BIOL-L 312 Cell Biology (Mehta) Book Notes - Test One
BIOL-L 312 Cell Biology (Mehta) Book Notes - Test One BIOL-L 312
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This 7 page Reader was uploaded by Ifeoma O'Gonuwe on Monday February 2, 2015. The Reader belongs to BIOL-L 312 at Indiana University taught by Sapna Mehta in Spring2015. Since its upload, it has received 152 views. For similar materials see Cell Biology in Biology at Indiana University.
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CHAPTER NINE VISUALIZING CELLS Looking at Cells in the Light Microscope 0 Cell doctrine proposal that all plants and animal tissues are aggregates of individual cells The Light Microscope Can Resolve Details 02 pm Apart 0 Because of the wave nature of light at high magnification a shadow of an edge appears as a set of parallel lines a circular spot appears as a set of concentric lines a single point appears as a blurred disc two objects close enough to each other can merge into one o The limit of resolution limiting separation at which two objects appear distinct depends on both the wavelength of the light and numerical aperture of the lens system used 0 The light microscope can theoretically achieve a limit of resolution of 02 pm I Objects smaller than this will be detected but they will not be distinguishable Living Cells Are Seen Clearly in a PhaseContrast or a DifferentialInterferenceContrast Microscope o Phasecontrast microscope and differentialinterferencecontrast microscope exploit the interference effects produced when light passes through a living cell 0 Due to the refractive index of the cell the light will shift differently based on the density of the cell 0 The microscopes create an image of the cell s structure produced when the sets of light recombine o Darkfield microscopes work by observing the light that is scattered by its various components because only this light enters the microscope lens 0 Result the cell appears as a bright object against a dark background 0 Brightfield microscopes work by passing light through a cell in culture Images Can Be Enhanced and Analyzed by Digital Techniques 0 CCD cameras increase our ability to observe cells in low light conditions 0 Using low light avoids the damaging effects of using heat and light 0 Images produced are electronic and can therefore be digitized for further analysis Intact Tissues are Usually Fixed and Sectioned before Microscopy 0 Use a fixative to form covalent bonds with the cell that crosslinks them into locking into position 0 Imbed tissues in a media to harden them 0 Use a microtome to section the cell 0 Stain the cell or use fluorescent probesmarkers to view the cell Specific Molecules can be Located in Cells by Fluorescence Microscopy o Fluorescent dyes used for staining cells are visualized with a fluorescence microscope o The microscope uses two filters One filters the light before it reaches the specimen to the wavelengths necessary to excite the fluorescent dye in the specimen The second filter filters through only the wavelengths emitted when the dye fluoresces 0 Fluorescence microscopy is used to detect specific proteins or molecules in cells and tissues Antibodies Can Be Used to Detect Specific Molecules 0 Each antibody has a binding sit that recognizes a specific target molecule antigen 0 There are primary and secondary antibodies 0 The primary antibody antigen link is very specific only one antibody antigen per link 0 Secondary antibodies are less specific Many secondary antibodies bin to one primary an body I Use secondary antibodies to amplify the signal called indirect immunocytochemistry Imaging of Complex ThreeDimensional Objects is Possible with the Optical Microscope o Thinner sections lead to crisper images but loss of three dimension The Confocal Microscope Produces by Excluding OutofFocus Light 0 Microscope is used with fluorescence optics then focuses light onto a single point at a specific depth in the specimen 0 The fluorescence emitted from the illuminated material is collected through a pinhole aperture that is confocal with the illuminating pinhole Fluorescent Proteins Can Be Used to Tag Individual Proteins in Living Cells and Organisms 0 Make a transgenic organism with GFPcoding sequence placed under the transcriptional control of the promoter belonging to a gene of interest 0 Tags the protein of interest and gives it the ability to fluoresce Protein Dynamics Can Be Followed in Living Cells 0 Fluorescence resonance energy transfer FRET 0 Two molecules of interest are each labeled with a different fluorochrome I Flurochromes are chosen so that the emission spectrum of one overlaps with the absorption spectrum of another 0 Photoactivation o Synthesize an inactive form of the fluorescent molecule of interest introduce it to the cell and activate it by focusing a spot of light on it 0 Fluorescence recovery after photobleaching FRAP o Extinguish GFP