Microtubule Dynamics in Mitotic Spindles of Living Cells a

Observations on living, dividing cells,’” together with more recent experiments with microtubules (MTs) assembled in vitro (for review see reference 4), suggest that MTs are dynamic structures and that an understanding of this dynamic behavior is critical for an understanding of mitosis. Early work on living cells clearly demonstrated the labile equilibrium between the MT-containing spindle fibers and a cellular pool of tubulin subunits. In addition, a role for this assembly and disassembly in chromosome movement was These studies showed that the assembly of spindle MTs and their subsequent disassembly are intimately linked to the mitotic process. The pathways and regulation of tubulin association and dissociation with spindle MTs, however, were not resolved. A variety of morphological studies, performed using immunofluorescence and electron microscopy, have revealed that spindles are composed, in part, of a bipolar arrangement of MTs that radiate from the spindle poles and overlap at the metaphase plate.612 Such techniques, however, yield only static images of fixed stained cells, so the contribution of MTs to chromosome movement has remained unclear. In an effort to investigate spindle M T dynamics, we have developed techniques to introduce fluorochrome-labeled tubulin subunits into living, dividing cells to monitor tubulin behavior. This work has been carried out in collaboration with the laboratory of J . R. McIntosh (this volume p. 566). Using this approach, termed fluorescence analogue cytochemistry,” the distribution and dynamics of the tubulin subunit can be investigated in living cells, avoiding the complexities of in vitro conditions. Similar methods were initially used by Keith et a1.,14 who observed the dynamic behavior of the interphase M T array in cultured cells. Subsequently, Wadsworth and Sloboda” demonstrated the rapid polymerization of fluorescent tubulin into M T arrays in sea urchin eggs during mitosis. These preliminary studies suggested the utility of these techniques to examine the behavior of tubulin in living cells. For our experiments, bovine brain tubulin has been covalently modified with the fluorochrome 5-(4,6-dichlorotriazin-2-yl) amino fluorescein (DTAF) and the resulting DTAF tubulin shown to retain the native characteristics of the tubulin molecule. (Details of the labeling procedures can be found in reference 16 and J. R. McIntosh, this volume p. 566). Following microinjection into suitable cells, the DTAF-tubulin has been shown to coassemble with all cytoplasmic M T containing structures in living cells.”.18 Thus the exogenous labeled subunits serve as “tracers” of the behavior of the endogenous tubulin pool. In initial experiments, mitotic cells were microinjected with DTAF tubulin, and