This document discusses evidence that membrane proteins have fluidity and freedom of movement within cell membranes. It provides three key points: 1) Classic indirect evidence from experiments with artificial lipid vesicles shows that membrane proteins require lipid fluidity to function properly. 2) Classic direct evidence from antibody labeling experiments demonstrates that some membrane proteins diffuse freely within the membrane. 3) However, membrane protein mobility can be restricted by interactions with other proteins, the cytoskeleton, surrounding lipids, and the lipid composition within membrane domains.

2. Lecture 3 Cell Biology Membrane Fluidity

3. “ Fluidity” is the capacity of individual molecules to move freely Lipids behave as fluids within the membrane

4. Lipid fluidity is temperature- sensitive Phase Transition Point (“melting temperature”)

5. Different lipids have different “melting points”

6. Artificial membranes made with just one lipid type have a sharp MP Real membranes with a mix of lipids have a broad MP

7. OK – the lipids can move But what about the proteins??

8. Classic Indirect Evidence that membrane proteins have and require freedom of motion within the membrane

9. Create artificial membrane vesicles made of pure dimyristoyl phosphatidyl choline

10. Add some familiar players Hormone Receptor Adenylate Cyclase (makes cAMP) “ G” protein These three work cooperatively so that a hormone signal turns on AC

11. Above 23 °C, this works just fine

12. Below 23 °C, it won’t work at all

13. This is why cold kills

14. What about fish that live in cold water?

15. More unsaturated fatty acids at low temperature In each pair below, the sat/unsat ratio is lower at low temp

16. Classic Direct Evidences that membrane proteins have freedom of motion within the membrane

17. Antibodies can be used to locate lymphocyte receptor proteins

18. Antibodies can be used to locate lymphocyte receptor proteins Initially spread all over the cell surface Becoming patchier over time Eventually all gathered together in one “cap” Called “Patching” & “Capping"

19. Patching can’t occur if the membrane lipids are gelled 4 ° C 37 ° C

20. Patching results because antibodies cross-link proteins Each Ab can bind more than one protein, and each protein can be bound by more than one Ab

21. Clearly these proteins must be free to move around Does the antibody induce the mobility, or did it pre-exist ???? Each Ab can bind more than one protein, and each protein can be bound by more than one Ab

22. Experiment to demonstrate that at least some proteins are free to diffuse naturally

23. Add some differently labeled antibody arm fragments (Fab) But if you let them sit a while . . .

24. Rate of blending is temperature-dependent

25. Wouldn’t it be nice if we could describe diffusion rates with greater quantitative accuracy ? FLIP: Fluorescence Loss In Photobleaching FRAP: Fluorescence Recovery After Photobleaching

26. FRAP

27. Read about FLIP on your own

28. Finally – nowadays it’s sometimes even possible to watch a single protein diffuse

29. The Immunogold Technique

30. So – multiple evidences demonstrate that membrane proteins are free to move within the membrane plane

31. BUT let’s not go too far with that thought

32. If all proteins are freely diffusible within the membrane, then why do some proteins have localized distributions ? (Guinea pig sperm labeled with fluorescent antibodies against different membrane proteins)

33. Integrin a highly localized protein The orange patches – localized with a fluorescent antibody

34. If some proteins have less than full freedom of motion What are the restraints upon mobility?

35. Integrins bind to cytoplasmic actin network This restricts their mobility

36. Answer #1 Sometimes membrane protein mobility is restricted by interactions with the cytoskeleton

37. But other restraints upon mobility are much more indirect

38. Let’s return to something we looked at a little earlier Band 3 Protein Diffusion Careful analysis of the film shows that Band 3 sometimes acts like it’s “corralled”

39. Careful trypsinization cleaves P-face of Band 3 protein – removes “boxed in”-type motion What are the main proteins that make up the network depicted in this diagram?

40. Answer #2 Sometimes protein mobility is restricted by interactions with other membrane proteins

41. Tight (Occluding) Junctions An example of membrane proteins restricting each other's motion

42. Tight Junctions form connective seals between epithelial cells Based on “claudin” transmembrane proteins (Another important protein type is occludins )

43. Motion of claudins is restricted by intra membrane and inter membrane interactions with other claudins

44. The multiple claudin strands fence off separate “pastures”

45. A similar example Septins in Yeast Cytokinesis

46. A similar example Septins in Yeast Cytokinesis Some membrane proteins are found only in the daughter cell

47. Septins Part of a peripheral protein network at the mother-daughter interface Are septins restricting mobility and creating separate membrane domains? How could you tell?

48. A temperature-sensitive septin mutant

49. Natural and engineered mutations are another tool for your box

50. FINALLY Sometimes protein mobility is restricted by surrounding lipids

51. Lipid Rafts Some “patches” of membrane stay together after mild detergent treatment Suggests their composition is different from the rest of the membrane

52. Rafts are enriched in sphingolipids and cholesterol Lipid rafts stained with Filipin (binds to cholesterol)

53. Lipid rafts are also enriched for certain membrane proteins Especially (but not only) proteins with fatty acid prosthetic groups

54. Some protein enrichment may be due to physical thickness of raft Rafts may act to “trap” wandering proteins of the right types Proteins with longer-than-standard membrane-spanning domains

55. Thus, rafts influence functional associations between membrane proteins The fungal pathogen Candida albicans (Filamentous fungal cells exhibit tip-growth ) Stained with filipin

56. For Next Lecture Core-Level Things To Review in Advance: What is the difference between "anabolism" and "catabolism"? What is the structure and what is the metabolic function of glycogen? What is the general effect in the body of epinephrine (adrenaline)? What is glucagon, and how does its general effect compare to that of epinephrine? What is the difference between ATP and cyclic AMP? In which of the following is a hydrolysis reaction involved: 1) removal of, or 2) addition of the terminal phosphate group of ATP? Does it require or release energy to remove the terminal phosphate of ATP? Is the delta-G (change in free energy) of that reaction a positive or a negative value? What is GTP? Have you ever run across a protein that binds to GTP? Advanced-Level Things to Learn Independently Before Class: What is the meaning of the term “signal transduction”? How would you define a “second messenger”? Be sure you do more than merely memorize a dictionary definition – would you recognize when something is or isn’t a second messenger? Who was Rube Goldberg, and what was his cultural contribution?

57. Also Read and understand the basic points made in pp. 169 – 177, paying particular attention to allosteric proteins and protein phosphorylation

58. NEXT TIME Signal Transduction How cells sense and respond to their environments