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

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