H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 80
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To aid in the imaging, the use of more fluorescent labels and tags will allowvisualization of five or six different types of molecules simultaneously. With more labeled proteins, the complex interactions among proteins and organelles will become betterunderstood.Finally, the electron microscope will become the dominant instrument for studying protein machines in vitro and insitu. Tomographic methods applied to single cells and molecules combined with automated reconstruction methods willgenerate models of protein-based structures that cannot bedetermined by x-ray crystallography.
High resolution threedimensional models of molecules in cells will help explain theintricate biochemical interactions among proteins.■Specimens for electron microscopy generally must befixed, sectioned, dehydrated, and then stained with electrondense heavy metals.■Surface details of objects can be revealed by transmissionelectron microscopy of metal-coated specimens. Scanningelectron microscopy of metal-coated unsectioned cells or tissues produces images that appear to be three-dimensional.■193KEY TERMSactin filaments 174apical 153basolateral 153chloroplast 172cytoskeleton 147cytosol 147cytosolic face 150endoplasmicreticulum (ER) 168endosome 165exoplasmic face 150fluorescentstaining 187glycolipid 151Golgi complex 169GPI anchor 161immunofluorescencemicroscopy 187194CHAPTER 5 • Biomembranes and Cell Architectureintegral membraneprotein 157intermediatefilament 174lipid raft 156lumen 147lysosome 165microfilament 174microtubule 174mitochondrion 171nuclear lamina 177nucleolus 171peripheral membraneprotein 157peroxisome 168phosphoglyceride 150phospholipid bilayer 149pleckstrin homology (PH)domain 163porin 160prenyl anchor 160resolution 184sphingolipid 151REVIEW THE CONCEPTS1.
When viewed by electron microscopy, the lipid bilayer isoften described as looking like a railroad track. Explain howthe structure of the bilayer creates this image.2. Biomembranes contain many different types of lipidmolecules. What are the three main types of lipid moleculesfound in biomembranes? How are the three types similar,and how are they different?use to isolate cells and organelles from complex mixtures,and how do these techniques work?10. Isolation of some membrane proteins requires the useof detergents; isolation of others can be accomplished withthe use of high-salt solutions. What types of membrane proteins require detergents as part of the isolation procedure?What types of membrane proteins may be isolated with highsalt solutions? Describe how the chemical properties ofdetergents and high salt facilitate the isolation process ofeach type of membrane protein.11.
Three systems of cytoskeletal filaments exist in mosteukaryotic cells. Compare them in terms of composition,function, and structure.12. Individual cytoskeletal filaments are typically organizedinto more complex structures within the cytosol. What twogeneral types of structures do individual filaments combineto form in the cytosol? How are these structures created andmaintained?13. Both light and electron microscopy are commonly usedto visualize cells, cell structures, and the location of specificmolecules.
Explain why a scientist may choose one or theother microscopy technique for use in research.3. Lipid bilayers are considered to be two-dimensional fluids; what does this mean? What drives the movement of lipidmolecules and proteins within the bilayer? How can suchmovement be measured? What factors affect the degree ofmembrane fluidity?14. Why are chemical stains required for visualizing cellsand tissues with the basic light microscope? What advantagedoes fluorescent microscopy provide in comparison to thechemical dyes used to stain specimens for light microscopy?What advantages do confocal scanning microscopy anddeconvolution microscopy provide in comparison to conventional fluorescence microscopy?4.
Explain the following statement: The structure of allbiomembranes depends on the chemical properties of phospholipids, whereas the function of each specific biomembrane depends on the specific proteins associated with thatmembrane.15. In certain electron microscopy methods, the specimenis not directly imaged.
How do these methods provide information about cellular structure, and what types of structuresdo they visualize?5. Name the three groups into which membrane-associatedproteins may be classified. Explain the mechanism by whicheach group associates with a biomembrane.6. Although both faces of a biomembrane are composed ofthe same general types of macromolecules, principally lipidsand proteins, the two faces of the bilayer are not identical.What accounts for the asymmetry between the two faces?7. One of the defining features of eukaryotic cells is thepresence of organelles. What are the major organelles of eukaryotic cells, and what is the function of each? What is thecytosol? What cellular processes occur within the cytosol?8. Cell organelles such as mitochondria, chloroplasts, andthe Golgi apparatus each have unique structures. How is thestructure of each organelle related to its function?9.
