Investigation in Microtubule Dynamic Instability

Title: Investigation in microtubule dynamic instability Introduction Microtubules are important for maintaining cell structure, intracellular transport, formation of mitotic spindle, as well as other cellular processes. Investigation of dynamics of microtubule assembly and disassembly allow us to understand the malfunction of mitotic spindle formation or other cellular processes. This experiment is divided into two parts; we are going to find out the critical parameters for achieving greatest average length of microtubules in part one and achieving the greatest number of microtubules in part two.Principle In this experiment, we used a simulation programme to explore how various factors change the way microtubules grow out from centrosome, and the shrink back. Growth rate, shrink rate, catastrophe rate, rescue rate, release rate, minus end end depolymerization rate, nucleation rate and nucleation site are the factors we can adjust to see how them affects the average length and number of microtubules. The simulation time acceleration is set to 5x real time. Each time a parameter is varied and others are controlled factors.The record is taken when the simulation has reached steady state and graphs are plotted. Results Part1 – How to achieving greatest average length of microtubules Fixed parameter| Shrink rate| Catastrope| Rescue Release| MED| Nuc rate| Nuc sites| Variable Growth rate| 0. 263| 0. 042| 0. 064 0. 024| 0. 8| 0. 02| 180| Result| 1| 2| 3| 4| 5| Mean| 0. 14| 32. 9| 21. 12| 23. 93| 23. 95| 27. 54| 25. 888| 0. 16| 33. 19| 36. 82| 32. 5| 28. 83| 30. 15| 32. 298| 0. 18| 29. 79| 39. 11| 41. 19| 40. 8| 31. 54| 36. 486| 0. 2| 40. 77| 41. 19| 45. 94| 38. 28| 47. 66| 42. 768| 0. 22| 38. 6| 47. 49| 48. 53| 48. 55| 47. 96| 46. 238| 0. 24| 42. 25| 45. 31| 45. 25| 46. 81| 40. 95| 44. 114| Table1 Figure1 Fixed parameter| Growth rate| Shrink rate| Catastrop/ Release| MED| Nuc rate| Nuc cites| Variable Rescue| 0. 12| 0. 263| 0. 042 0. 024| 0. 8| 0. 02| 180| Resu lt| 1| 2| 3| 4| 5| mean| 0. 084| 23. 76| 22. 77| 26. 56| 30. 78| 25. 12| 25. 798| 0. 104| 18. 88| 19. 07| 17. 82| 20. 08| 17. 55| 18. 68| 0. 124| 19. 96| 16. 69| 17. 37| 19. 37| 22. 38| 19. 154| 0. 144| 21. 34| 19. 53| 20. 54| 21. 44| 21. 95| 20. 96| 0. 164| 20. 65| 18. 76| 21. 76| 16. 33| 19. 73| 19. 446|Table2 Figure 2 Discussion Each free tubulin dimer contains one tightly bound GTP molecule that is hydrolyzed to GDP after the subunit is added to a growing microtubules. When polymerization is proceeding rapidly, tubulin molecules add to the end of the microtubule faster that the GTP they carry is hydrolyzed, and the microtubule growth. [1] Varied the growth rate and kept other factors constant, the average length of microtubules should always increase. However, the average length of microtubules rises as growth rate increase from 0. 14 to 0. 22Â µm/sec and stop increasing at 0. 2Â µm/sec. It tends to level off rather than increase at 0. 22Â µm/sec. It means the growth rate is no longer the limiting factor. Some factors other than growth rate, may be the rescue rate, limited the increase of the average length. Rescue rate is the rate at which a shrinking microtubule switches to growing state. We assume the greatest rescue rate, the more the microtubules undergo polymerization. So that the proportion of growing microtubules would increase and the average length rise. Instead of increase, the average length of microtubules drops from 0. 084 to 0. 104Â µm/sec.Increase the rescue rate may trigger the mechanism that lowers the average length of microtubules. It remains at around 20Â µm from 0. 104 to 0. 164Â µm/sec means that that there is no correlation between rescue rate and the average length beyond a point among 0. 084 and 0. 