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  1. 1. Lipids, Membranes & the First Cells Chapter 6
  2. 2. Evolution of the Membrane  The evolution of the plasma membrane was a momentous event because it separated life from non-life  Before the cell RNA molecules clung to clay particles, building copies as nucleotides washed over them randomly
  3. 3. Lipid Membrane  The formation of the membrane performed three important tasks  Separated the chemical composition of the inside from the chemical composition of the outside  Chemical reactions became more efficient as reactants could collide more frequently  Membrane could form a collective barrier
  4. 4. Lipid Formation  Spark discharge experiments succeeded in production at least two types of lipids  How did these lipids behave when they are immersed in water  Transmission electron microscope  Lipids spontaneously formed enclosed compartments filled with water
  5. 5. What Characterizes a Lipid  Lipids are defined by a physical property  Solubility in water  Not strictly characterized by structure  Therefore the structure of lipids varies widely  Hydrocarbons  Hydrophobic
  6. 6. Lipids Found In Cells  There are three types of lipids found in cells  Lipids for energy storage  Triacylglycerols  Lipids for cell membranes  Phospholipids  Lipids for steroids  Cholesterol, estrogen, & testosterone
  7. 7. Triglycerides  Via condensation reactions one glycerol molecule is connected to three fatty acids  The glycerol and fatty acid are linked by an ester linkage
  8. 8. Steroids  Family of lipids that has a four ring structure  Cholesterol is an important membrane component of plasma membranes is some organisms  Others are important hormone signals
  9. 9. Phospholipids  Structure  Glycerol linked to phosphate group  Glycerol linked to two fatty acids  For Archaea bacteria  Glycerol linked to phosphate group and two isoprene units
  10. 10. Lipid Classification
  11. 11. What is a Sphingolipid
  12. 12. Lipids & Disease  Membrane lipids undergo constant metabolic turnover  Breakdown is performed by hydrolytic enzymes in lysosomes  Impaired degradation by a defect enzyme leads to the accumulation of partial breakdown products  Brain, liver & spleen  Genetic basis for Niemann-Pick disease and Tay- Sachs disease  Metal retardation, paralysis, blindness, early death
  13. 13. Lipids in Membranes  In order to spontaneously form a lipid bilayer lipid must have  Charges and polar bonds in the head region to interact with water  Long fatty acid tails to interact with each other  Amphipathic
  14. 14. Lipid Bilayer Formation & Energy  Lipid structures form spontaneously  No energy input is required  Energy Concepts  Independent phospholipids are unstable in water high potential energy  Hydrophobic tails disrupt hydrogen bonds  When tails interact with one another reach a lower potential energy state  Formation of these structures clearly decreases entropy  But overall ∆H outweighs ∆S leading to a negative ∆G
  15. 15. What Will Form  Micelles  Short tails  Bilayers  Long tails
  16. 16. Artificial Membranes  Researchers produced many types of vesicles by using many different types of phospholipids  When phospholipids are agitated by shaking the layers break and re- form small water enclosed vesicles
  17. 17. Evolution of Membrane Models  Today cell membranes are characterized by what is known as a fluid mosaic model  Over 100 years of research was performed before this model  Historical Perspectives
  18. 18. History & Membranes
  19. 19. Cell water soluble lipid soluble Charles Overton, 1890’s  Question  What is the composition of the cell’s membrane?  Experiment  Added both water soluble (hydrophilic) and lipid soluble (hydrophobic) substances to cells to determine if those substances could enter cell  Conclusion  Cells have a lipid ‘coat’ on their surface
  20. 20. Gorter and Grendel, 1925 Langmuir trough  Question:  What is the arrangement of lipids in the plasma membrane  Experiment  Measure surface area of RBC  Measure surface area of lipid monolayer  Compare areas  Why use RBCs
  21. 21. Gorter and Grendel, Results Results: Surface area of monolayer = 2x the SA of RBCs, therefore lipid is oriented as a bilayer Conclusions:
