1 2 3 Two - step interphase microtubule disassembly 4 aids spindle morphogenesis 5 6 7

29 Entry into mitosis triggers profound changes in cell shape and cytoskeletal 30 organisation. Here, by studying microtubule remodelling in human flat mitotic cells, 31 we identify a two-step process of interphase microtubule disassembly. First, a 32 microtubule stabilizing protein, Ensconsin, is inactivated in prophase as a 33 consequence of its phosphorylation downstream of Cdk1/CyclinB. This leads to a 34 reduction in interphase microtubule stability that may help to fuel the growth of 35 centrosomally-nucleated microtubules. The peripheral interphase microtubules that 36 remain are then rapidly lost as the concentration of tubulin heterodimers falls 37 following dissolution of the nuclear compartment boundary. Finally, we show that a 38 failure to destabilize microtubules in prophase leads to the formation of microtubule 39 clumps, which interfere with spindle assembly. Overall, this analysis highlights the 40 importance of the stepwise remodelling of the microtubule cytoskeleton, and the 41 significance of permeabilization of the nuclear envelope in coordinating the changes 42 in cellular organisation and biochemistry that accompany mitotic entry. 43 44 45 Introduction 46 The goal of mitosis is the equal segregation of genetic material into two daughter 47 cells. To achieve this, animal cells undergo profound changes in cell organisation. 48 Cells round up, chromosomes condense, and the permeability of the nuclear 49 envelope increases (a process we term NEP), leading to mixing of nucleoplasm and 50 cytoplasm. At the same time, the array of long interphase microtubules is replaced by 51 a population of short and highly dynamic centrosome-nucleated microtubules, which 52 go on to form the mitotic spindle – the structure responsible for chromosome 53 segregation. 54 55 2 . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/126342 doi: bioRxiv preprint first posted online May. 29, 2017;