fluorescence in a region of the cell and analyze the amount of time the remaining fluorescent protein molecules move into the bleached area Summary Many lightmicroscope techniques are available for observing cells Cells that have been fixed and stained can be studied in a conventional light microscope whereas antibodies coupled to fluorescent dyes can be used to locate specific molecules in cells in a fluorescence microscope Living cells can be seen with phasecontrast differentialinterferencecontrast darkfield or brightfield microscopes All forms of light microscopy are facilitated by digital imageprocessing techniques which enhance sensitivity and refine the image Confocal microscopy and image deconvolution both provide thin optical sections and can be used to reconstruct three dimensional images Techniques are now available for detecting measuring and following almost any desired molecule in a living cell Fluorescent indicator dyes can be introduced to measure the concentrations of specific ions in individual cells or in different parts of a cell Fluorescent proteins are especially versatile probes that can be attached to other proteins by genetic manipulation Virtually any protein of interest can be genetically engineered as a fluorescent fusion protein and then imaged in living cells by fluorescence microscopy The dynamic behavior and interactions of many molecules can now be followed in living cells by variations on the use of fluorescent protein tags in some cases at the level of single molecules Radioactive isotopes of various elements can also be used to follow the fate of specific molecules both biochemically and microscopically Looking At Cells and Molecules in the Electron Microscope The Electron Microscope Resolves the Fine Structure of the Cell 0 With electrons the limit of resolution can be made very small 0 At best the limit is 1nm 200x better than the resolution in the light microscope 0 To use transmission electron microscope TEM the specimen must be stained with electron dense material Biological Specimens Require Special Preparation for the Electron Microscope o Specimen is preserved by fixation with glutaraldehyde and then osmium tetroxide o Osmium tetroxide binds to and stabilizes lipid bilayers as well as proteins 0 The specimen is then dehydrated and permeated with a resin to create a solid form of plastic 0 The specimen is finally cut with fine glass or diamond knife 0 To ensure that the prepared specimen is an actual representation of the live specimen scientists developed rapid freezing 0 Cool the aqueous specimen fast enough to force the water and other components to create a rigid but noncrystalline state vitreous ice Specific Macromolecules Can Be Localized by Immunogold Electron Microscopy o lmmuogold electron microscopy is analogous to immunocytochemistry used with fluorescence microscopy Images of Surfaces Can Be Obtained by Scanning Electron Microscopy o A scanning electron microscope produces an image of the three dimensional structure of a surface of a specimen 0 SEM scans the specimen with a very narrow beam of electrons which creates an image of the surface 0 This technique is used to study whole cells and tissues rather than subcellular organelles Different Views of a Single Object Can Be Combined to Give a ThreeDimensional Reconstruction o Electronmicroscope EM tomography the specimen is tilted in different orientations to achieve a threedimensional reconstruction by combining a set of different views of a single object in the microscope s field of view 0 Each individual view will contain a lot of noise but when the different sections are combined in three dimensions and an average is taken 9 much of the noise is eliminated Summary Determining the detailed structure of the membranes and organelles in cells requires the higher resolution attainable in a transmission electron microscope Specific macromolecules can be localized with colloidal gold linked to antibodies Threedimensional views of the surfaces of cells and tissues are obtained by scanning electron microscopy The shapes of isolated macromolecules that have been shadowed with a heavy metal or outlined by negative staining can also he readily determined by electron microscopy Using computational methods either multiple images or views from different directions can be combined to produce detailed reconstructions of macro molecule and molecular complexes through the techniques of electron tomography and single particle reconstruction often applied to cryopreserved specimens The resolution obtained with these methods means that atomic structures of individual macromolecules can often be fitted to the images derived by electron microscopy and that the TEM is increasingly able to completely bridge the gap between structures determined by xray crystallography and those determined in the light microscope CHAPTER 10 MEMBRANE STRUCTURE The Lipid Bilayer Phosphoglycerides