Fig 6. Cooperation between Bgs1 and the SH3 Domain of Cdc15 is essential for CAR maintenance and septum formation.

(A) Fluorescence micrographs of cdc15 _ΔSH3-GFP Pnmt81-bgs1 ⁺ cells stained with CW and carrying Cdc15_ΔSH3-GFP. Cells were grown to early log-phase in EMM+S (time 0 h), shifted to the same medium plus thiamine, EMM+S+T (times 15, 24 and 40 h, bgs1 ⁺ repressed) and imaged at the indicated times. Arrow: Cell with an open septum without the ring of Cdc15. Arrowhead: Cell with a disorganized ring of Cdc15. (B) Histograms showing the indicated percentages of septa and Cdc15 structures in the strains Pnmt81-bgs1 ⁺ (n = 150 cells or hypha units were quantified for each time) and cdc15 _ΔSH3-GFP Pnmt81-bgs1 ⁺ (n = 100 cells or hypha units were quantified for each time). Note that Pnmt81-bgs1 ⁺ strains after 24 h of bgs1 ⁺ repression appear as hypha units, each being equivalent to several single cells. (C) Fluorescence micrographs of Pnmt81-bgs1 ⁺ and cdc15 _ΔSH3-GFP Pnmt81-bgs1 ⁺ cells stained with CW and carrying Ags1-RFP or RFP-Bgs4 after the indicated times of repression of bgs1 ⁺. (D) Fluorescence micrographs of Pnmt81-bgs1 ⁺, Pnmt81-bgs1 ⁺ pxl1Δ and Pnmt81-bgs1 ⁺ cdc15 _ΔSH3 cells carrying the hypomorphic version of Bgs1 GFP-Cps1-191, and wild-type cells carrying GFP-Bgs1 used as Bgs1 localization control. Since cps1-191 allele is lethal in pxl1Δ and cdc15 _ΔSH3 backgrounds, strains are maintained alive with an inducible version of bgs1 ⁺ (Pnmt81-bgs1 ⁺). Cells were grown at 25°C (permissive temperature for cps1-191 cells) in EMM+S (time 0 h, bgs1 ⁺ induced) and shifted to EMM+S+T (time 24 h +T, bgs1 ⁺ repressed) to visualize cell phenotype and GFP-Cps1-191 localization. Scale bars, 5 μm.