[The dynamics of microtubule repolymerization in a cell: rapid growth from the centrosome and slow recovery of free microtubules].

According to the current view, the microtubule system in animal cells consists of two components: microtubules attached to the centrosome (these microtubules stretch radially towards the cell margin), and free microtubules randomly distributed in the cytoplasm without visible association with any microtubule-organizing centers. The ratio of the two sets of microtubules in the whole microtubule array is under discussion. Addressing this question, we have analysed the recovery of microtubules in cultured Vero nucleated cells and cytoplasts, with and without centrosomes in these. Cells were fixed at different time points, and individual microtubules were traced on serial optical sections. During a slow recovery after cold treatment (4 degrees C, for 4 h; recovery at 30 degrees C) polymerization of microtubules started mainly from the centrosome. At early stages of recovery the share of free microtubules made about 10% of all microtubules, and their total length increased slower than the lenght of centrosome-attached microtubules. During a rapid recovery after nocodazole treatment (10 microg/ml, 2 h; recovery in drug-free medium at 37 degrees C), the share of free microtubules was about 35%, but their total length increased slower than the length of centrosome-attached microtubules. In 6-8 min (rapid recovery) or 12-16 min (slow recovery), tips of centrosomal microtubules reached the cell margin, and their increased density made it impossible to recognize individual microtubules. However, under the same conditions in cytoplasts without centrosomes the normal number of microtubules recovered only in 60 min, which enabled us to suppose that the complete recovery of microtubule system in the whole cells may be also rather long. When the first centrosomal microtubules reached the cell margin, the optical density of microtubules started to decrease from the centrosome region towards the cell margin, according to the exponential curve. Later on, the optical density in the centrosome region and near the cell margin remained at the same level, but microtubule density increased in the middle part of the cell, and in 45-60 min the plot of the optical density vs the distance from the centrosome became linear, as in control cells. Since no significant curling of microtubules occurs near the cell margin, the density of microtubules in the endoplasm may increase due only to polymerization of free microtubules. We suppose that in cultured cells the microtubule network recovery proceeds in two stages. At the initial stage, a rapid growth of centrosomal microtubules takes place in addition to the turnover of free microtubules with unstable minus ends. At the second stage, when microtubule growth from the centrosome becomes limited by the cell margin, a gradual extension of free microtubules occurs in the internal cytoplasm.