During prophase, centrosomes are positioned on the shortest nuclear axis. This process, which depends on Arp2/3 and nuclear envelope Dynein, is essential for efficient spindle assembly.

As cells prepare to divide, they must ensure that enough space is available to assemble the mitotic machinery without perturbing tissue homeostasis. To do so, cells undergo a series of biochemical reactions regulated by cyclin B1-CDK1 that trigger cytoskeletal reorganization and ensure the coordination of cytoplasmic and nuclear events. Along with the biochemical events that control mitotic entry, mechanical forces have recently emerged as important players in cell-cycle regulation. However, the exact link between mechanical forces and the biochemical pathways that control mitotic progression remains unknown. Here, we identify a tension-dependent signal on the nucleus that sets the time for nuclear envelope permeabilization (NEP) and mitotic entry. This signal relies on actomyosin contractility, which unfolds the nucleus during the G2-M transition, activating the stretch-sensitive cPLA2 on the nuclear envelope and regulating the nuclear translocation of cyclin B1. Our data demonstrate how nuclear tension during the G2-M transition contributes to timely and efficient mitotic spindle assembly and prevents chromosomal instability.

Results: 55 Centrosomes position on the shortest nuclear axis at nuclear envelope breakdownTo characterize mitotic spindle assembly at high spatiotemporal resolution, we performed 4D imaging in HeLa cells. We observed that when cells are seeded on a substrate that does not activate integrin signaling (poly-L-lysine; PLL), centrosomes separate independently of NEB ( Fig. S1A), as reported previously [2, 25]. However, 60 when seeded on integrin-activating fibronectin (FBN), ~82% of the cells separate their centrosomes to opposite sides of the nucleus before NEB. Moreover, cells that have an increased spreading area at NEB show longer inter-centrosome distances ( Fig. S1B), suggesting that centrosome separation prior to NEB is a function of the adhesion area.To normalize cell area and shape in 2D, we seeded cells on defined FBN micropatterns 65 and monitored centrosome dynamics, cell membrane and nuclear shape ( Fig. 1A), which were subsequently reconstructed using specifically developed computational algorithms ( Fig. S2). Centrosome dynamics relative to the micropattern was defined by two angles theta and phi, reflecting movements in xy (azimuth) and xz (inclination), respectively (Fig. 1B). These vary between 0 o (aligned with the long axis of the pattern) and 90 o 70 (perpendicular to the pattern). We anticipated that separated centrosomes should align with the long axis of the micropattern, due to the distribution of retraction fibers imposed by extracellular matrix organization [17,20]. However, during mitotic entry, centrosomes deviated from the underlying micropattern, as observed by the high variability of theta and phi (Fig. 1B). This was accompanied by a rotation of the nucleus relative to the long 75 axis of the pattern, as well as a decrease in cell area (Fig. 1C). Due to the shape asymmetry of the line micropattern, we could calculate cell membrane eccentricity, which varies between 1 (completely elongated cell) and 0 (spherical cell). As cell progressed towards NEB, membrane eccentricity decrease (Fig. 1D) due to a retraction of the long cell axis (Fig. 1E, 0 o ) and a simultaneous increase in cell width, perpendicularly to the 80 pattern ( Fig. 1E, F; 90 o ; *** p<0.001). Interestingly, during the rounding process, the