During mitosis, the cell forms a spindle that segregates chromosomes symmetrically into two daughter cells. The mitotic spindle consists of several classes of microtubules, including kinetochore fibers that interact with chromosomes via kinetochores, and overlap or interpolar bundles that consist of antiparallel microtubules extending from the opposite spindle poles. A precise spatial organization of microtubule bundles and kinetochores is necessary for spindle function, and it has been to a large extent described for a metaphase spindle. However, during prometaphase, microtubules are unevenly distributed over the area of the future metaphase spindle, whereas kinetochore fibers and the associated overlap bundles are not yet evident. A central question is how during prometaphase these unevenly distributed microtubules become rearranged into discrete well-organized bundles, which position kinetochores into the metaphase plate. Interestingly, our pilot experiments show that the number of overlap bundles increases during prometaphase, and that new bundles can form by lateral splitting of the existing ones. To dissect the molecular mechanisms of bundle formation, we will develop an approach based on new CRISPR-edited human cell lines, which will be studied by live-cell confocal and superresolution microscopy. To identify key molecular players in bundle formation, we will modify the expression of candidate kinetochore proteins, motor proteins, and non-motor microtubule crosslinkers. Moreover, we will develop an optogenetic approach to remove a crosslinker protein from the spindle, and an approach based on bioactive peptides to modulate motor velocity. Finally, we will identify the sites of kinetochore fiber and overlap bundle formation and detyrosination, as well as the time of their formation and interaction. This project will reveal how microtubule bundles form, giving rise to the highly organized architecture of the mature metaphase spindle.