104Â µm/sec. Part2 – How to achieve the greatest number of microtubules Fixed parameter| Growth rate| Catastrop| Rescue Release| MED| Nuc rate| Shrink rate| Variable #nuc site| 0. 12| 0. 042| 0. 064 0. 024| 0. 8| 0. 02| 0. 263| Result| 1| 2| 3| 4| 5| mean| 180| 47| 65| 42| 57| 68| 55. 8| 200| 70| 77| 66| 53| 68| 66. | 220| 71| 73| 86| 70| 68| 73. 6| 240| 82| 88| 85| 81| 84| 84| 260| 90| 93| 80| 81| 84| 85. 6| 280| 87| 107| 100| 97| 91| 96. 4| 300| 90| 101| 110| 92| 96| 97. 8| Figure3 Fixed parameter| Growth rate| Shrink rate| Catastrop| Rescue Release| MED| Nuc cites| Variable nuc rate| 0. 12| 0. 263| 0. 042| 0. 064 0. 024| 0. 8| 180| Result| 1| 2| 3| 4| 5| mean| 0. 02| 62| 57| 49| 54| 50| 54. 4| 0. 04| 95| 107| 85| 80| 86| 90. 6| 0. 06| 103| 110| 107| 113| 114| 109. 4| 0. 08| 120| 99| 112| 113| 115| 111. 8| 0. 1| 124| 134| 126| 116| 113| 122. 6| 0. 12| 120| 131| 130| 119| 136| 127. | 0. 14| 136| 128| 127| 130| 136| 131. 4| Table4 Figure4 Discussion Centrosomes contain ring-shaped structures formed from ? -tubulin, and each ? -tubulin ring serves as the starting point, the nucleation site, for the growth of one microtubule. The nucleation site acts as a preexisting microtubule structure for -tubulin dimers assembly. [1] We assume the more the nucleation site, the more the microtubules present. According to table3, the number of microtubules is always increasing with the number of nucleation site. There is no sign of level off or decline of the curve.It always is the limiting factor of the number of microtubules. The nucleation rate is the rate at which new microtubules are nucleated at the centrosome. The number of microtubules should be raised if the nucleation rate increase since new microtubules generated. Indeed, the number of microtubules is raised as the nucleation rate increased. From 0. 02 to 0. 06Â µm/sec, the increase of microtubules is sharp and starts to slow down afterward. The trend shows that the curve would level off at certain level eventually. It means there are some factors other than nucleation rate control the number of microtubules.The number of nucleation site may be the limiting factor as all nucleation sites are occupied by the microtubules, so that n o new microtubules generated. Limitations In actual cell, the number of tubulin dimer is limited. This factor is not shown in this simulation programme. The temperature and the pH may affect the configuration and polymerization of the microtubules. There are some microtubules not attached to the centrosome, but present in cilia and flagella. It is not clearly stated by the simulation programme whether these microtubules is counted. ConclusionsBesides the growth rate, there are other limiting factors controlling the average length of microtubules. We cannot achieve the greast average length of microtubules by consider growth rate is the only factor. We found that we should keep the rescue rate at 0. 084Â µm/sec or below. Also, more information about the rescue rate below 0. 084Â µm/sec should be obtained. Both nucleation site and nucleation rate are the factors controlling the number of microtubules. But the nucleation site is more critical than the nucleation site. The above show the nucleation rate is restricted by other factors but the nucleation sites does not.We should examine another set of data by varying the nucleation rate with more nucleation site. If the plateau of new obtain curve is above the original curve, nucleation site is limiting factor of the number of microtubules. Similar experiment should be established with different combination of parameters in order to obtain the best curve. In short, there is not enough information for us to draw conclusion for how to achieve the greatest average length and greatest number of microtubules unless we obtain more data. Reference 1. Alberts et al,. (2010) Essential Cell Biology, 3rd Garland Science, p. 579-580