Sphingolipids and Sterols Are the Major Lipids in Cell Membranes o All lipid molecules in cell membranes are amphiphillic 0 They have a hydrophilic and hydrophobic end 0 Most abundant membrane lipids are phospholipids 0 Have a polar head group and two hydrophobic hydrocarbon tails tails are usually fatty acids 0 Main phospholipids in animal cell membranes are phosphoglycerides o Sphingolipids are important lipids in the membrane as well 0 Cholesterol is a major component of lipids as well Phospholipids Spontaneously Form Bilayers 0 Shape and amphiphillic nature of phospholipid molecules causes them to form bilayers spontaneously in aqueous environments 0 When dispersed in water the phospholipids arrange in a way to shield the hydrophobic hydrocarbon tails from the water I The phospholipids are cylindrical 9 form a bilayer o If they were cone shaped they would form a micelle The Lipid Bilayer is a TwoDimensional Fluid 0 Liposomes synthetic lipid bilayers made in the form of spherical vesicles 0 Studies of synthetic bilayers shoe that phospholipids rarely flipflop from one monolayer to the next 0 Cholesterol is an exception 0 Instead phospholipids will undergo rapid lateral diffusion meaning that the phospholipids will exchange place with each other within the same monolayer o Phospholipids will also rotate rapidly along their long axis The Fluidity of a Lipid Bilayer Depends on Its Composition 0 If hydrocarbon chains are short or have double bonds the membrane is more fluid 0 Shorter chain reduces the tendency of the hydrocarbon tails to interact with one another 0 Cisdouble bonds produce kinks that make the chains harder to pack together 0 When cholesterol is added to the membrane it interacts with and partly immobilizes the regions of the hydrocarbon chain closest to the polar head groups 0 Decreases the permeability of the lipid bilayer to small watersoluble molecules 0 Tightens the packing of lipids 9 does not make membrane less fluid 0 Prevents hydrocarbon chains from coming together and crystalizing at high temperatures Despite Their Fluidity Lipid Bilayers Can Form Domains of Different Compositions o Lipid rafts occur when lipid molecules transiently assemble into specialized domains thought to be stabilized by protein 0 Hydrocarbon chains of sphingolipids are longer and straighter 9 thicker raft domains and better accommodate certain membrane proteins 0 Lipid rafts help organize membrane proteins o Concentrate them either for 1 transport in membrane vesicles or 2 for working together in protein assemblies The Asymmetry of the Lipid Bilayer is Functionally Important 0 Lipid compositions of the two monolayers within the bilayer are strikingly different 0 Lipid asymmetry is important especially in converting extracellular signals into intracellular ones Summary Biological membranes consist of a continuous double layer of lipid molecules in which membrane proteins are embedded This lipid bilayer is fluid with individual lipid molecules able to diffuse rapidly within their own monolayel The membrane lipid molecules are amphiphilic When placed in water they assemble spontaneously into bilayers which form sealed compartments Cells contain 5001000 different lipid species There are three major classes of membrane lipids phospholipids cholesterol and glycolipids and hundreds of minor classes The lipid compositions of the inner and outer monolayers are different reflecting the different functions of the two faces of a cell membrane Different mixtures of lipids are found in the membranes of cells of different types as well as in the various membranes of a single eukaryotic cell Inositol phospholipids are a minor class of phospholipids which in the cytosolic leaflet of the plasma membrane lipid bilayer play an important part in cell signaling in response to extracellular signals specific lipid lltinases phosphOlylate the head groups of these lipids to form docking sites for cytosolic signaling proteins whereas specific phospholipases cleave certain inositol phospholipids to generate small intracellular signaling molecules Membrane Proteins Membrane Proteins Can Be Associated with the Lipid Bilayer in Various Ways 0 Transmembrane proteins are amphiphillic proteins that pass through the lipid bilayer 0 Their hydrophobic regions interact with the hydrophobic tails of the lipid molecules in the interior of the bilayer 0 Their hydrophilic regions are exposed to water on either side of the membrane 0 Lipidlinked proteins are proteins made as soluble proteins in the cytosol that are subsequently anchored to the membrane by the covalent attachment of a lipid group 0 While in the ER part of the protein is cleaved and a glycosylphosphatidylinositol GPI anchor is added leaving the protein bound to the noncytosolic surface of the membrane 0 Peripheral membrane proteins are proteins bound to either face of the membrane which interfere with proteinprotein interactions but leave the