  22. 22. Gorter and Grendel, Conclusion  Lipids are two layers thick in the membrane.  bilayer  Proposed that lipids were in bilayer with polar groups toward the aqueous compartments and non- polar fatty acid parts toward the center of the bilayer  Phospholipids can rotate, diffuse and flip in the lipid sea
  23. 23. protein protein Davson and Danielli, 1935  A role for proteins  Surface tension
  24. 24. Davson and Danielli  1st model  Evidence  surface tension of oil droplets is high  surface tension of cell membranes is low  using starfish eggs  Surface tension of oil droplets coated with protein is low
  25. 25. Davson and Danielli  2nd model  Realized that there was a problem with the Davson/Danielli 1st model  Transport  Model was revised to allow for pores
  26. 26. Protein Coat Lipid This was believed to confirm the Davson/Danielli model of the plasma membrane structure. Robertson, 1959
  27. 27. Singer and Nicholson, 1972  Disproved Robertson, Davson, & Danielli  Lipid bilayer kept  Protein coat lost  This model had two important components  Fluid  all components are free to diffuse in the plane of the membrane  Mosaic  heterogeneity in the membrane – proteins and lipids interspersed  AND, because of fluidity, randomly distributed
  28. 28. Evidence for Fluid Mosaic  Mixing of fluorescently tagged proteins on hybrid cells  Frye & Edidin, 1970  Florescence recovery after photobleaching  FRAP  Mosaic  Freeze fracture
  29. 29. Frye and Edidin, 1970  Journal of cell science  Used fluorescently labeled antibodies Fluorophore
  30. 30. Membrane proteins Mouse cell Human cell Hybrid cell Mixed proteins after 1 hour + Frye and Edidin, 1970  Explanations  Proteins are free to diffuse in the membrane  Newly synthesized membrane proteins are inserted into the membrane  Process is ATP dependent
  31. 31. Note: Experiment done at 37°C Frye and Edidin (1970)
  32. 32. Frye and Edidin, 1970  Is protein synthesis of new membrane proteins responsible for intermixing  Add protein synthesis inhibitor cyclohexamide  Mixing still occurred  Is intermixing an ATP dependent process  Block ATP production with DNP, cyanide  Mixing still occurred  Conclusion  Mixing is due to fluidity
  33. 33. (fusing) virus Mouse cell (a) Incubation temperature (ºC) Mosaics (%) 0 + + + + + + + + 50 100 5 15 25 35 + + + + (c) Frye and Edidin, 1970  If intermixing is due to membrane fluidity, then intermixing should be temperature dependent
  34. 34. Fluorescence Recovery After Photobleaching  FRAF  Measures lateral diffusion of molecules (lipids/proteins) in cell membranes  Method allows us to look at populations of molecules  Information obtained addresses whether components are, in fact, free to diffuse
  35. 35. 3 2 1  Measure Recovery  All labeled components are free to diffuse  Slow diffusion  A fraction of the population is not mobile  Anchored proteins  Conclusion  Most but not all components are free to diffuse
  36. 36. Mosaic Evidence  Scanning electron micrographs showed pits and mounds studding the inner surfaces of the bilayer
  37. 37. Proteins in the Bilayer  Integral Membrane Proteins (IMPs)  Cell surface receptors  Adhesion molecules  Transporters  Ion channels  Peripheral Membrane Proteins (PMPs)  Associate non-covalently with the membrane  Interact with IMPs of phospholidip head groups  Can be inner or outer leaf of PM
  38. 38. IMPs Hydrophobic Regions  Usually α helical structure is found in the transmembrane space  There can be one α helix or several  These α helical structures can also form pores or tubes
  39. 39. IMPs Extracted Using Detergents  Detergents are small amphipathic molecules  They disrupt plasma membranes and hydrophobic regions of the detergents binds to hydrophobic region of the protein  Purification of protein product will allow you to test its function
  40. 40. Glycosylation of Proteins  Never occurs on cytoplasmic proteins  Uses specific enzymes of ER and golgi  Two types  N-linked = carbohydrates are attached to asparigine (terminal amino group) synthesis of CHO side chain begins in ER and is completed in Golgi  O-linked = carbohydrates are attached to serine or threonine (hydroxyl groups) synthesis of CHO side chain begins and ends in Golgi  For review of the function see chapter 5 notes
