Rate Of Tubulin Polymerization Research Articles

Experimental manipulation of the amount of tubulin available for assembly into the spindle of dividing sea urchin eggs.

Spindle assembly is studied in the eggs of the sea urchin Lytechinus variegatus by experimentally varying the amount of polymerizable tubulin within the egg. Aliquots of fertilized eggs from the same female are individually pulsed for 1-6 min with 1 X 10(-6) M Colcemid at least 20 min before first nuclear envelope breakdown. This treatment inactivates a portion of the cellular tubulin before the spindle is formed. Upon entering mitosis, treated eggs form functional spindles that are reduced in length and birefringent retardation but not width. With increased exposure to Colcemid, the length and retardation of the metaphase spindles are progressively reduced. Similar results are obtained by pulsing the eggs with Colcemid before fertilization, which demonstrates that the tubulin found in unfertilized sea urchin eggs is later used in spindle formation. Spindles, once assembled, are responsive to increases in the amount of polymerizable tubulin within the cell. Rapid increases in the amount of polymerizable tubulin within a Colcemid-treated cell can be experimentally effected by irradiating the cells with 366-nm light. This treatment photochemically inactivates the Colcemid, thereby freeing the tubulin to polymerize. Upon irradiation, the small prometaphase spindles of Colcemid-treated cells immediately increase in length and retardation. In these irradiated cells, spindle length and retardation increase as much as four times faster than they do during prometaphase for normal spindles. This suggests that the rate of the normal prometaphase increase in retardation and spindle size may be determined by factors other than the maximum rate of tubulin polymerization in the cell.

Interaction of nerve growth factor with tubulin. Studies on binding and induced polymerization

The interaction of the nerve growth factor with the neurotubule protein has been studied with the aim of elucidating the nature of the large complexes that they form when incubated together and the factors that control this event. The results show that the binding of nerve growth factor to tubulin is followed by the formation of large structures that, in certain experimental conditions, accelerate the rate of tubulin polymerization to form microtubules or catalyze their assembly in conditions where this process does not occur spontaneously. The formation of large nerve growth factor-tubulin complexes starts to occur only at a molar ratio of 1.0–1.5 NaCl or GTP strongly inhibit this process without a detectable effect on NGF binding. Two hypotheses are postulated to explain these finding. Firstly, that tubulin has two sites with different affinity for nerve growth factor and the polymerization occurs only when the second NGF molecule has interacted with the microtubule protein. Alternatively, free tubulin in solution is the limiting factor of the polymerization by hindering a site of tubulin-factor complexes present in solutio at a 1 : 1 molar ratio. In both cases, GTP, Na + or H + will affect the formation of large unsoluble, tubulin-NGF complexes, by changing their conformation or by decreasing electrostatic interactions.