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Midterm 1 Compliation Notes.pdf

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Western University
Biology 2290F/G
David Vollick

Introduction Cell Culture Requirements: ● Media which contains amino acids, vitamins, minerals, salts, sugars; all the nutrients the cell would normally have within the body ● Serum which contains insulin and growth factors; helps cell take in sugars, and keeps cell growing and dividing respectively ● Temperature of 37 C and the culture is grown in a CO incuba2or; incubator necessary to mimic the gas ratio as it is in the body---need to balance CO and O as it is in the body 2 2 ● The culture should be coloured red; colour is a pH indicator---yellow would be too low pH, purple would mean too high pH Primary vs Immortalized Cells: ● Primary cell cultures are taken directly from an organism, they have a limited number of times that they can divide (Hayflick limit) due to formation of confluent monolayers as a result of contact inhibition ○ still have regulatory mechanisms that tell the cell to stop dividing or kill itself ● Immortalized cells (cell lines) are cells that are transformed and are able to grow and divide indefinitely ○ these cells have lost contact inhibition (will grow on top of each other), and lost the regulatory mechanisms that tell the cell to kill itself or stop dividing ○ HeLa was the first cell line Stem cells, adult stem cells, iPS cells: ● Stem cells have two major properties: they can divide and give rise to two identical daughter cells (can go on indefinitely; called cell renewal or cell lineage), and it can undergo differentiation to perform a specific function ● Embryonic stem cells are derived from embryos; they are pluripotent meaning they can be grown indefinitely and when under the correct conditions, can give rise to the three germ layers ● Adult stem cells are the type found in us (we are no longer embryos); are required to maintain and repair the tissue ○ they are not pluripotent as they can give rise to only one or two types of differentiated cells; their goal is to simply replace other cells ○ a balance between making enough cells to differentiate for tissue function, and maintaining the reservoir of stem cells must be kept ○ are located in stem-cell niche: adjacent helper cells provide signals in the form of growth factors to tell stem cells to proliferate or differentiate ● Stem cells made from reprogramming of differentiated cells are called induced pluripotent stem cells (iPS cells) ○ by introducing certain genes (transcription factors) to differentiated cells, and then overexpressing the transcription factors, the differentiated cells begin to adopt stem cell-like features ○ the transcription factors introduced and overexpressed are normally expressed in embryonic stem cells during embryogenesis; normally activate genes required for pluripotency, or deactivate genes needed for differentiation ○ can be used as personalized medicine Cancer cells: ● Cancer cells are any cells that have uncontrolled growth or avoid apoptosis ● They can arise from normal differentiated cells, or from stem cells ● Cancerous stem cells are even more dangerous than normal cancer cells as there can be so many different types---treating the stem cell-like features of cancer stem cells would be more effective in treating them rather than targeting all the different types of cells ● There is a danger in reprogramming normal cells back into stem cells because to get iPS cells, we have to reprogram certain transcription factors which deregulate features in cell cycle and cell growth; deregulation of these features could possible lead to promoting the transformation process and forming cancer cells Bioimaging Types of microscopes and their applications: ● Bright-field microscopy: uses light to illuminate source and depends on structures with high refractive index to view the specimen---ability to discern detail not very good ● Phase-contrast microscopy: uses increased differences in phase and interference, and exploits differences in refractive index and thickness in cell to view contrast of specimen and provide high detail images ○ results in outer edges of cells to be brighter (phase halos) and thus this method isn't good to view detail on outer peripheries of cell ● Differential interference contrast microscopy: Uses polarized light and small differences in refractive index and thickness within cells to convert them into contrast visible to eye ○ DIC microscopy is equipped with polarizers which allows for only one plane of light to pass through; by combining images from all the polarized light together, we can see topographical features of the cell---3D features of the cell ○ Defines the outline of large organelles and provides better detail of cell edges ● Fluorescence microscopy: uses property of certain molecules to fluoresce when they absorb light at specific wavelengths ○ allows us to see very precise detail of proteins or structures inside or cells at higher resolutions that white light can provide ○ dyes or fluorophores absorb energy kicking electrons into a higher orbital, which when dropping to its normal orbital, releases energy as visible light (fluorescence) ○ Light is focused through an excitation filter (filters broad range of wavelengths into specific wavelength that is optimal for specimen) onto specimen which will emit fluorescence ● Immunofluorescence Microscopy: Uses antibodies made to specific proteins, and secondary antibodies with attached fluorophores which bind to the primary antibodies; and then using a unique excitation wavelength, we can observe where certain proteins are in cells as the secondary antibodies will fluoresce and this can be detected with appropriate filters in microscope ● Dual Label Fluorescence Microscopy: Add in chemicals which will fluoresce a certain colour and will bind to a specific protein or structure; combined with immunofluorescence, we overlay the images from each ● Laser Scanning Confocal Microscope: focuses on just a single plane of the cell; suited to take good resolution images of fluorescently