Figure 7.

Microtubules are required for emergence of large iMTOC structures upon entry into the new cell cycle. (A) When control Mto1p-GFP mad2Δ cells underwent septation at 18°C, we detected several large Mto1p-GFP–positive structures around the NE of daughter cells. However, Mto1p-GFP was uniformly distributed around the NE of Mto1p-GFP mad2Δ nda3-KM311 cells that septated at the restrictive temperature of 18°C. The SPB localization was comparable in both strains. (B) When Mto1p-GFP–expressing cells exiting mitosis were treated with MBC, septation proceeded normally, but we could not detect formation of the iMTOCs in daughter cells (75%; n = 38 cells). (C) When we prepared daughter cells lacking iMTOC structures in the manner described above and washed out the drug, we observed an emergence of Mto1p-GFP–positive distinct dots around nuclei and along microtubules, presumably the iMTOCs (100%; n = 20 cells). Microtubules were subsequently (3 min) depolymerized to allow better visualization of the emergent structures. In A–C, the single maximum intensity reconstructions of z-stacks are shown. (D) A model of the iMTOC formation in S. pombe cells. Fluctuations in initial distribution and/or activity of γ-tubulin complexes are positively reinforced when additional complexes are delivered to the nucleating sites via attached microtubules. This positive feedback loop is disrupted in mia1Δ cells due to the lack of microtubule attachment to the nucleating sites, leading to a defect in coalescence of γ-tubulin complexes into larger MTOC structures.