STEPWISE RECONSTITUTION OF INTERPHASE MICROTUBULE DYNAMICS IN PERMEABILIZED CELLS AND COMPARISON TO DYNAMIC MECHANISMS IN INTACT-CELLS

Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using thi s model system we have established conditions for stepwise reconstitut ion of microtubule dynamics in permeabilized interphase cells when sup plemented with various cell extracts. When permeabilized cells are sup plemented with mammalian cell extracts in the presence of protein phos phatase inhibitors, microtubules become sensitive to nocodazole. Depol ymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensi tive microtubules do not exhibit subunit turnover. By contrast, when p ermeabilized cells are supplemented with Xenopus egg extracts, microtu bules actively turn over. This involves continuous creation of free mi crotubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. WI: provide evidence that similar microtubule fragmentation and minus end-directed disassembly occur at the whole-cell level in intact cells . These data suggest that microtubule dynamics resembling dynamics obs erved in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data su pport recent work suggesting that microtubule tread-milling is an impo rtant mechanism of microtubule turnover.