lipid bilayer intact o Integral membrane proteins are proteins embedded within the membrane In Most Transmembrane Proteins the Polypeptide Chain Crosses the Lipid Bilayer in an alpha Helical Conformation o The hydrogenbonding between peptide bonds is maximized if polypeptide chain forms a regular helix as it crosses the bilayer 0 Hydrogen bonds occur between peptide bonds because they are in a hydrophobic environment 0 They form an alpha helix that places the hydrophobic R groups in contact with the hydrophobic hydrocarbon tails of the membrane and allows the polar amino acid side chains to interact with each other 0 In singlepass transmembrane proteins the polypeptide chain crosses only once whereas in multipass transmembrane proteins the polypeptide chain crosses multiple times o Multipass transmembrane proteins create beta sheets rolled into a closed barrel beta barrel to form as many hydrogen bonds as possible Some Beta Barrels Form Large Transmembrane Channels 0 Multipass transmembrane proteins that have their transmembrane segments arranged as a beta barrel are more rigid than alpha helices formed with singlepass transmembrane proteins and tend to crystallize readily 0 Beta barrel proteins are abundant in the outer membrane of mitochondria chloroplasts and many bacteria 0 Some are poreforming proteins and form waterfilled channels that allow selected small hydrophilic molecules to cross the lipid bilayer of the bacterial outer membrane Many Membrane Proteins Are Glycosylated 0 Because most plasma membrane proteins are glycosylated carbohydrates coat the surface of all eukaryotic cells 0 The carbohydrate layer can be visualized by stains or by labeling lectins carbohydratebinding proteins Membrane Proteins Can Be Solubilized and Purified in Detergents o Detergents are small amphiphillic molecules of variable structure that can solubilize transmembrane proteins 0 Hydrophobic ends of detergents bind to the hydrophobic regions of the membrane proteins leading to a detergentprotein complex 0 Strong ionic detergents can solubilize hydrophobic membrane proteins by binding to the internal hydrophobic cores of proteins results in denaturation 0 They are inactive but once separated and purified from the strong ionic detergent the protein might refold and recover functional activity Many Membrane Proteins Diffuse in the Plane of the Membrane o Membrane proteins do not flipflop across the lipid bilayer but they will exhibit rotational diffusion and will also laterally move within the membrane 0 Experiment to prove lateral diffusion occurs I Fuse two cells mouse and human cells to create a heterocaryon I Tag antibodies against the proteins on the membrane I Allow the proteins to diffuse given time 0 Lateral diffusion rates of membrane proteins can be measured by using the technique of fluorescence recovery after photobleaching FRAP Cells Can Confine Proteins and Lipids to Specific Domains Within a Membrane 0 There is often an asymmetric distribution of membrane proteins amongst the apical basal and lateral surfaces of the cell 0 There is a prevention of diffusion of lipidmolecules between the domains 0 Tight junctions maintain the separation of both protein and lipid molecules The Cortical Cytoskeleton Gives Membranes Mechanical Strength and Restricts Membrane Protein Diffusion o Spectrin is a filamentous protein within the red cell cytosllteleton that maintains the structural integrity and plasma of the plasma membrane Summary Whereas the lipid bilayer determines the basic structure of biological membranes proteins are responsible for most membrane functions serving as specific receptors enzymes transport proteins and so on Many membrane proteins extend across the lipid bilayer Some of these transmembrane proteins are single pass proteins in which the poly peptide chain crosses the bilayer as a single a helix Others are multipass proteins in which the polypeptide chain crosses the bilayer multiple timeseither as a series of a helices or as a 3 sheet in the form of a closed barrel All proteins responsible for the transmembrane transport of ions and other small watersoluble molecules are multipass proteins Some membraneassociated proteins do not span the bilayer but instead are attached to either side of the membrane Many of these are bound by noncovalent interactions with transmembrane proteins but others are bound via covalently attached lipid groups In the plasma membrane of all eukaryotic cells most of the proteins exposed on the cell surface and some of the lipid molecules in the outer lipid monolayer have oligosaccharide chains covalently attached to them Like the lipid molecules in the bilayer many membrane proteins are able to diffuse rapidly in the plane of the membrane However cells have ways of immobilizing specific membrane proteins as well as ways of confining both membrane protein and lipid molecules to particular domains in a continuous lipid bilayer