stained cells or cells that are expressing fluorescent fusion proteins---instead of using a broad wavelength of light, lasers are used ○ focuses light at a small point of the cell, gets an image, then scans along that entire focal plane eventually resulting in highly focused image ○ blocks influence of out-of-focus light that would otherwise degrade the image by means of a specific pinhole located in front of the detector ● Deconvolution Microscopy: Computationally intensive math procedure to remove fluorescence contributed from out-of-focus parts of stained sample; uses point spread function which determines degree of blurring by comparison to reference set of tiny fluorescent beads ○ images are taken at different focal planes (called a Z-stack) ○ images are restored by deconvolution display; impressive details and no blurring ○ by taking all the Z-stack images and recombining them, we get a 3D image; these can be created from both deconvolution microscopy and confocal microscopy ● Electron microscopy: a wire filament is the electron source (when heated); magnetic condenser focuses electrons onto specimen; specimen is stained with electron-dense heavy metals ○ when electrons hit the specimen, they will bounce off the metals giving us dark images, and when the electrons go straight through, gives us light images ○ Transmission electron microscope (TEM) images are formed from electrons passing through specimen; specimen is usually a thin layer or section of cell ○ Scanning electron microscope (SEM) instead uses a whole cell or tissue whcih is coated with electron dense metals ○ TEM allows us to see inside of cells, SEM allows us to see 3D surface details of cells (outside of cell) Difference between microscopes: ● In brightfield microscopy, you shine the light through the specimen, which is then magnified and focused to produce the image, but in fluorescence microscopy, the light is focused onto the specimen and its emission (fluorescence) is observed ● Phase contrast microscopy uses differences between phases of light, but differential interference contrast microscopy uses differences in polarized light ● Phase contrast microscopy creates phase halos, but is higher resolution than bright-field; DIC allows us to see topographical features; fluorescence microscopy allows us to see high detail organelles and structures inside of the cell ● Bright-field, phase contrast, and DIC microscopy all are used to examine live “unstained” cells; fluorescence microscopy can use stained cells ● Dual label Fluorescence microscopy must use dead cells that are frozen---only provides a fixed picture ● Confocal and deconvoluted microscopes are both fluorescence microscopes, however confocal microscopy works on principle of using pinholes of shining light at very focused points; deconvolution microscopy uses broad light and then digitally removes light that isn’t in focus ○ both types can give Z-stacks ● Resolution for electron microscopy is very fine; equation becomes: D = 0.61λ / α because no N as light is replaced by electrons in vacuum, and sin α is now α since electron scatter is almost 0 Resolution: ● Resolution (D) is the distance resolved between 2 points; we want as low a number for resolution as possible ● D = 0.61λ / Nsin α ○ λ is the wavelength of light ○ Nsin α is the numerical aperture (higher the better) ○ N is the refractive index of medium between specimen and objective lens ○ α is 1/2 the angle of light entering objective lens ● To get optimum resolution, we can increase the refractive index (use oil), increase the angle α to be as wide as possible, and we could decrease the λ of light we’re using Monoclonal antibody production (medium, properties of cells): ● Once B-cells that produce antibodies in mice are extracted, we need to make them into a cell line so that we have indefinite antibody production ● To do this, we take mouse myeloma cells, and fuse them with primary B-cells to form a hybridoma cell line ● To select for the fusion of these B-cells and myeloma cells, we grow the cells in a HAT medium (selection medium) which is toxic for just myeloma cells; thus only successful hybrid cells which have a missing gene from spleen cells (due to them fusing) ● We can not establish cell lines from these hybrid cells and then look to those lines for antibody production by testing them with the corresponding antigen Fluorescent Fusion Proteins: ● A gene that makes a protein that fluoresces when excited can be isolated and heavily modified and then recombined with gene of interest into an expression plasmid ● The plasmin can then be inserted into a live cell where it’ll be recognized by mRNA transcription machinery and then the protein will be expressed ● Everytime the gene vof interest is expressed, because the fluorescent fusion protein was attached to it, that gene will also be expressed resulting in fluorescence ● This way, we can tell when our gene of interest is being expressed and thus, fluorescence imaging in live cells is possible Methods Fluorescence activated cell sorting: Centrifugation: ● Two different types of centrifugation: differential, and equilibrium density-gradient ● When we spin out samples, we homogenize them and get a mixture of broken and unbroken cells, which is then filtered to remove the unbroken cells and connective tissue ● Differential Centrifugation: spinning homogenate yields pellet and supernatant; increasing centrifugal force is used to isolate organelles based on mass ○ spinning at different speeds will allow us to isolate different organelles ○ smaller organelles (ribosomal subunits) require very spinning and high centrifugal force whereas large organelles (nucleus) require lower speeds ● Equilibrium Density-Gradient Centrifugation: separation based on density ○ Say we want to isolate just a single organelle; differential centrifugation results in pellets left over which still contain all kinds of organelles (eg. at 15000g force, you’ve got mitochondria, chloroplasts etc.)