into the same cell I Shine light at CFP excitation wavelength 480nm I CFP will emit at 480 nm I If CFP is close enough to YFP excitation wavelength 480nm then YFP will emit 535nm I Yellow uorescence X and Y are close enough to interact O 0 Doesn t necessarily mean that they are interacting 0 Measures conformational changes of a single protein I Only 1 protein ex calmodulin tagged to CFP and YFP at the same time I Looking for conformational changes I Ex without calcium ions CFP and BFP are not close enough to transfer energy but with calcium ions protein bends and they become close enough Preparing samples for light and electron microscopy 0 Fix with formaldehyde 0 Formaldehyde crosslinks amino groups on adjacent molecules 0 Dehydrates sample 0 Can also be glutaraldehyde same family of molecules 0 Embed in paraffin for light or liquid plastic for electron 0 Section 0550 micrometers for light 50100 nm for TEM 0 Do not section for SEM I Stain with hematoxylin binds to basic AA and eosin binds to acidic molecules Electron microscopy 0 Fixed cells only 0 Transmission EM TEM 0 Used for clear 2D images I Reveals surface details Practical resolution 01nm 1nm 2000x than light microscopy Highvelocity beam passes through the sample 50100nm thick sections Can simulate a 3D image by getting different sections from different areas and putting together using a computer program 0 Samples need to be fixed and stained I Cannot image live cells 0 Stained with heavy metals such as uranium lead osmium tetroxide I Stains the membrane I Evaporate platinum layers platinum on top I Then evaporate carbon layers carbon on top I Creates a metal replica of the surface that is visualized I Shows fine structural details 0 Detecting a protein using gold particles that are coated with protein A I Use an antibody that binds specifically to the protein of interest ex catalase I Protein A that is attached to gold particles binds to a generic Fc domain in the antibody I Results in a complex that is attached to gold show up as black dots in TEM 0000 0 For catalase show up only in peroxisomes 0 Scanning EM SEM 0 Used for 3D images 0 Resolution about lOnm 0 Used to view unsectioned metalcoated specimens Cryoelectron microscopy 0 Allows visualization of specimens without fixation or staining 0 Looks at sample in native hydrated state 0 Useful when dehydrating the protein changes its structure I 5nm resolution 0 Method an aqueous suspension of the sample is applied on a grid frozen in liquid nitrogen and held be a special mount 0 Variation Cryoelectron tomography O Allows determination of the 3D structure Puri cation of cell organelles Cell disruption breaking open of cells I Should be done in isotonic sucrose 0 Isotonic similar to cytoplasm environment maintains ionic strength and pH 0 Can break open by O O O Sonication Homogenization similar to mortarpestle Putting in hypotonic solution Separation of different organelles using centrifugation 0 Differential centrifugation 0 0 Filter homogenate to remove unbroken cells etc Spin at 600g X 10 min I Pellet nuclei Spin supernatant and spin at 100000g X 60 min I Pellet mitochondria chloroplasts lysosomes and perOXisomes Spin supernatant 300000g X 2 hours I Pellet now ribosomal subunits small polyribosomes I Supernatant cytosol 0 Density gradient centrifugation 0 00000 Used after differential centrifugation I EX if you want a mitochondrial protein you would spin twice but then have to get mitochondria out of the pellet vs lysosomes chloroplasts etc Pour the gradient Increasing density from top 109 to bottom 125 Centrifuge organelles will migrate to their density When looking at the tube will see 3 fuzzy bands across Top to bottom I Lysosomes 112 gcm3 I Mitochondria 118 gcm3 I PerOXisomes 123 gcm3 To extract the bands needle After extraction Lysosomes and mitochondria have similar densities so after removing can t be sure that you have 100 pure organelle So then have to run a western blot using markers enzyme markers that are specific to the 3 organelles I Catalase perOXisomes I Cyt C or cytochrome oxidase mito I Acid phosphatase lysosomes I Plasma membrane amino acid permease I Rough ER ribosomal RNA I Smooth ER cytidylyl transferase 0 Example of western blot run from 3 samples I Goal to isolate and purify mitochondria I Have 3 tubes each band extracted from differential centrifugation I Run western blots I From western blot 1 alone I Can NOT conclude that M is pure 0 Can say that L and P are not contaminated with M I From western blot 2 as well 0 M is contaminated with L 0 L is not contaminated with M 0 If your mitochondrial fraction is contaminated you can purify by using differential centrifugation using a deepermore spread out density gradient I Rerun western blot Using antibodies to purify