MICROTUBULE DYNAMICS AT THE G(2) M TRANSITION - ABRUPT BREAKDOWN OF CYTOPLASMIC MICROTUBULES AT NUCLEAR-ENVELOPE BREAKDOWN AND IMPLICATIONSFOR SPINDLE MORPHOGENESIS/

We recently developed a direct fluorescence ratio assay (Zhai, Y., and G.G. Borisy. 1994. J. Cell Sci. 107:881-890) to quantify microtubule (MT) polymer in order to determine if net MT depolymerization occurred upon anaphase onset as the spindle was disassembled. Our results show ed no net decrease in polymer, indicating that the disassembly of kine tochore MTs was balanced by assembly of midbody and astral MTs. Thus, the mitosis-interphase transition occurs by a redistribution of tubuli n among different classes of MTs at essentially constant polymer level . We now examine the reverse process, the interphase-mitosis transitio n. Specifically, we quantitated both the level of MT polymer and the d ynamics of MTs during the G(2)/M transition using the fluorescence rat io assay and a fluorescence photoactivation approach, respectively. Pr ophase cells before nuclear envelope breakdown (NEB) had high levels o f MT polymer (62%) similar to that previously reported for random inte rphase populations (68%). However, prophase cells just after NEB had s ignificantly reduced levels (23%) which recovered as MT attachments to chromosomes were made (prometaphase, 47%; metaphase, 56%). The abrupt reorganization of MTs at NEB was corroborated by anti-tubulin immunof luorescence staining using a variety of fixation protocols. Sensitivit y to nocodazole also increased at NEB. Photoactivation analyses of MT dynamics showed a similar abrupt change at NEB, basal rates of MT turn over (pre-NEB) increased post-NEB and then became slower later in mito sis. Our results indicate that the interphase-mitosis (G(2)/M) transit ion of the MT array does not occur by a simple redistribution of tubul in at constant polymer level as the mitosis-interphase (M/G(1)) transi tion. Rather, an abrupt decrease in MT polymer level and increase in M T dynamics occurs tightly correlated with NEB. A subsequent increase i n MT polymer level and decrease in MT dynamics occurs correlated with chromosome attachment. These results carry implications for understand ing spindle morphogenesis. They indicate that changes in MT dynamics m ay cause the steady-state MT polymer level in mitotic cells to be lowe r than in interphase. We propose that tension exerted on the kMTs may lead to their lengthening and thereby lead to an increase in the MT po lymer level as chromosomes attach to the spindle.