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Problem Sets due tomorrow ! Dropbox on course website is open and will close at midnight tomorrow.

Problem Sets due tomorrow ! Dropbox on course website is open and will close at midnight tomorrow. Late assignments can be emailed to Allegra until Monday morning. 1. Cytoskeleton. What is the cytoskeleton? Why do cells need a cytoskeleton?.

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Problem Sets due tomorrow ! Dropbox on course website is open and will close at midnight tomorrow.

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  1. Problem Sets due tomorrow! Dropbox on course website is open and will close at midnight tomorrow. Late assignments can be emailed to Allegra until Monday morning. 1

  2. Cytoskeleton • What is the cytoskeleton? • Why do cells need a cytoskeleton?

  3. Cytoskeleton Regulate Cell Shape and Cell Motility

  4. Three Major Classes of Cytoskeletal Proteins Cytoskeletal Proteins Microfilaments- polymers of G-actin monomers Microtubules- tubes of alpha/beta-tubulin dimers Intermediate filaments- various proteins 4

  5. Intermediate Filaments

  6. Microtubules • Extend throughout the cell • Provide organizational framework for organelles • Motors include kinesin and dyneins

  7. Microfilaments • Polymers of actin • Organization of the plasma membrane

  8. Dynamic changes to cytoskeleton mediate cellular shape changes Microfilament Organization of the PM Tracks for Myosin Motors Microtubule organization of organelles Cilia & flagella Mitotic spindle Tracks for Kinesin and Dynein Motors Intermediate Filaments Variety of roles Structural support for nuclear membrane Tissue integrity Structural and barrier functions in skin 9

  9. Actin and Microfilaments • Actin microfilaments • Myosin motors • Cell motility

  10. Actin • Actin proteins polymerize • Compose the cell cortex

  11. Actin • Migrating cells: • Cytokinesis

  12. Locations/Functions of Actin

  13. Actin • Building block proteins of microfilaments: • α-actin: associated with contractile structures • γ-actin: in stress fibers • β-actin: in cell cortex and leading edge 13

  14. Structure of Actin • Actin exists in two forms: • G-actin • F-actin 15

  15. F-actin • F-actin is a linear chain of G-actinsubunits

  16. Actin polarity • (+) end is where monomers are readily added to growing filament • Barbed end • (-) end is favored for dissociation • Pointed end

  17. Microfilament growth from actin-ATP monomers Actin was fixed on a glass slide at (-) end, so we are only seeing (-) end growth 18

  18. F-actin growth occurs in 3 steps Nucleation- This is the rate-limiting step due to lag period Elongation- addition of G-actin to both the (+) and (-) ends until the concentration of G-actin and F-actin equilibrates Steady State- monomer removal and addition are steady 19

  19. Step 1: Nucleation • Long lag period • Come together in short, unstable small polymers

  20. Step 2: Elongation Phase • Rapid increase in filament length on both ends

  21. Step 3: Steady-State Phase • G-actin subunits exchange, but there is no net change in the total mass of filaments

  22. F-actin growth movie • Animation 17.7 in Lodish 22

  23. Actin polymerization • How much G-actin is needed to form filaments? • Critical Concentration: The concentration of G-actin where filaments with form

  24. F-actin growth occurs in 3 steps • In vitro assay for F-actin growth • fixed concentration of G-actin in solution • measured change in filament mass over time • perform with or without nucleation step • What does this tell us about the lag period? 24

  25. Growth of Actin is different at (+) and (-) ends The Cc of the total filament is between the Cc of the (+) and (-) ends Rate of growth at (+) end is ~10X faster, rate of dissociation is ~2X faster at the (-) end ATP hydrolyzes to ADP and Pi ADP-actin has lower affinity than ATP actin Cc(+ end) < 0.12uM Cc(- end) < 0.6uM Treadmilling = Cc(+ end) < [G-actin] < Cc(- end) 28

  26. Treadmilling Movie

  27. Regulation of Actin Polymerization Binding proteins important for dynamic actin changes Profilin Binds ADP-actin and enhances exchange for ATP Binds to proline-rich sequences Cofilin F-actin severing protein Leads to more free (-) end-- enhances disassembly Thymosin beta 4 Sequesters free ATP-actin Acts as a reservoir for ATP-actin 30

  28. Profilin Profilin Binds • Small protein that binds G-actin

  29. Cofilin • Disassembles actinfilaments

  30. Actin monomer sequestering • Excess ATP bound G-actin in the cell • How are they prevented from forming actin filaments?

  31. Actin Binding Proteins

  32. Capping of filaments • Capping of filaments blocks assembly and disassembly • Both (+) and (-) end caps exist • CapZ • Tropomodulin • Gelsolin

  33. Filament assembly • Actin nucleating proteins help initiate the nucleation process • Formin: Helps to form long filaments • Arp2/3: Forms branched filaments

  34. Formin • FH1 and FH2 domains • Form a doughnut like circular complex

  35. Formin • FH1 domain is rich in proline residues • What binds proline rich amino acids?

  36. Formin

  37. Formin

  38. Formin • Activity must also be regulated • G protein-Rho

  39. Arp2/3 • Actin Related Protein • WASp: stands for Wiskott-Aldrich Syndrome protein

  40. Arp2/3

  41. Arp2/3 • WASpregulates Arp2/3

  42. Listeria monocytogenes • Bacterium that causes intestinal disease • Harnesses Arp2/3 and actin to promote pathogenesis • Protein called ActA on bacterial cell surface binds activates the Arp2/3 complex • Also interacts with the intracellular protein VASP that recruits profinin and holds onto the new filament

  43. Tools that help studying actin • CytochalasinD: binds (+) end to prevent further polymerization, promotes depolymerization • Latrunculin: Binds G-actin and prevents filament binding, • cell movements and cytokinesis are inhibited, no new actin assembly • Jasplakinolide: Enhances nucleation by stabilizing actindimers and lowers critical concentration • Phalloidin: Binds between subunits of F-actin and completely prevents depolymerization

  44. Organization of Actin Cell Structures • Crosslinking proteins organize filaments into bundles • Fimbrin • Alpha-actinin

  45. Organization of Actin Cell Structures • Spectrin: Tetramer with two actin binding sites • Filamin: flexible region between binding sites • forms a meshwork of actin filaments • Arp2/3 also facilitates crosslinking

  46. Actin crosslinking

  47. Microvilli • Microfilaments provide support for microvilli • (+) end is at the tip

  48. Actin Motors • Proteins that move along filaments • Powered by ATP • Convert energy released by ATP hydrolysis into physical work

  49. Myosin motor proteins move along actin filaments Myosin Uses energy of ATP hydrolysis to power movements Structure: 3 domains Head motor binds to actin binds to ATP Neck lever arm length helps dictate walk Tail cargo binding vary in different myosins sequence determins cargo 54

  50. Myosin Motors • Class I: Large family, have a variable number of light chains associated with neck region • Class II: Assemble into bipolar filaments involved in contractile function 50

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