---what if you just want one organelle? ○ Through equilibrium density gradient centrifugation, you can separate based on density of that organelle ○ Pouring homogenate onto a gradient of sucrose and then spinning at high speed for several hours will result in the organelles migrating to the sucrose layer equal to their own density and will remain there Detergents: ● All the detergents used are amphipathic detergents, meaning they have both hydrophobic and hydrophilic domains ● There are two types of detergents: ionic and non-ionic detergents ● We use ionic detergents (SDS) if we simply want to isolate any and all proteins; this will break apart and denature the membrane, but will denature the proteins as well ● We use non-ionic detergents if we want to preserve protein functions and interactions; break apart the membrane but will not denature proteins SDS-Page: ● Electrophoretic separation of proteins is commonly performed in polyacrylamide gels and called polyacrylamide gel electrophoresis (PAGE) ● PAGE is usually carried out in presence of negatively charged detergent SDS called SDS-PAGE ● SDS is a negatively charged detergent, and using its hydrophobic region, it will bind to the inner hydrophobic regions of proteins which opens (denatures) the proteins up; the SDS sticks out its negative sulfate entity ● The proteins are now all denatured and are equally negatively charged; only difference between them is their size ● Running them through a gel will separate them in terms of their molecular size---smaller proteins will be attracted to the positive pole faster as they can travel through the gel faster; larger proteins will move slower as it will take longer to get through the gel Western Blotting: ● Also called immunoblotting; is a technique which is used to detect specific proteins by using the specificity of antibodies for the protein of interest ● Before we can administer the antibodies, we have to transfer the protein bands from the SDS-PAGE gel to a membrane; antibodies can’t penetrate the gel ● Once the protein bands are on a membrane, we first incubated with the primary antibody which will detect the protein, then we incubate with a secondary antibody that recognizes the primary antibody and is attached to an enzyme that can react with a substrate, converting it from its non-luminescent form to its luminescent form ● We administer chemiluminescence substrate which reacts with the enzyme on the secondary antibody and using X-rays, we will be able to visualize our protein of interest ● Western Blotting can be used to see expression of a certain protein as it changes over time Rough Endoplasmic Reticulum Secretory vs nonsecretory pathways: ● The nonsecretory pathway has to do with proteins that will be made in the cytosol and will remain the cytosol after completion ○ They can be targeted to itnracellular organelles such as the mitochondria, chloroplasts, peroxisomes, and nucleus ● Secretory pathway has to do with proteins that will end up outside of cell and are usually synthesized initially in the cytosol, but complete synthesis by ribosomes attached to the RER ○ The proteins in the secretory pathway reside in ER and are sorted to the plasma membrane, golgi complex, and lysosomes ○ Proteins often go under multiple modifications and further processing in this pathway Signal sequence, SRP, SRP receptor, translocon, Cotranslational translocation: ● Cotranslational translocation refers to the fact that translational and translocation of proteins in the secretory pathway occur simultaneously---the proteins are not completely made in the cytosol, rather they are translated slightly, transported to the RER and complete translation while being stuffed into the RER all at the same time ● The method by which this occurs is that there is a signal sequence on the N-terminus of the nascent polypeptide which is recognized by a signal recognition protein (SRP) ● The SRP binds to the signal sequence and moves the entire complex towards the RER membrane where the SRP receptor is located ● SRP binds to the SRP receptor; both bind GTP, which is then hydrolyzed causing the translocon on the membrane to open, and SRP to dissociate from SRP receptor ● The translocon provides an opening allowing the nascent protein through; translation of mRNA continues and once the signal sequence of the protein is inside the lumen of ER, it is cut off by a signal peptidase ● Eventually, translational is completed and the folded protein is left in the lumen of the RER Protein modifications in ER: Glycosylation, PDI, disulfide bonds, protein folding: ● Glycosylation (putting sugars on the protein) is important in that proteins found outside of the cell on the membrane need to be sticky to help cells stick together---the sugars allow this stickiness ○ An enzyme, oligosaccharyl transferase, transfers a 14-residue oligosaccharide (composed of glucose, manose, and N-acetylglucosamine) from dolichol carrier onto asparagine residues of a nascent polypeptide ○ The protein is then further processed by glycosidases which will cleave off certain portions to get the right combination of carbohydrates ○ Glycosylation helps with protein folding, stability, and cell adhesion ● The outside of the cell is a harsh environment and the protein needs to be as stable as possible---disulfide bonds help stabilize protein structure and are important for secreted proteins (occur between cysteine residues) ○ Disulfide bonds only form in oxidized conditions, but the ER lumen is an oxidizing environment, thus to prevent inappropriate disulfide bonds forming, the process is regulated by protein disulfide isomerase (PDI) ○ PDI will form the correct disulfide bonds by acting as the oxidizing agent and oxidizing thiol groups to form the bonds ○ PDI is recycled by a protein called Ero1 ● As proteins enter the ER lumen, it is very vulnerable as there are several proteins that will be attracted to the hydrophobic domains of the newly translated protein and can alter its shape---normally those domains are not exposed but the protein hasn’t had time to fold as it is not fully translated yet ○ Chaperone proteins called BiP will bind to the hydrophobic domains of the protein and prevent any other prote
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