Micromanipulation

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Micromanipulation

  1. 1. Micromanipulation Studies of Chromatin Fibers in Xenopus Egg Extracts Reveal ATP-dependent Chromatin Assembly Dynamics<br />Presented To:<br /> Ms. NaziaJamil<br />Presented By:<br />MazharHussain<br /> MMG-07-37<br /> BS-6<br />Microbiology & Molecular Genetics<br />
  2. 2. Micromanipulation Studies of Chromatin Fibers in Xenopus Egg Extracts Reveal ATP-dependent Chromatin Assembly Dynamics<br />Jie Yan<br />Thomas J. Maresca<br />DunjaSkoko<br />Christian D. Adams<br />Botao Xiao<br />Morten O. Christensen<br />Rebecca Heald<br />John F. Marko<br />
  3. 3. Introduction<br />
  4. 4. What is Chromatin?<br />Chromatin is DNA molecule which is associated with different types of proteins…<br />Provides basic structure for chromosome formation<br />Histone is one of those proteins.<br />
  5. 5. Nucleosome<br />Consist of 160-180 nucleotides wound up on octamer of histone proteins…<br />H2A, H2B, H3, H4<br />Nucleotides make 2 left hand turns on core proteins…<br />
  6. 6. Advantages of Biophysical Experiments<br />Chromatin dynamics can be visulised by this appreach under light microscope<br />Dynamics include : Assembly and Disassembly reactions<br />Reveal details about structures<br />Details about procedures like chromatin remodeling, mitotic chromosome condensation.<br />
  7. 7. ΔG for Nucleosome Assembly<br />ΔG is free energy for nucleosome formation<br />Its value measured to be 3.5pN<br />This is the force which is required to stall assembly and disassembly for nucleosome formation.<br />ΔG is counted as equilibrium point<br />
  8. 8. Why Xenopus?<br />Containing Enzymes which help in chromatin assembly in vitro conditions.<br />Competent in many physiological condtions<br />
  9. 9. Materials & Methods<br />
  10. 10. Single DNA Tethering<br />Paramagnetic Bead (Streptoviridin)<br />Dioxygen Labeled Plastic Bead<br />Glass Slide<br />DNA<br />Biotin Labeled<br />
  11. 11. Interphase Xenopus Egg Extracts<br />1<br />2<br />Activated Eggs <br />55000rpm for 2h at 4⁰C<br />Cyclohexamide 50 µg/ml<br />10200rpm 15mins<br />HB-6<br />3. Middle Layer Extracted Using 18 Gauge Niddle<br />4. Again Centrifuged at 55000rpm for 30mins at 4 ⁰C<br />5. Aliquoted Immediately, Flash Frozen at -80 ⁰C.<br />
  12. 12. Real Experiments<br />
  13. 13. Micrococcal Nuclease Gel Assay for Nucleosome Assembly by Extracts<br /><ul><li>Assembly reactions were run in extracts diluted 1:4 in HEPES buffer
  14. 14. With energy mix 5µl
  15. 15. Without Energy Mix</li></ul>Performed to check Viability of Extracts to Perform Assembly in vitro conditions<br />
  16. 16. Results<br />
  17. 17. <ul><li>Digital Video Microscopy
  18. 18. Bead Tracking System
  19. 19. Focusing Microscope to Single DNA molecule</li></ul>MT Setups Allow Precise Observation and Control of Chromatin Assembly Under Force Control<br />
  20. 20. Magnetic Tweezers Employed<br />
  21. 21. ATP-depleted Extracts Assemble Chromatin Onto DNA in Two Phases Below 1.2pN<br />Exponential Condensation Phase<br />Slow Condensation<br />
  22. 22. Extension Reduction Occur at Same Rate, No Matter What is Size of Molecule…<br />Graph Shows Same Pattern For Different Molecules<br />
  23. 23. 4pN- A Dead-Line for Assembly… Disassembly Starts<br />If force increase beyond this limit, chromatin molecule disassemble and DNA molecule show disassembly<br />
