CYTOPLASMIC ASSEMBLY OF MICROTUBULES IN CULTURED-CELLS

The origin of non-centrosomal microtubules was investigated in a varie ty of animal cells in culture by means of time-lapse digital fluoresce nce microscopy, A previous study (Keating et al, (1997) Proc. Nat, Aca d, Sci, USA 94, 5078-5083) demonstrated a pathway for formation of non -centrosomal microtubules by release from the centrosome, Here we show a parallel pathway not dependent upon the centrosome, Correlative imm unostaining with anti-tubulin antibodies and electron microscopy estab lished that apparent free microtubules observed in vivo were not growi ng ends of long stable microtubules. Free microtubules appeared sponta neously in the cytoplasm and occasionally by breakage of long microtub ules. Estimates of the frequencies of free microtubule formation sugge st that it can be a relatively common rather than exceptional event in PtK1 cells and may represent a significant source of non-centrosomal microtubules. The observation of free microtubules permitted analysis of the microtubule minus end. Unlike the plus end which showed dynamic instability, the minus end was stable or depolymerized. Breakage of l ong microtubules generated nascent plus and minus ends; the nascent mi nus end was generally stable while the plus end was always dynamic, Th e stability of microtubule minus ends in vivo apparently provides the necessary condition for free microtubule formation in the cytoplasm. P arameters of the dynamic instability of plus ends of free microtubules were similar to those for the distal ends of long microtubules, indic ating that the free microtubules were not exceptional in their dynamic behavior. Random walk analysis of microtubule end dynamics gave appar ent diffusion coefficients for free and long microtubules which permit ted an estimate of turnover half-times, The results support the concep t that, in PtK1 cells, a pathway other than plus end dynamics is neede d to account for the rapidity of microtubule turnover.