Molecular control of bipolar spindle assembly

Abstract

Cell division has long been used as a therapeutic target for cancer treatment. Many agents that are clinically beneficial in the treatment of cancer block chromosome segregation during cell division and thereby inhibit the ability of cancer cells to divide. Thus, a better understanding of cell division and knowledge of all the proteins involved in cell division will hopefully allow the identification of novel therapeutic targets in the treatment of cancer. During cell division, duplicated sister chromatids must attach to microtubules coming from opposite spindle poles in order to be segregated equally over the two daughter cells during anaphase. For successful biorientation of kinetochores to occur, the mitotic spindle must form a bipolar structure. In addition, individual kinetochores must attach to microtubules very tightly to maintain attached for many minutes under continuous movement of microtubules and chromosomes, but at the same time these attachments must be very dynamic to allow for correction of erroneous attachments and turnover of individual microtubules in a kinetochore-microtubule bundle. In this thesis, the molecular mechanisms underlying kinetochore-microtubule attachment and bipolar spindle assembly are addressed. Using a combination of candidate and unbiased RNAi screens, several novel proteins are identified that are involved in the formation of kinetochore-microtubule attachments. Mechanistically, these proteins appear to have distinct functions in the process of kinetochore-microtubule attachment. While CLIP-170 is likely involved in the initial capture of microtubules at kinetochores, RAMA1 (also known as Ska3) is likely involved in the stable interaction between microtubules and kinetochores. Finally, GAK promotes the formation of new microtubules in the vicinity of kinetochores, which facilitates the process of kinetochore-microtubule attachment. The kinesin-5 motor Eg5 has been viewed as the major motor that drives bipolar spindle assembly in many different experimental systems by sliding microtubules apart. However, it is possible that additional motors are involved in spindle bipolarity that cooperate with or antagonize Eg5. Here, this possibility is tested using a systematic assay in which the role of all kinesins and dynein in bipolar spindle assembly is analyzed. This analysis revealed that indeed multiple additional microtubule motors cooperate with Eg5 to promote bipolar spindle assembly and that one motor, dynein, antagonizes Eg5’s function. These results not only yield novel insights into the mechanism of bipolar spindle assembly, but also have clinical relevance. Currently, inhibitors of Eg5 are tested in clinical trials as novel anti-cancer agents, as Eg5 was generally thought to be indispensable for bipolar spindle formation and thus for cell division. Results described in this thesis show that other motors are involved as well, and that at least one of these motors, Kif15, can take over all the essential functions of Eg5. These results suggest that the effectiveness of Eg5-inhibitors in cancer therapy may depend on the functionality of additional motors.