1. BIOL 200 (Section 921) Lecture # 6-7; June 26/27, 2006 UNIT 5: MEMBRANES Reading: ECB (2nd ed.) Chap 2, pp 70-74 (to review carbohydrate and lipid chemistry); Chap 11, pp 365 –386; Chap 12 pp. 389-410 (essential) 411- 421 (optional), and related questions [11- 9a,b,c,d,e,f, 11-10 to 11-14, 11-17, 11-18; 12- 9abce; 12-11; 12-12; 12.13; 12-15; 12-18]

2. MEMBRANES - LIPIDS, LIPID BILAYERS: OBJECTIVES 1.Explain the condensation reactions that occur to assemble lipids and to form glycolipids 2.Explain the connection between the fluid mosaic model and the evidence supporting it. 3.Recognize that models of membranes have to be revised constantly to account for new experimental data. 4.Understand the properties and general synthesis of phospholipids, glycolipids, cholesterol, and various glycosides in membrane structure 5.Understand the properties of integral and peripheral proteins in membrane structure

3. MEMBRANE PROTEINS - OBJECTIVES 1.Explain the difference between peripheral and integral membrane proteins 2.Explain the forces that anchor proteins of each of these classes to membranes. 3.Explain how proteins can form 'aqueous pores' for transport of water, ions and other charged molecules. 4.Explain how membranes differ from bimolecular phospholipid leaflets. 5.Explain the evidence for fluid mobility of proteins within the plane of the membrane

4. MEMBRANE FUNCTION -TRANSPORTES, CHANNELS AND MEMBRANE POTENTIAL LEARNING OBJECTIVES 1.Each membrane has specific functions which are reflected in the functioning systems located in it. 2.To understand fundamental transport processes across membranes and the role of membrane proteins. 3.To understand the linkages between electrical forces, ATP hydrolysis and specific ion transport pumps. 4.To understand ion selectivity, gated channels and membrane potential. Nernst equation 5.To understand how a membrane potential is generated and propagated. 6.To understand the link between ion channels and nerve impulses. 7.Understand the control of directed secretion and its relation to nerve impulse transmission

5. Functions of membranes [Becker et al. The World of the Cell Fig. 7-2]

6. Biological membranes [Fig. 11-4]

7. Fluid Mosaic Model of Biological Membranes [Becker Fig. 7-5]

8. Key features of Fluid Mosaic Model Membrane lipids: arranged in a bilayer fluid (free to move in plane of bilayer) asymmetrically arranged (different lipid components on one face of the bilayer than the other) Membrane proteins: globular units with hydrophobic domains embedded in the hydrophobic core of the membrane, "fluid", in the lipid bilayer, unless anchored by interactions with other proteins.

9. [Becker et al. The World of the Cell]

10. LIPID CHEMISTRY Read pp. 73-74 for a review of chemical structures and terminology of different lipid molecules

11. Triacylglycerols form oil droplets Oil seed embyro

12. 11_10_Fat_phospholip.jpg Fat molecules are hydrophobic, whereas Phospholipids are amphipathic [Fig. 11-10]

13. 11_07_amphipathic.jpg Membrane lipids have both polar and non-polar regions (amphipathic) [Fig. 11-7]

14. 11_06_Phosphatidylch.jpg Phosphatidylcholine is the most common phospholipid in cell membranes [Fig. 11-6]

15. Sphingolipids: serine backbone, 2 acyl tails (Often glycolipids)

16. [Becker et al. The World of the Cell]

17. Fig. 11-8: hydrophilic, water forms H-bonds with atom carrying uneven charge distribution Fig. 11-9: hydrophobic, water doesn’t interact with solute; forms cage- like structures

18. 11_11_bilayer.in.H20.jpg Amphipathic phospholipids form a bilayer in water. What interactions stabilize the bilayer structure?

19. Three characteristics of membrane lipids 1.Fluidity 2.Asymmetry 3.Permeability

20. Membrane lipid fluidity The ease with which the lipid molecules move in the plane of the bilayer Depends on temperature and the phospholipid composition [higher the temperature, lipids with longer tails and fewer double bonds are synthesized in temp.- adapting bacteria/yeast] Depends on the length and unsaturation of fatty acids –Shorter hydrocarbon tails = increased fluidity –Increased unsaturation (# double bonds) = increased fluidity

21. Movement of phospholipid molecules within Membranes [Becker Fig. 7-5]

22. The effect of chain length and the number of double bonds on the melting point of fatty acids [Becker et al. Fig. 7-13]

23. 11_16_Cholesterol.jpg What is the role of cholesterol in animal cell membrane fluidity?

24. Membrane flexibility (fluidity) can be studied by either phospholipid vesicles or a synthetic phospholipid bilayers

25. 11_36_Photobleaching_techniqu es.jpg FRAP – fluorescence recovery after photobleaching Membrane Fluidity: Experimental evidence

26. Below are results from two FRAP experiments. What can you conclude about the membrane in cell 2 compared to cell 1? What characteristics would you predict that cell 2’s membrane lipids would have? Cell 2Cell 1

27. Lipid asymmetry – The two halves of the bilayer are different. Membranes have a distinct lipid profile in inside and outside layers of bilayer [Fig. 11-17] extracellular space cytosol PC sphingomyelin cholesterol glycolipid PSPI PE

28. How is lipid asymmetry created? All membrane lipids are made in the SER. Enzymes in the SER join fatty acids and glycerol and phosphate and head groups to make phospholipids The phospholipid is inserted into one of the monolayers Enzymes called FLIPPASES flip some of these lipids into the other bilayer, so that the whole membrane will grow. They only transfer specific lipids. Glycolipids get their sugar groups in the Golgi only on the non-cytosolic monolayer of the membrane. It then travels as a vesicle and fuses with the plasma membrane, where it maintains the orientation of glycolipids in the non- cytosolic monolayer [see Fig. 11-15]. It makes the membrane asymmetrical.