  41. 41. Transport Proteins  Facilitated Diffusion  Requires no ATP moves with a concentration gradient  Channels  Ion channels  Ions move according to an electrochemical gradient  Usually specific to one type of ion  Aquaporins  Transporters  Glucose transporter  Changes shape  Active Transport  Requires ATP moves against concentration gradient  Ion pumps
  42. 42. Gramicidin – Ion Channel  Ions carry charge movement of ions produces and electric current  Can carry H+, K+ and Na+ Ions travel down this pore
  43. 43. Aquaporins  Very specific/selective channel  Only water goes through  Water is able to cross the plasma membrane 10X faster than without aquaporins
  44. 44. Gated Channels  Aquaporins and ion channels are gated  Open or close in response to signal  Voltage gated channels  Open in response to depolarization of membrane  Ligand gated channels  Open in response to a chemical signal  Remember no energy is needed for transport  Powered by diffusion along an electrochemical gradient
  45. 45. Carrier Proteins  Lipid bilayer is not permeable to glucose, yet glucose is a main source of cellular energy  Researchers used RBCs to extract and purify a glucose transporter
  46. 46. GLUT – 1  After isolating and analyzing many proteins from RBCs ghosts researchers found one protein that increased membrane permeability to glucose  This protein was added to liposomes (artificial membranes) and it transported glucose at the same rate as a living cell
  47. 47. Active Transport Pumps  Requires energy – ATP  ATP ADP + P  The phosphate group is transiently and covalently attached to a protein in a process known as protein phosphorylation  Phosphorylation of proteins often leads to a change in protein shape or protein conformation  Leads to a decrease in entropy  Movement is against a concentration gradient  Important for establishing electrochemical gradients
  48. 48. Cell Membrane
  49. 49. Selective Permeability  Artificial membrane systems proved to be invaluable in determining the permeability of membranes  Allowed researches to change one parameter at a time and asses the effect  How rapid is diffusion  What happens when a different type of phospholipid is used  Does permeability change with cholesterol or other molecules
  50. 50. Membranes are Highly Selective  Small nonpolar molecules move across quickly  CO2, O2, N2, hydrophobic molecules  Small polar molecules have intermediate permeability  H2O, glycerol, urea  Large uncharged polar  Glucose  Ions  Na+, K+, Cl- Permeability cm/sec
  51. 51. Does Lipid Composition Affect Permeability  Two properties affect permeability  Number of double bonds  Saturated vs. unsaturated  Packing  Double bonds produce spaces between tails  Atoms are in one plane locked in place  Spaces reduce strength of hydrophobic interactions  Tail length
  52. 52. Fluidity and Double Bond Character  Degree of hydrophobic interactions increases with saturated fats  Fluidity increases with double bonds
  53. 53. Fluidity and Double Bond Character
  54. 54. Cholesterol and Permeability  Cholesterol is a large bulky ring structure  Cholesterol should increase the density of hydrophobic interactions
  55. 55. Cholesterol and Permeability
  56. 56. Diffusion  Diffusion is a process which occurs spontaneously due to an increase in entropy  Diffusion occurs from an area of high concentration to an area of low concentration  Diffusion across a plasma membrane
  57. 57. Osmosis  Osmosis is the diffusion of water from higher concentration to lower concentration  Only occurs when solutions are separated by a membrane that is permeable to some molecules and not others  Movement is spontaneous  Driven by an increase in entropy when the solute becomes more dilute Entropy decreased 5X on one Side up increased 10X on the other The system had a net gain of entropy
  58. 58. Osmosis  If water is more concentrated on one side of a membrane then there will be a net movement of water
  59. 59. Osmosis & Diffusion  Osmosis and diffusion reduce differences in chemical composition between the inside and outside of membrane bound vesicles  Therefore, it is unlikely that interiors differed radically from the external environment  Lipid bilayers become capable of creating a specialized internal environment due to specialized protein transporters