vesicles 0 Cannot use density gradient centrifugation if the density of the vesicles are the samesimilar I Use antibody for protein that is unique to that vesicle ex clathrin I Then use a bacterial cell which has protein A on the surface 0 Protein A binds to Fe domain in the antibody 0 A single bacterial cell can bind to multiple coated vesicle antibodies 0 Bacterial cell is very heavy quick spin will bring down the entire complex 0 Easy to use with clathrin because it forms a cagelike structure on the outsidesurface so it s easy for the bacterial cell to bind to 0 Could not use with cytc to purify mitochondria because cytc is not a surface protein Screening for drugs that affect specific biological processes 0 EX screening for drugs that specifically affect spindle morphology 0 Start with 16320 chemical compounds 0 Screen for those that arrest cells in mitosis I 139 compounds 0 Screen for those that do not affect microtubule formation I 86 0 Screen for those that specifically affect spindle morphology I 5 siRNA small inhibitory RNA Knockdown the expression of a specific protein Targets the degradation of specific mRNAs in cultured cells Can start with synthetic siRNA antisense to target mRNA 0 Directly introduced into cell goes to RISC complex Or DNA expressing shRNA small heteroduplex RNA 0 Integrated into cell as a DNA construct O Goes into nucleus 67825 0 shRNA is made and is a hairpin loop useless bc double stranded 0 Dicer an RNA endonuclease cleaves the loop region 0 Then goes to RISC complex RISC RNA induced silencing complex then is introduced to the cell and allows the siRNA to bind to the target mRNA Target mRNA is degraded 0 To look for effect of siRNA knockdown 0 Use an antibody that recognizes another gene in the intended area 0 Ex knockdown of EBP50 component of microvilli then use ab against erzin other protein in microvilli 0 Genomic screens use siRNA O RNAi screens explore the function of all the genes in cultured cells 0 RNAi is used to suppress genes in specific tissues only 0 Parent A introduce shRNA downstream of promoter Parent B introduce tissuespecific promoter upstream of promoter element 0 Progeny have both constructs in all cells but shRNA only transcribed in that tissue because only in target tissue is the promoter made ex GAL4 that acts on the upstream promoter element to make the shRNA RNAi O AnimalPlant Cell Organelles Plasma membrane controls movement of molecules in and out functions in cellcell signaling and cell adhesion Mitochondria generate ATP by oxidation of glucose and fatty acids 0 Inner and outer membrane I Intermembrane space in between the two I Cristae folded sections 0 Has own DNA but cannot make all the proteins it needs to function has to import proteins Lysosomes degrade material internalized by the cell and womout cellular membranes and organelles O Acidic lumen pH 52 O Autophagy Digest cellular debrisdamaged organelles 0 To digest wraps membrane around damaged organelle fuses with it digests it using acidic hydrolases 0 Can bring soluble or insoluble material from outside endocytosis I Soluble pinocytosis I Insoluble phagocytosis Nuclear envelope encloses the contents of the nucleus the outer nuclear membrane is continuous with the rough ER Nucleolus site of most rRNA synthesis Nucleus chromatin DNA proteins site of mRNA and tRNA synthesis Smooth ER synthesize lipids and detoxify certain hydrophobic compounds Rough ER synthesize process and sort proteins Ribosomes Made of proteins and RNA 2 subunits large and small 0 There is an equilibrium between free large subunits free small subunits and ribosomes in the cytoplasm O Ribosome assembly occurs during protein synthesis 0 Synthesize proteins in the nucleus Golgi Process and sort proteins synthesized by rough ER 0 3 parts cis medial trans Secretory vesicles store secreted proteins and fuse with the plasma membrane to release their contents Peroxisomes detoxify molecules and break down fatty acids Cytoskeletal fiber form networks and bundles that support cellular membranes help organize organelles and participate in cell movement Microvilli animal only increase surface area for absorption of nutrients from surrounding medium Cell wall plants only composed largely of cellulose beta glucose units joined together helps maintain the cell s shape and provides protection against mechanical stress allows higher pressure Vacuole plants only stores water ions and nutrients degrades macromolecules and function in cell elongation during growth Chloroplasts plants only photosynthesis proteins are separated by different membranes Protein trafficking 0 Made in rough ER goes to Golgi then leave via vesicles Mitochondria nucleus and chloroplasts have a double membrane
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