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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