Microtubule Dynamics at the G2/M Transition: Abrupt Breakdown of Cytoplasmic Microtubules at Nuclear Envelope Breakdown and Implications for Spindle Morphogenesis Ye Zhai, Paul J. Kronebusch, Patrick M. Simon, and Gary G. Borisy Journal of Cell Biology 135: 201-214: 1996

We recently developed a direct fluoroscence 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 diassembled. Our results showed no net decrease in polymer, indication that the diassembly of kinetochore MTs was balanced by assembly of midbody and astral MTs. Thus, the mitosis-interphase transition occurs by a redistribution of tubulin among different classes of MTs at essentially constant polymer level.

We now examine the reverse process, the interphase-mitosis transition. Specifcally we qantitated both the level of MT polymer and the dynamics of MTs during the G2/M transition using the fluorescence ratio assay and a fluorescence photoactivation approach, respectively. Prophase cells before nuclear envelope breakown (NEB) had high levels of MT polymer (62%) similar to that previously reported for random interphase populations (68%). However, prophase cells just after NEB had significantly reduced levels (23%) which recovered as MT attachments to chromosomes were made (prometaphase, 46%; metaphase, 56%). The abrupt reorganization of MTs and NEB was corroborated by anti-tubulin immunoflourescence staining using a variety of fixation protocols. Sensitivity to nocodazole also increased at NEB. Photoactivation analyses of MT dynamics showed a similar abrupt change at NEB, basal rates of MT turnover (pre-NEB) increased post-NEB and then became slower later in mitosis.

Our results indicate that the interphase-mitosis (G2/M) transition of the MT array does not occur by a simple reditribution of tubulin at constant polymer level as the mitosis-interpase (M/G1) transition. Rather, an abrupt decrease in MT polymer level and increase in MT dynamics occurs tightly correlated with NEB. A subsequent increase in MT polymer level and decrease in MT dynamics occurs correlated with chromosome attachment. These results carry implications for understanding spindle morphogenesis. They indicate that changes in MT dynamics may cause the steady-state MT polymer level in mitotic cells to be lower than in interphase. We propose that tension extered on the kMTs (kinetochore MT) may lead to their lengthening and thereby lead to an increase in the MT polymer level as chromosomes attach to the spindle.