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bioRxiv · 10.1101/2025.09.05.674440

Role of the Arp2/3 Complex in Regulating Mitotic Entry and Progression in S. pombe

Kalathil, D.·Flores Reyes, E.·Das, M.
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Abstract

Fission yeast undergoes closed mitosis, wherein chromosome segregation occurs inside an intact nucleus. The microtubule organizing center, Spindle Pole Body (SPB) is duplicated in the G2 phase followed by insertion into a fenestra on the nuclear envelope (NE). The SPB then nucleates spindle microtubules at mitotic onset. Here we show that the branched actin network promotes mitotic entry by enabling NE fenestration. Inhibiting the branched actin nucleator Arp2/3 complex with the drug CK666 fail mitotic entry and the few cells that enter mitosis show significant delays. Upon CK666 inhibition, most proteins are correctly recruited to the SPB at the onset of mitosis however they fail to properly recruit the POLO-like kinase Plo1 and the SPB docking protein Cut11, suggesting a failure in SPB insertion. The start of NE fenestration is marked by the formation of a Sad1 ring, a SUN domain protein followed by a Cut11 ring. Super-resolution structured illumination microscopy indicates that CK666-treated cells show impaired Sad1 or Cut11 rings. Furthermore, these cells do not leak NLS-GFP, a hallmark of fenestration failure. In agreement with the role for branched actin in NE fenestration, CK666 treatment after SPB insertion does not show any mitotic entry defects as observed with preprophase-arrested tubulin mutants. We observed a component of the Arp2/3 complex Arc5, and actin-binding proteins Fimbrin and Coronin, on the nuclear envelope within minutes of mitotic onset. In CK666-treated cells where mitosis is delayed, in the absence of branched actin the SPB aberrantly interacts with the actomyosin ring, preventing its proper dynamics. This results in phenotypes such as the formation of multinucleate structures, asymmetric nuclear division, floppy nuclear membrane, and severe bending of spindle microtubules. Our findings demonstrate a novel role for Arp2/3 complex-dependent branched actin in promoting NE fenestration and SPB insertion during mitosis.

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Kalathil, D., Flores Reyes, E., Das, M.. 2025-09-05. Role of the Arp2/3 Complex in Regulating Mitotic Entry and Progression in S. pombe. https://doi.org/10.1101/2025.09.05.674440

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