  24. 24. Bit Confused Here…<br />What is the Thing , I am Searching For?<br />What is Key to Understand Chromatin Dynamics?<br />
  25. 25. Quest for Force at Which Rate of Assembly stand Equivalent to Rate of Disassembly<br /><ul><li>1pN…
  26. 26. 2.8pN…
  27. 27. 3.5pN…
  28. 28. 4.5pN…
  29. 29. 15pN…</li></ul>Cannot Be Assembly Reaction is There…<br />
  30. 30. Quest for Force at Which Rate of Assembly stand Equivalent to Rate of Disassembly<br /><ul><li>1pN…
  31. 31. 2.8pN…
  32. 32. 3.5pN…
  33. 33. 4.5pN…
  34. 34. 15pN…</li></ul>Figure a showed that reaction is in equilibrium… But it was’nt… Because Using Fresh Extracts again showed Assembly…<br />
  35. 35. Quest for Force at Which Rate of Assembly stand Equivalent to Rate of Disassembly<br /><ul><li>1pN…
  36. 36. 2.8pN…
  37. 37. 3.5pN…
  38. 38. 4.5pN…
  39. 39. 15pN…</li></ul>When Force Increased to 3.5pN… Ideal Equilibrium Maintained…<br />
  40. 40. Quest for Force at Which Rate of Assembly stand Equivalent to Rate of Disassembly<br /><ul><li>1pN…
  41. 41. 2.8pN…
  42. 42. 3.5pN…
  43. 43. 4.5pN…
  44. 44. 15pN…</li></ul>Extension in DNA fiber started… Equilibrium Passed…<br />
  45. 45. Quest for Force at Which Rate of Assembly stand Equivalent to Rate of Disassembly<br /><ul><li>1pN…
  46. 46. 2.8pN…
  47. 47. 3.5pN…
  48. 48. 4.5pN…
  49. 49. 15pN…</li></ul>Now Rapid Expension Starts… Very Sharp Plateau of Extension Observed where Physical Noise is Minimum…<br />
  50. 50. Quest for Force at Which Rate of Assembly stand Equivalent to Rate of Disassembly<br /><ul><li>1pN…
  51. 51. 2.8pN…
  52. 52. 3.5pN…
  53. 53. 4.5pN…
  54. 54. 15pN…</li></ul>3.5pN<br />Was the equilibrium point for ATP less Extracts…<br />
  55. 55. 180 Steps Observed<br />50nm<br />25nM<br />75nm<br />
  56. 56. Experiment Containing ATP<br />In graph (a), pattern was quite linear and Gradual Contraction of DNA molecule was observed…<br />Graph (a) was further resolved in graph (b), showing very dynamic ups and downs in the presence of ATP…<br />
  57. 57. Experiment Containing ATP<br />ATP less Extract<br />But Still ATP containing extract show linear attribute… Not exponential as shown in ATP less Extract graph…<br />
  58. 58. Addition of ATP Caused Large V and ^ Steps<br />
  59. 59. DISCUSSION<br />
  60. 60. 1. Chromatin Assembles onto Naked DNA in Cytoplasmic Extracts in the Absence of ATP<br />Evidence:<br />Every ATP depleted reaction tend to go for DNA assembly below 3.5pN force… Never Extension Appeared without force…<br />2. Are Nucleosomes Present in the Assembled Fibers?<br />Evidence:<br />Nucleosomes were there because extension steps observed were exactly 50nm and total number of steps were equal to total number of Nucleosomes estimated…<br />
  61. 61. 3. Initial Assembly of Nucleosomes onto DNA by ATP-depleted Reaction Is Not Processive<br />Evidence:<br />Assembly of Nucleosome occur in random manner. Assembly and disassembly procedures continue side by side. <br />4. Stall Force of Assembly Reaction Indicates Nucleosome<br />Assembly Free Energy<br />5. Nucleosome Disassembly require slightly larger force than 3.5pN. This force is much less then previously reported force.<br />
  62. 62. “It was not only a revenge from those who made me bore by their presentations, but also a humble return for the knowledge, they poured on me.”<br />NeverAsk.com<br />

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