Requirement for ‘Factor(s)’ for Tubulin Assembly during Brain Development

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Abstract
The polymerisation of neurotubulin to neurotubules in vitro was studied using brain supernatants from rats of different ages (from the 15th day of gestation till 30 days post‐partum). When the concentration of tubulin was kept low the initial rates of polymerisation of both fetal and new born material were markedly lower than that of the adult. Both the critical concentration and the lag period were, however, increased. When higher tubulin concentrations were used the initial rates of the young stages ressembled those of the adult (3 days after birth) or were even higher (9 days after birth). These differences in the relationship between the initial assembly velocities and tubulin concentration were interpreted as resulting either from the presence of an inhibitor of microtubule assembly in the young brain or the fact that a factor is required for the formation of nucleating centers and for tubulin polymerisation, which may be limiting in early developmental stages. The first possibility has been disproved by mixing small amounts of fetal supernatants with adult preparations; no inhibition was found under these conditions. Support for the second possibility has been gained by adding small quantities of adult supernatant to young brain preparations. Clear stimulation of the initial rates of polymerisation and overall turbidity was found. These results suggest that a factor (and therefore the number of nucleating centers) is in excess in the adult and limiting in the young brain. Further experiments were performed to better characterize the limiting entity(ies). Additions of sonicated microtubule fragments, of isolated microtubule associated proteins and of purified τ protein, all produced a clear increase in assembly rate and a decreased lag period. These results suggest that the τ protein plays an important role in promoting the initiation and the elongation of the microtubule. The overall turbidity, however, was never completely restored. Moreover, the more the ‘factor’ were purified the more the overall polymerisation was diminshed, suggesting that another factor involved in the elongation process is probably present in the brain but is eliminated during the purification of the τ protein. In conclusion these results demonstrate that tubulin alone is unable to assemble into microtubules in the absence of one (or several) factors which are limiting in the early stages of brain development.