29. ER New phospholipids added to cytoplasmic leaflet Phospholipid translocator Membrane protein Both sides enlarged Phospholipids moved between leaflets by translocator proteins ATP

30. Different organelles have different lipid compositions

31. Relative permeability of lipid bilayer [Fig. 12-2] Cell membrane acts as barriers Rate of crossing the membrane varies with the size and the solubility of the molecule Small, hydrophobic molecules cross the membrane most rapidly Other moleules require special transport proteins Synthetic bilayers have been used to study permeability

32. Functions of membrane proteins [Fig. 11-20] e.g. Na+ pump Structural linkers e.g integrin receptors

34. 11_21_proteins.associ.jpg Membrane proteins associate with the lipid bilayer in several different ways [Fig. 11-21]

35. Fig. 11-23:  -helix spans the bilayer (why?) H-bonded polypeptide chain inside, Hydrophobic R-groups outside.

36. 11_24_hydrophl.pore.jpg Five transmembrane  helices form a water channel across the lipid bilayer. Where are the hydrophillic and hydrobobic aa sidechains?

37. 11_28_Bacteriorhodop.jpg Bacteriorhodopsin acts as a proton pump. Two polar aa side chains are Involved in proton transfer process.

38. Fig. 11-27: solubilizing membrane proteins with detergent like TritonX-100

39. Fig. 11-27: solubilizing membrane proteins -the results Water-soluble complex of membrane protein and detergent Water soluble complex of lipid + detergent

40. Panel 4-5: protein electrophoresis

41. [Becker et al. The World of the Cell]

42. 11_31_spectrin_network.jpg Spectrin-based cell cortex of human red blood cells provides mechanical strength and shape to the cell [Fig. 11-32] Genetic abnormalities in spectrin result in abnormal, spherical and fragile red blood cells causing anemia

43. Glycocalyx: sugar coat Fig. 11-32 Glycocalyx functions in cell-cell recognition, protection, lubrication and adhesion

44. - Surface proteins of cultured cells are labeled with antibodies coupled to fluorescent dyes (red and green). - The "red" and "green" cells are then mixed and can fuse. - In time, labeled proteins from each cell mix showing membrane fluidity Can plasma membrane proteins move? Figure 11-34 (p.383) CELL FUSION EXPT.

45. What restricts lateral mobility of plasma membrane proteins? Cell cortex Extracellular matrix Intercellular protein-protein interactions Diffusion barriers

46. Tight Junctions: Gut epithelium cells showing apical and basolateral domains [Fig. 11-39]

47. Fig. 21-22: tight junctions seal epithelia Epithelium is sheet of cells Tight junctions wrap around the apical face of each cell Inject dye into either apical face or baso- lateral face-can’t cross tight junction

48. [Becker et al.] Membrane transport

49. ECB 2 nd Ed. Table 12-1

50. 12_02_diffusion_rate.jpg Membrane permeability

51. Carrier protein Channel protein Carrier has conformational change, carries solute [Fig. 12-3] channel opens, selectively allows ions in.

52. Passive vs. active transport [Fig. 12-4] Passive transport Active transport

53. 12_08_electroch_gradient.jpg Two components of an electrochemical gradient = Concentration gradient + membrane potential

54. Three ways of active transport of molecules via the cell membranes [Fig. 12-9]

55. The Na+/K+ pump uses ATP hydrolysis to pump Na+ out and K+ in to the cell both against their electrochemical gradient [Fig. 12-10]

56. 12_12_Na_K_cyclic.jpg Mechanism of the Na+/K+ pump [Fig. 12-12]

57. 12_13_Carrier_proteins.jpg Carrier protein-mediated transport [Fig. 12-13]

58. 12_15_glucose_gut.jpg Transport of glucose across the gut lining is mediated by two types of glucose carriers

59. Fig. 12-5: Each organelle has its own subset of unique channels and carriers ER- Ca2+ transporters Ca

60. Fig. 12-15: hypotonic‘ghost’ Osmosis=movement of water molecules across a semi-permeable membrane in response to a concentration gradient

61. Cells use different ways to prevent osmotic swelling [Fig. 12-16]

62. Animal cell Similarities and differences in carrier-mediated transport in animal and plants [Fig. 12-18] Plant cell

64. 12_19_selectivity_filter.jpg A K+ channel protein

65. 12_21_Venus _flytrap.jpg The quick closing of the leaves of Venus Flytrap in response to touch is due to the mechanical stimulation of ion channels changing the turgor pressure [Fig. 12-21]

66. 12_22_patch_clamp_record.jpg The patch-clamp technique

67. 12_23_current _measured.jpg Measurement of ion channel activity as current by the Patch-clamp technique [Fig. 12-23]

68. 12_24_Gated _ion_chan.jpg Gated ion channels [Fig. 12-24]

69. Fig. 12-26: Touch-induced opening of voltage-gated Ion channels generate (1) an electric impulse followed by a rapid water loss by hinge cells, causing (3) folding of Mimosa leaflets.

70. Fig. 12-27: The unequal distribution of ions across thebBilayer produces the membrane potential (electrical potential difference across the membrane).

71. Potassium ion channels are important in generating the membrane potential across the plasma membrane [Fig. 12-28]