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Fin1p over-expression results in Cdc16p disassociation from the SPB.

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posted on 05.08.2015 by Anastasia Chasapi, Paulina Wachowicz, Anne Niknejad, Philippe Collin, Andrea Krapp, Elena Cano, Viesturs Simanis, Ioannis Xenarios

(A) Cells expressing the labeled tubulin marker leu1::m-Cherry-atb2 and cdc16-GFP were induced to express fin1 from the medium strength nmt41 promoter [80]. Cells transformed with empty vector served as control. Cells were grown in medium without thiamine for 27h at 25°C. Cells were imaged and the intensity of SPB associated cdc16-GFP signal was analyzed as described in [12]. The panel shows m-Cherry-atb2 leu1::cdc16-GFP(ura4+) cells bearing REP41 or REP41-fin1. The scale bar is 10 μm. (B) The SPB associated signal was determined in interphase cells in each strain. Since REP41-fin1 eventually leads to a mitotic arrest [87] interphase cells were identified by the presence of an interphasic microtubule array. The box shows 25%-75% range for the population, the line indicates the median. The bars indicate 10% and 90% range for the population, and dots indicate more extreme individual values. The y-axis shows fluorescence intensity on an arbitrary scale. (C) Cells bearing the leu1::cdc16-HA allele were induced to express fin1 (ON) by growing them in defined minimal medium [81] in the presence (OFF) or absence (ON) of 2mM thiamine. Protein extracts were prepared 27h after induction and analyzed by western blotting using monoclonal antibody 12CA5. The anti-α-tubulin monoclonal antibody TAT-1 [90] was used as a control.