Description:
This cumulative PhD thesis covers in five chapters (chapters 2, 3, 4 and 5 consisting of published papers and chapter 6 consisting of a submitted manuscript) new findings on different aspects of the regulation of kinesin-5 motor proteins. Outside the topic of kinesin-5 motor proteins, chapter 7 consists of a paper about cellular motility of neuronal receptors to which I contributed TIRF-microscopy in-vivo measurements in neurons. In chapter 2, the factors that cause directional switching in Cin8, one of the two kinesin-5 motor proteins from S. cerevisiae, were studied. Until know the directionality of kinesin-5 motor proteins was believed to be fixed and unidirectional to the plus-end of the microtubule (MT). The findings presented here, and an independent recent report from Roostalu et al., showed that surprisingly Cin8 is able to switch directionality. In the work presented here Cin8 directionality was examined using single-molecule fluorescence motility experiments and live-cell microscopy. Single-molecule fluorescence experiments revealed that individual Cin8 motor proteins could be switched by ionic strength in the buffer solution from fast and processive minus-end to slow plus-end motion on single MTs. An even more interesting finding was that Cin8 motility was strongly affected by binding to a second MT: at high ionic strength in the buffer solution, Cin8 motor proteins moved fast and processive on single MTs and switched to rapidly alternated directionalities when bound between antiparallel MTs, while at the same time driving steady plus-end relative sliding. This extraordinary behaviour of Cin8 probably plays a crucial role in the regulation of its different functions inside the mitotic spindle ...