Multiple sites for the initiation of microtubule assembly in mammalian cells

The pattern of microtubule regrowth in mammalian fibroblast and epithelial cells has been examined by immunofluorescence of cytoskeletal preparations with antibody to tubulin. After reversal of treatment with colcemid, vinblastine or low temperature, microtubules appear to grow simultaneously from several distinct initiation sites located within 5 microns of the nucleus of mouse and human fibroblasts. Each site initiates the growth of 10-30 microtubules. More than 70% of the mouse fibroblasts have between 5 and 10 initiation sites with an average of 8. The human fibroblasts have an average of 5 sites per cell. The average number and numerical distribution of sites per fibroblast cell are not affected by time of exposure to colcemid or the concentration of colcemid applied to the cells. Multiple microtubule initiation sites are also observed during the process of microtubule depolymerization. In addition to growth from these complex initiation sites, microtubules appear to grow singly from the perinuclear region of human fibroblasts. The regrowth of individual microtubules from the perinuclear growth is especially prominent in epithelial cell lines from rat kangaroo and pig. These epithelial lines have only a single complex initiation site per cell. Two classes of complex initiation sites can be distinguished in microtubule regrowth experiments in human and mouse fibroblasts after exposure to griseofulvin. Microtubules first grow extensively from a single distinct site, which has approximately 20 microtubules growing from it and may be the centriole or centriolar pair. Subsequently, microtubules regrow from other perinuclear complex initiation sites. It thus appears that at least three distinct classes of initiation sites can be observed in mammalian cells: primary sites, which regrow microtubules first after griseofulvin treatment; secondary sites, which are distinct perinuclear sites and recover from griseofulvin treatment more slowly than the primary sites; and tertiary sites or sites of growth of single microtubules, also located near the cell nucleus.