THE DYNAMICS OF MICROTUBULE REPOLYMERIZATION IN VIVO: RAPID GROWTH FROM THE CENTROSOME
AND SLOW RECOVERY OF FREE MICROTUBULES
O. A. Chernobelskaya,1 I. B. Alieva,1 I. A. Vorobjev2
1 A. N. Belozcrsky Institute of Physico-Chemical Biology, Moscow State University,
and 2 Biology Faculty, Moscow State University, Moscow, Russia;
1 e-mail: olga_chern99@mail.ru
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 °C, for 4 h; recovery at 30 °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 μg/ml, 2 h; recovery in drug-free medium at 37 °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.
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