| When the cell undergoes mitosis, cell cycle regulators affect important changes to microtubules and centrosomes. In order to study two of these changes, I developed an in vivo microtubule end-marking assay that can be used to monitor growing microtubule ends and, consequently, microtubule nucleation. My research has contributed to the understanding of changes in microtubule dynamics and organization at the time of NEB. I have also helped elucidate cell cycle regulated changes in centrosomal nucleation rates.; I have shown, as discussed in Chapter 2, that prophase microtubules no longer grow to the periphery of the cell. Microtubules in prophase cells grow 1.5 times faster than interphase cells, and these prophase growth rates continue at this same rate through metaphase. Prophase cells also have a high density of growing microtubule ends that co-localize with dynein in a ring surrounding the nuclear envelope. I found that injection of these cells with p50/dynamitin to disrupt the dynein/dynactin complex on the nuclear envelope significantly reduced dynein localization to this ring and growing ends appeared to lose attachment to the nuclear envelope.; In Chapter 3,1 show that 4 times more microtubules were nucleated during prophase than in interphase cells. In metaphase, this rate increased to 5 times interphase rates, which was in agreement with classic electron microscopy studies. Post-metaphase nucleation rates have never been measured in any system, thus I am the first to show a continued increase in microtubule nucleation to maximum levels in anaphase and telophase (almost 7 times interphase rates). Remarkably, during cytokinesis, nucleation rates drop to 40% anaphase and telophase rates; but are still high, similar to the prophase rate of 4 times interphase levels. I have also shown that γ-tubulin recruitment to the centrosome, centrosome size, and microtubule nucleation from the centrosome are correlated from G1 through metaphase, but do not correlate from metaphase through G1. |
