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Problem Sets due tomorrow! Office Hours will start at 4:30 next week

Problem Sets due tomorrow! Office Hours will start at 4:30 next week. 1. Cytoskeleton. What is the cytoskeleton? Why do cells need a cytoskeleton?. Cytoskeleton Regulate Cell Shape and Cell Motility. Three Major Classes of Cytoskeletal Proteins. Cytoskeletal Proteins

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Problem Sets due tomorrow! Office Hours will start at 4:30 next week

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  1. Problem Sets due tomorrow! Office Hours will start at 4:30 next week 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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