Centrosome-nuclear axis repositioning drives the assembly of a bipolar spindle scaffold to ensure mitotic fidelity.

During the initial stages of cell division, the cytoskeleton is extensively reorganized so that a bipolar mitotic spindle can be correctly assembled. This process occurs through the action of molecular motors, cytoskeletal networks and the nucleus. How the combined activity of these different components is spatiotemporally regulated to ensure efficient spindle assembly remains unclear. To investigate how cell shape, cytoskeletal organization and molecular motors crosstalk to regulate initial spindle assembly, we use a combination of micropatterning with high-resolution imaging and 3D cellular reconstruction. We show that during prophase, centrosomes and nucleus reorient so that centrosomes are positioned on the shortest nuclear axis at nuclear envelope (NE) breakdown. We also find that this orientation depends on a combination of centrosome movement controlled by Arp2/3-mediated regulation of microtubule dynamics and Dynein-generated forces on the NE that regulate nuclear reorientation. Finally, we observe this centrosome configuration favors the establishment of an initial bipolar spindle scaffold, facilitating chromosome capture and accurate segregation, without compromising division plane orientation. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].