THE SPATIAL ORGANIZATION OF CENTROSOME-ATTACHED AND FREE MICROTUBULES IN 3T3 FIBROBLASTS
I. B. Alieva,1 G. G. Borisy,2 I. A. Vorobjev 1
1 A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 2 Department of Cell and Molecular Biology,
Feinberg School of Medicine, and Northwestern University, Chicago, USA;
1 e-mail: irina_alieva@belozersky.msu.ru
Microtubules spatial organization is essential for different cellular processes to proceed normally. It is supposed traditionally, that the fibroblasts have radial
microtubule array consisting of long microtubules running from the centrosome. However, the detailed analysis of the microtubule array in the internal cytoplasm has
never been performed. In the current study we used laser photobleaching for the analysis of the spatial organization of microtubules in the internal cytoplasm of
cultured 3T3 fibroblasts. Cells were injected with Cy-3-labeled tubulin, and then in the bleached zone growth of microtubules in the centrosome region and in the
peripheral parts of cytoplasm was analyzed. In most cases microtubules growth in the bleached zone occurred rectilinearly, on the distance up to 5 μm they
seldom bend more than 10-15°. We considered a growing fragment of the microtubule as a vector with the beginning in the point of occurrence and with the end in a
point where growth terminated (or the end point after 30 s if microtubule's persistent growth proceeded longer). We defined the direction of microtubules growth in
different parts of the cell using these vectors and measured the angle of their deviation from the vector of comparison. In the area of the centrosome we directed the
vector of comparison inside of the bleached zone from the centrosome to the beginning of the growing microtubule segment; in fibroblast lamella and in fibroblast
trailing part we used, the vector of comparison was directed along the long axis of the cell from its geometrical center to periphery. The microtubules growing
immediately from the centrosome grew along the cell radius. However at a distance of 10 μm from the centrosome radially growing microtubules gave 40 %
from the overall number, and at a distance of 20 μm - only 25 %. The rest of microtubules grew in different directions, with the preferred angle between their
growth direction and cell radius around 90°. Fibroblast lamella and trailing part 80 % of all microtubules grew along the cell long axis or at the angle no more than 20°,
and 10-15 % of microtubules grew along cell axis but towards the centrosome. Thus, in 3T3 fibroblasts the radial system of microtubules is perturbed starting from
the distance of several microns from the centrosome. In the internal cytoplasm the microtubule system is completely disordered, and in the stretched parts of the
polarized cell (lamella, trailing edge) the microtubule system again becomes well organized - microtubules are preferentially oriented along the long cell axis. From
the results obtained we conclude that orderliness of microtu-bules at the periphery of the fibroblast is not a consequence of their growth from the centrosome, but
their orientation is preset by local factors.
Key words: centrosome, centrosome-attached microtubules, free microtubules, the microtubule system organization, videomicroscopy
Back
Contents
Main
|
