Molecular Cell Biology

1. Molecular Cell Biology Actin, including Principles of Assembly Cooper

2. Introduction • Handouts • Readings • Text • MiniReviews - PDF files online • Homework

3. Reading • Textbook Chapters • Lodish et al., Molecular Cell Biology, 6th ed., 2008, Freeman. Chaps. 17, 18. • Pollard & Earnshaw, Cell Biology, updated ed., 2004, Saunders. Chaps. 35-42, 47. • Articles on the Course Web Site • Original Articles • Reviews

4. Older Advanced / Reference Materials • 1. Cell Movements, 2nd ed. ,Dennis Bray, 2001, Garland. • 2. Guidebook to the Cytoskeletal and Motor Proteins. Kreis and Vale, eds. 1999, Oxford Univ. Press. • 3. Video Tape of Motility. Sanger & Sanger, Cell Motility & the Cytoskeleton, Video Supplement 2, 1990. A one-hour tape of examples of microtubule-based motility. Short segments shown in class. Available at the Media Center in the Becker (medical) library.

5. Chemotaxis of neutrophil to bacteria

6. Phagocytosis of bacteria by Dictyostelium amoebae

7. Biological Scope of Cell Motility & the Cytoskeleton • Shape • Translocation • Contraction • Intracellular Movements • Mechanical & Physical Properties

8. Elements of the Cytoskeleton • Structural • Filaments - Actin, Microtubules, Intermediate Filaments, Septins • Crosslinkers • Motors • Actin - Myosin • Microtubules - Dynein, Kinesin • Regulators

9. Higher Order Structures and Functions • Actin • Muscle sarcomere • Epithelial cell brush border • Cortex of motile cells • Microtubules • Cilia & Flagella • Mitotic spindle apparatus • Radiate from MTOC - organize membranes • Septins - cytokinesis • Major Sperm Protein in nematode sperm

10. Self-Assembly by Proteins -Entropy & the Hydrophobic Effect • High Order in Assembled State Implies Lower Entropy, which is Unfavorable • ∆G = ∆H - T∆S must be <0 for a Reaction to Occur • But ∆H>0, ∆S>>0 ! • Higher Entropy => Disorder in Assembled State • Ordered Water on Hydrophobic Surface of Protein Subunit is Released

11. Self-Assembly by Proteins - Specificity • Hydrophobic Surfaces of Proteins Must Fit Snugly to Exclude Water • Assorted Non-covalent Bonds • Van der Waals • Coulombic • H-bond

12. Why Use Subunits to Make Large Molecules? • Efficient Use of the Genome • Error Management • Variable Size • Disassembly / Reassembly

13. Equivalence and Quasi-Equivalence • Subunits in Polymer Must be Indistinguishable from Each Other • Helical Arrangement Produces Linear Filament • Some Flexibility in Structure Produces Loss of Equivalence • Quasi-Equivalence: Similar with Distortion

14. Assembly of Helical Filaments • Add & Lose Subunits Only at Ends • ON Rate = k+ c1 N • OFF Rate = k- N c1 = Concentration of Monomers N = Concentration of Filament Ends

15. Assembly of Helical Filaments • At Steady State, by Definition • ON Rate = OFF Rate • k+ c1 N = k- N • c1 = k- / k+ • Subunit Concentration is Constant?!

16. Critical Concentration [Polymer] Steady-state Concentrations of Polymer & Monomer [Monomer] [Total]

17. Critical Concentration and Binding Affinity A1 + Nj Nj+1 [Nj+1] Ka = _ c1 [Nj]

18. Critical Concentration and Binding Affinity 1 Ka = _ c1 _ c1 _ c1 [Nj] Kd = = [Nj+1]

19. Treadmilling • Polar Filaments have Two Different Ends • Can Have Different Critical Concentrations at the Two Ends • Steady State Critical Concentration is an Intermediate Value • Net Addition at One End, Net Loss at the Other End

20. Microtubule PhotobleachingExperiment In Vivo Fluorescent Tubulin Microinjected into Cell as Tracer Laser Bleaches a Vertical Stripe

21. Cells Regulate Polymers • Cells Have Unexpectedly High Concentrations of Subunits • Cells Change their Subunit / Polymer Ratio Dramatically • Filament Lengths in Cells are Short

22. How do Cells Regulate the Level of Polymerization? • Total Concentration of Protein • Covalent Modification of Subunits • Binding of Small Molecules • Binding of Another Protein

23. How do Cells Regulate the Number and Length of Filaments? • Limit Growth • Intrinsic to Protein • Deplete Subunits • Capture by Capping End • Template • Create New Filaments • Nucleation - End or Side • Bolus of Subunits - High Concentration

24. Nucleation • Creation of New Filament from Subunits is Unfavorable • Subunit Prefers End of Filament to One or Two Other Subunits • Allows Cell to Control Where & When Filaments Form

25. “Dynamic Instability” of Microtubules GFP-tubulin in Cells Pure proteins in vitro

26. Nucleotides Can Generate “Dynamic Instability” • The Basic Facts... • Tubulin Binds GTP or GDP • GTP Tubulin Polymerizes Strongly • GDP Tubulin Polymerizes Poorly • Subunits Exchange w/ Free GTP • GTP on Tubulin Hydrolyzes to GDP over Time after Addition to Microtubule

27. The Implication of All those Facts, taken together is... • At Steady State, at any given time... • Most Ends have a GTP “Cap” and Grow Slowly • A Few Ends • Lose their GTP Cap • Exposing GDP-tubulin subunits • so the Microtubule Shrinks Rapidly • Occurs In Vitro and In Vivo for Tubulin - Extensive and Relevant

28. Steps in Cell Movement Extension Adhesion Retraction Lodish et al. Molecular Cell Biology

29. Types of Actin Structures in a Migrating Cell

30. Scanning EM of the Front of a Migrating Cell

31. Small G-Proteins Regulate Different Assemblies of Actin Stress Fibers Lamellipodia Filopodia

32. GFP-Actin in a Migrating Melanoma Cell Text

33. Fish Keratocyte - Gliding Across a Surface 0.1 - 1 µm per second

34. Fish Keratocytes Stationary Moving

36. End-to-Side Branches Svitkina et al. 1997.

37. Free Ends toward Direction of Movement Svitkina et al. 1997.

38. Arp2/3 Complex at Filament Branches in vitro in vivo

39. Arp2/3 Complex Structure, at a Filament Branch Point Hanein, Robinson & Pollard. 2001.

40. Creation & Growth

41. Termination

42. Destruction & Recycling

43. Model for Listeria Actin Motility Jon Alberts. Center for Cell Dynamics, Friday Harbor, U Wash. CellDynamics.Org.

44. Model for Listeria Actin Motility Jon Alberts. Center for Cell Dynamics, Friday Harbor, U Wash. CellDynamics.Org.

45. Fluorescence Microscopy of Living Cells • GFP technology - colors, aggregation, multiple labels, FRET • Sensitive video cameras - increased time until bleaching • Speed and sensitivity • Confocality • Laser scanning •Spinning disk • Two-photon •TIRF

46. Speckles to Single Molecules

47. Evidence for Single Molecules Fluorescence Intensity of Single Speckles over Time

48. Speckle Microscopy in Living Cells

49. Two-Color Speckle Microscopy Microtubules Actin

50. TIRF (Total Internal Reflection Fluorescence) Microscopy