Mitotic spindle organization by dynein & kinetochores

Abstract

The human body contains an estimated 37 trillion cells. Although most cells in the body are non-dividing, approximately 300 billion new cells are produced every day. This is important for the maintenance of our tissues but also for the repair of tissues when they are damaged. To prepare for cell division, a cell goes through a cycle of four stages; G1-, S-, G2- and M-phase. M-phase or mitosis, is the phase where the DNA is physically separated over the two new daughter cells. Mitosis is the shortest phase of the cell cycle and occurs within approximately 1 hour. Within this short time period the cell needs to build a complex microtubule-based spindle, generate stable and correct connections between the kinetochores and this spindle, spatially and temporally control protein localization and degrade/stabilize specific proteins that are important for mitotic progression. To accomplish all these processes within this narrow time-window (without making mistakes), these processes must be extremely tightly controlled. Indeed, the list of proteins participating in mitosis is enormous and is still expanding. These include motor proteins, microtubule-associated proteins (MAP’s), kinetochore proteins, DNA-binding proteins, kinases, membrane-associated proteins and cyclins. One previously characterized mitotic player is cytoplasmic dynein, a large minus-end-directed microtubule motor complex. In mitosis, dynein is implicated in multiple processes, amongst which centrosome positioning, centrosome separation, spindle pole organization, spindle positioning and mitotic checkpoint silencing. Dynein consists of multiple subunits and interacts with multiple adaptor proteins for its regulation. Besides providing a comprehensive overview of the currently available literature on dynein regulation in mitosis in this thesis, we investigated how the different dynein subunits and adaptor proteins contribute to the regulation of dynein in both space and time. Using a systematic siRNA-based approach, we were able to deplete each individual dynein subunit/adaptor protein and test their contribution to the different mitotic processes. Strikingly, we found that dynactin, previously thought to be essential for all dynein functions, was dispensable for dynein-mediated spindle organization. Furthermore, we identified the subunits that specifically contribute to the targeting of dynein to different subcellular structures and the subunits that are more important for the general regulation of dynein-activity. In the next part of this thesis, we focused on some specific functions of dynein in more detail. Firstly, we describe a novel function for dynein in prophase centrosome separation. Furthermore, we studied a previously proposed function for dynein in mitotic checkpoint silencing and we found that dynein is essential for APC/C activation rather then for checkpoint protein ‘stripping’ from kinetochores, as was previously proposed. Besides, we studied the role of dynein in spindle positioning by using cells grown on adhesive micropatterns. We confirmed a role for dynein in correct spindle positioning, but more importantly, we found that chromosomes that are closely positioned to the cell membrane disperse cortically localized dynein. Finally, we describe the identification of a novel mitotic protein ‘RAMA1’ using an siRNA screening approach. We find that RAMA1 is localized to the kinetochore where it regulates kinetochore-microtubule attachments.