The role of microtubules during cytokinesis and other post-anaphase events in PTK1 cells.

摘要

Cytokinesis is the physical division of one cell into two daughter cells. The regulation of cytokinesis is not well understood. It is known, however, that microtubules play an important role in this process. Specifically it is thought that microtubules in the midzone complex, a bipolar anti-parallel array of microtubules that forms at the cell equator at the site of furrow ingression, are responsible for furrow positioning. In this dissertation, I will focus on the role of microtubules in cytokinesis as well as in other post-anaphase events in mammalian cultured cells.; It is known that cytokinesis can only occur during a discrete period of the cell cycle. In Chapter II, I characterize this stage and define it as the cytokinetic phase of the cell cycle, or C-phase. I also provide evidence that microtubules are not essential for C-phase. Finally, I show that midzone complex microtubules can form de novo and without the previous existence of a pre-anaphase spindle.; The spindle checkpoint monitors kinetochore/microtubule attachment and prevents anaphase onset until all chromosomes form bipolar attachments. Therefore, functional disruption of key mitotic proteins results in a cell cycle arrest prior to anaphase and cytokinesis. As many proteins that are thought to play a role in cytokinesis (such as tubulin) also play an important role in mitosis, it is important to be able to overcome the spindle checkpoint and allow cells to progress through anaphase in order to determine the function of these proteins during cytokinesis. In Chapter III, I review the current methods for inducing precocious anaphase in mammalian cells. I also introduce two novel ways to induce anaphase based on dominant negative spindle checkpoint component constructs.; During mitosis, many kinetochore/centromere binding proteins are removed from the chromosomes before or just after anaphase onset. In Chapter IV, I show that microtubules, and not just a change in the cell cycle environment, are required for a significant reduction at kinetochores in anaphase of spindle checkpoint proteins Mad2 and BubRl, the 3F3/2 phosphoepitope, the motor protein cytoplasmic dynein, and the centromeric protein INCENP.; As stated above, microtubules are essential for cytokinesis. However, due to technical difficulties the organization of microtubules during cytokinesis has not been well characterized. In Chapter V, I overcome the previous technical difficulties and provide a high-resolution description of the organization of microtubules within the mammalian midzone complex that are likely to position the cytokinetic furrow. I also show that differential microtubule stability may play a role in furrow positioning. Finally, I also show that microtubule plus ends interact with the cell cortex in the region of the cytokinetic furrow.; The normal bipolarity of the midzone complex is thought to be important for cytokinesis. However, the role of bipolarity has not been well tested in mammalian cells. In chapter VI, I show that, in fact, cytokinesis can occur in cells with a monopolar spindle. Furthermore, I provide evidence that the furrows in cells with monopolar spindle are associated with a bipolar midbody.