Much of what we know about cellular function dependson experiments utilizing specific cells and specific parts (e.g.,organelles) of cells. What techniques do scientists commonlyA N A LY Z E T H E DATAMouse liver cells were homogenized and the homogenatesubjected to equilibrium density-gradient centrifugation withsucrose gradients. Fractions obtained from these gradientswere assayed for marker molecules (i.e., molecules that arelimited to specific organelles). The results of these assays areshown in the figure. The marker molecules have the following functions: Cytochrome oxidase is an enzyme involved inthe process by which ATP is formed in the complete aerobicdegradation of glucose or fatty acids; ribosomal RNA formspart of the protein-synthesizing ribosomes; catalase catalyzesdecomposition of hydrogen peroxide; acid phosphatasehydrolyzes monophosphoric esters at acid pH; cytidylyltransferase is involved in phospholipid biosynthesis; andamino acid permease aids in transport of amino acids acrossmembranes.ReferencesA100BCD ESprong, H., P.
van der Sluijs, and G. van Meer. 2001. How proteins move lipids and lipids move proteins. Nature Rev. Mol. CellBiol. 2:504–513.Tamm, L. K., V. K. Kiessling, and M. L. Wagner. 2001. Membrane dynamics. Encyclopedia of Life Sciences. Nature PublishingGroup.Vance, D. E., and J. E. Vance. 2002. Biochemistry of Lipids,Lipoproteins, and Membranes, 4th ed. Elsevier.Yeager, P. L. 2001. Lipids. Encyclopedia of Life Sciences.
Nature Publishing Group.F% of maximum8060Biomembranes: Protein Componentsand Basic Functions402001950510Fraction number50%Curve A = cytochrome oxidaseCurve B = ribosomal RNACurve C = catalase1520Sucrose 0%Curve D = acid phosphataseCurve E = cytidylyl transferaseCurve F = amino acid permeasea. Name the marker molecule and give the number ofthe fraction that is most enriched for each of the following: lysosomes; peroxisomes; mitochondria; plasma membrane; rough endoplasmic reticulum; smooth endoplasmicreticulum.b. Is the rough endoplasmic reticulum more or less densethan the smooth endoplasmic reticulum? Why?c.
Describe an alternative approach by which you couldidentify which fraction was enriched for which organelle.d. How would addition of a detergent to the homogenateaffect the equilibrium density-gradient results?REFERENCESGeneral Histology Texts and AtlasesCross, P. A., and K. L. Mercer. 1993. Cell and Tissue Ultrastructure: A Functional Perspective. W. H. Freeman and Company.Fawcett, D.
W. 1993. Bloom and Fawcett: A Textbook of Histology, 12th ed. Chapman & Hall.Kessel, R., and R. Kardon. 1979. Tissues and Organs: A TextAtlas of Scanning Electron Microscopy. W. H. Freeeman andCompany.Biomembranes: Lipid Compositionand Structural OrganizationSimons, K., and D. Toomre. 2000. Lipid rafts and signal transduction.
Nature Rev. Mol. Cell Biol. 1:31–41.Cullen, P. J., G. E. Cozier, G. Banting, and H. Mellor. 2001.Modular phosphoinositide-binding domains: their role in signallingand membrane trafficking. Curr. Biol. 11:R882–R893.Lanyi, J. K., and H. Luecke. 2001. Bacteriorhodopsin. Curr.Opin. Struc. Biol. 11:415–519.MacKenzie, K. R., J. H. Prestegard, and D. M. Engelman. 1997.A transmembrane helix dimer: structure and implications. Science276:131–133.Minor, D.
L. 2001. Potassium channels: life in the post-structuralworld. Curr. Opin. Struc. Biol. 11:408–414.Schulz, G. E. 2000. -Barrel membrane proteins. Curr. Opin.Struc. Biol. 10:443–447.Organelles of the Eukaryotic CellBainton, D. 1981. The discovery of lysosomes. J. Cell Biol.91:66s–76s.Cuervo, A. M., and J. F. Dice. 1998. Lysosomes: a meeting pointof proteins, chaperones, and proteases. J. Mol. Med. 76:6–12.de Duve, C. 1996. The peroxisome in retrospect. Ann. NY Acad.Sci.
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