Instructor: Katie L Kathrein, Ph.D. kkathrein@endersh.harvard - PowerPoint PPT Presentation

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Instructor: Katie L Kathrein, Ph.D. kkathrein@endersh.harvard
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Instructor: Katie L Kathrein, Ph.D. kkathrein@endersh.harvard

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  1. E16/W: Cell Biology Thursdays 5:30-7:30pm, Science Center Hall E Section: 7:35-8:35pm, Science Center, Rms 101b and 103b Instructor: Katie L Kathrein, Ph.D. kkathrein@enders.tch.harvard.edu Office: Children’s Hospital Karp Family Research Building One Blackfan Circle, RB07005H Boston, MA 02115 Office hours: Wednesday 4:15-5:15pm in Café in Science Center Office phone: (617) 919-2078 Undergraduate Teaching Assistant: Allegra Lord alord@fas.harvard.edu Office: Karp Building, RB05007G Office phone: (617) 355-9086 Graduate Teaching Assistant: Alison Taylor alison_taylor@dfci.harvard.edu Dana Building, 1526 Office phone: 617-632-2930

  2. Organization of the cell

  3. Plasma membrane Functions: Compartmentalization Scaffold Barrier Gatekeeper Monitoring of outside signals Energy Transduction

  4. Plasma membrane • The plasma membrane serves many functions for the cell • skinof the cell • gatekeeperof the cell • shapeof the cell.

  5. Lipids -exoplasmic -cytoplasmic -Organelles

  6. Plasma Membrane • Semi-permeable barrier • Prevents : • Stability is maintained by the interactions of components

  7. Plasma membrane • Amphipathic chemical structure • In mechanic dissociation in aqueous solutions • Spontaneously form sealed, closed compartments • useful for laboratory studies

  8. Plasma MembranePhospholipid Bilayer

  9. Plasma membrane:Components Modified Lipids Proteins (Phosphoglycerides)

  10. Plasma MembranePhospholipids Modified Lipids • Three types in most memebranes: • Amphipathic molecules

  11. Phospholipids • comprise ~50% by mass of animal cells • 2 chain fatty acid tail • Linked to phosphate group via glycerol linker • Capped by a polar head group • Phospholipids include: • Negative charged: • phosphotidylserine (PS) • phosphotidylinositol (PI) • Neutral: • phosphatidylcholine (PC) • phosphatidylethanolamine (PE) • sphingomyelin Polar Head group Phosphate group 2 Fatty Acid Chains

  12. Phospholipids • Phospholipids have a glycerol linker • The phosphate group is often modified • serine • inositol • Choline • Ethanolamine

  13. PhospholipidsHead Groups • Phosphatidylethanolamine (PE) • Phosphatidylcholine(PC) • Phosphatidylserine(PS) • Phosphotidylinositol(PI)

  14. Plasma membrane Saturation and length • Saturated fatty acids • Unsaturated fatty acids

  15. Other membrane lipids • Sphingolipids • sphingosine • amino alcohol with a long carbon chain

  16. Glycolipids • Polar Head linked to lipid

  17. Cholesterol • Steroids • Mainly in eukaryotes • Amphipathic hydrophobic Polar head group

  18. Cholesterol • Enhances barrier properties • Modulates fluidity • Temperature dependence • Concentration dependence

  19. Membrane Asymmetry • Different leaflets have different compositions

  20. Membrane Asymmetry Sphingolipids PS and PI PC and PE Cholesterol

  21. Membrane Fluidity:Lipids • Membranes are extremely mobile • Lipids • Flip flop is rare

  22. Membrane Fluidity: FRAP Study the movement of membrane components Fluorescence Recovery After Photobleaching Free diffusion of labeled molecule- faster recovery Membrane anchored protein- little to no recovery http://www.youtube.com/watch?v=LicQb_SnCSI

  23. Membrane Fluidity: FRAP • How do we fluorescently label a transmembrane protein? • GFP fusion protein • Fluorescently labeled antibody 3) Amphipathic molecule with fluorochrome

  24. Membrane Fluidity: FRAP • fluorescent microscopy • “bleach” the fluorescent molecule • Watch the movement of non-bleached proteins

  25. Membrane Fluidity: FRAP Study the movement of membrane components Fluorescence Recovery After Photobleaching Free diffusion of labeled molecule- faster recovery Membrane anchored protein- little to no recovery http://www.youtube.com/watch?v=LicQb_SnCSI

  26. FRAP

  27. Membrane Fluidity: FRAP

  28. Plasma membrane:Components Modified Lipids Proteins (Phosphoglycerides)

  29. Membrane Proteins • Proteins in the plasma membrane • Integral • Peripheral

  30. Proteins in membrane Peripheral Integral Exo Cyto Integral 1 - Single-pass 2 - Multi-pass Peripheral 3 - Fatty acid chain attachment to membrane 4 - Oligosaccharide-linked attachment to membrane 5, 6 - Noncovalent attachment to protein anchor

  31. Integral Membrane Proteins • Integral proteins • stretches of amino acids that span the membrane • extracellular and cytoplasmic components

  32. Peripheral MembraneProteins • Peripheral proteins

  33. Peripheral Membrane Proteins • fatty acid chain attachments • covalent linkage • Integral membrane proteins

  34. Peripheral Membrane Proteins • membrane proteins have limited movements

  35. Lipid Rafts • In lab models of bilayers, lipids can come together transiently • Suggests the existence of “lipid rafts”

  36. Membrane Permeability Passive Diffusion • Gases and small uncharged molecules will • diffuse across the bilayer unassisted • Passive Diffusion • - Follows concentration gradient • - No energy input • - No transport proteins • Depends on relative hydrophobicity

  37. Membrane TransportProteins • Membrane proteins act as transporters • Two basic types of transport: • Active and passive

  38. Membrane Transport Active ATP-powered pump Channel Transporter Passive

  39. Channels • Channels allow movement of a large number of molecules fast

  40. Passive Transporters Three types of passive transporters • Uniporter, symporter and antiporter

  41. Passive Transporters Uniporter • moves a single molecule down its concentration gradient

  42. GLUT1 • Uniporter • Transports glucose into cells

  43. GLUT1 • GLUT1 transports glucose from the outside in or inside out

  44. Studying Uniporters in the lab • How do we characterize these proteins in the lab?

  45. Passive Transporters Uniporter • moves a single molecule down its concentration gradient Coupled Transport • Moves against concentration gradient • Symporter • Antiporter

  46. Symporters and Antiporters • Undergo the same conformational changes as uniporters • Move molecules against their concentration gradient

  47. Glucose/Na+ Symporter [Glucose] high inside cell [Na+] high outside cell

  48. Na+ linked Ca2+ Antiporter • Ions move down their respective concentration gradients

  49. Na+ H+ antiporter • The pH in cells must be maintained as close to neutral (~7.2)

  50. Active Transport • ATP-powered pump • couples ATP hydrolysis with movement