Author response: Nuclei determine the spatial origin of mitotic waves [peer_review]

Felix E Nolet, Alexandra Vandervelde, Arno Vanderbeke, Liliana Piñeros, Jeremy B Chang, Lendert Gelens
2020 unpublished
Traveling waves play an essential role in coordinating mitosis over large distances, but what determines the spatial origin of mitotic waves remains unclear. Here, we show that such waves initiate at pacemakers, regions that oscillate faster than their surroundings. In cell-free extracts of Xenopus laevis eggs, we find that nuclei define such pacemakers by concentrating cell cycle regulators. In computational models of diffusively coupled oscillators that account for nuclear import, nuclear
more » ... import, nuclear positioning determines the pacemaker location. Furthermore, we find that the spatial dimensions of the oscillatory medium change the nuclear positioning and strongly influence whether a pacemaker is more likely to be at a boundary or an internal region. Finally, we confirm experimentally that increasing the system width increases the proportion of pacemakers at the boundary. Our work provides insight into how nuclei and spatial system dimensions can control local concentrations of regulators and influence the emergent behavior of mitotic waves. Nolet et al. eLife 2020;9:e52868. DOI: https://doi.org/10.7554/eLife.52868 1 of 28 RESEARCH ARTICLE Results Nuclei serve as pacemakers to organize mitotic waves We reconstituted mitotic waves in vitro according to Chang and Ferrell (Chang and Ferrell, 2013; Chang and Ferrell, 2018) . We loaded cycling extracts in a 100 mm wide Teflon tube and used green fluorescent protein with a nuclear localization signal (GFP-NLS) to image mitotic waves (see Box 2). This approach allows visualization of regular oscillations between interphase and mitotic phase. In interphase, nuclei form spontaneously in the extract supplemented with sperm chromatin. These nuclei then import GFP-NLS. In mitosis, the nuclear envelope breaks down and GFP is no longer localized to nuclei. Mitotic waves can be observed by the disappearance of nuclei in a wave-like fashion. Waves become apparent after a couple of cell cycles and they self-organize so that they emerge from more clearly defined foci (see Figure 1A , Figure 1-video 1) . The origin of the wave (point P) was determined as the intersection of straight lines drawn through the points where the nuclei disappear (see orange curve and Figure 1-figure supplement 1) . The wave at cell cycle 5-6 was found to propagate with a speed of~20 mm/min. We noticed that the mitotic wave originated close to a nucleus that is considerably brighter than the surrounding nuclei ( Figure 1A) . We hypothesized that a region with higher GFP-NLS intensity correlates with a higher local oscillation frequency, serving as a pacemaker that organizes the mitotic wave. We therefore analyzed the spatial GFP-NLS intensity profile, the spatial profile of cell cycle periods, and the internuclear distance ( Figure 1B) . As a brighter nucleus has taken up more GFP-NLS, we reasoned that it similarly concentrates cell cycle regulators that lead to a local increase in the cell cycle frequency. We directly correlated this with the local period, which indeed showed that this region oscillated faster ( Figure 1B) . To further understand why certain nuclei were brighter, we explored whether their environment had any particular characteristics. We characterized the distance between the different nuclei and found that they were typically separated by 150-200 mm (Figure 1-figure supplement 2) . However, we found that the brightest nucleus is also most separated from its neighboring nuclei ( Figure 1B) . This finding is consistent with the idea that nuclei increase their oscillation frequency by concentrating cell cycle regulators, as they have a larger pool of regulators in their surroundings to import. We analyzed the spatial GFP-NLS intensity profile and the internuclear distance for nine other experiments where we could clearly identify nuclei and mitotic waves. Overall, in 90% of the analyzed experiments the pacemaker location was well predicted by the region with the highest GFP-NLS intensity and/or the region where nuclei were most separated from their neighboring nuclei ( Figure 1A ,B, Figure 1-figure supplement 3, Figure 1 -figure supplement 4). The total nuclear GFP-NLS intensity was also found to be a better indicator of the Nolet et al. eLife 2020;9:e52868.
doi:10.7554/elife.52868.sa2 fatcat:naxcp2j2pbdp7loedafduxft5y