Drug screening to isolate pharmacological suppressors of lethality of an msp1^P300S strain grown at restrictive temperature in a galactose-based medium. (A) Experimental strategy: a yeast strain expressing a thermosensitive form of the Msp1p protein (msp1^P300S) was grown at the permissive temperature (25°C) and then spread on agar-based solid medium containing galactose. Then, filters were deposited onto the agar surface and individually loaded with single pharmacological compounds from repurposed drug libraries (3 µl at 10 mM or DMSO as a control onto the top left filter) and Petri plates were then incubated at the restrictive temperature (37°C) for 5-7 days and photographed. (B) The presence of a white halo around the filter indicates yeast growth. The presence of a dark halo indicates the absence of growth and therefore toxicity of the compound at high concentration (close to the filter). The names of the compounds and their chemical structures are indicated. The indicated quantities of drugs were added onto the filters. (C) Drops containing 800 cells expressing WT (msp1^WT) or thermosensitive (msp1^P300S) Msp1p protein were deposited on agar-based solid medium containing either galactose (gal) without (-) or with 6 μM vanoxerine, 30 μM hexestrol, 15 μM clomifene, 1 μM ketoconazole or 9 μM terconazole, or dextrose (dex) without (-) or with 1 μM vanoxerine, 10 μM hexestrol, 15 μM clomifene, 1 μM ketoconazole or 1 μM terconazole, as indicated. The plates were then incubated at 37°C for 3 days and photographed. C is representative of three independent experiments.

Effects of hexestrol and clomifene on mitochondrial morphology and maintenance of mtDNA. (A,B) Yeasts expressing the mitochondrial protein Arg11p fused to the fluorescent mCherry protein (Arg11p-mCherry), together with either WT (strain msp1^WT) or mutated (strain msp1^P300S) Msp1p protein, were cultured at 37°C for 18 h on dextrose liquid medium, without (-) or with 15 μM hexestrol (Hex) or 4 μM clomifene (Clo) as indicated, and then fixed and labeled with DAPI before being observed with a fluorescence microscope. Scale bar: 5 μm. High magnifications are shown in insets (×1.7). A and B are representative of five experiments. (C) The number of mitochondrial nucleoids was counted in the msp1^WT and msp1^P300S strains cultured at 37°C in dextrose liquid medium for 18 h without (-) or with 15 μM hexestrol (Hex) or 4 μM clomifene (Clo), as indicated. Data represent the mean±s.d. of two independent experiments with 50 cells per condition. They were statistically analyzed using Kruskal–Wallis Dunn's multiple comparison tests to compare, for each strain, the values obtained with drugs (Hex, Clo) with that obtained for the control (-) (****P<0.0001). (D) qPCR was performed on DNA extracted from the msp1^WT and msp1^P300S strains cultured at 37°C on dextrose liquid medium for 18 h without (-) or with 15 μM hexestrol (Hex) or 4 μM clomifene (Clo), as indicated. The results of three independent experiments performed in triplicate are expressed as a percentage relative to those obtained for the msp1^WT strain cultured at 37°C (Student's t-tests; **P<0.01; ***P<0.001).

Mechanisms of action of hexestrol and clomifene. (A) Yeast strains of the indicated genotypes (Table 1) were cultured on dextrose minimal medium without (-) or with 50 μM hexestrol (Hex) or 15 μM clomifene (Clo) at 25°C for 6 days and then photographed. Top panel: strains with deletions of msp1⁺ (Δmsp1⁺) ectopically expressing msp1⁺, or not, under the control of the nmt1 promoter. Msp1p is produced in the absence of thiamine (Msp1p), but not in its presence (no Msp1p). Middle panel: WT strains ectopically overexpressing a WT form of msp1⁺, or a form containing a mutated GED domain, under the control of the nmt1⁺ promoter. WT Msp1p (OP Msp1p), Msp1p with the L876P mutation (OP Msp1pL876P) or Msp1p with a deletion of the last 50 amino acids of the protein (OP Msp1pΔGED) were overexpressed in the absence of thiamine. Bottom panel: strains with deletions of fzo1⁺ (Δfzo1⁺) ectopically expressing fzo1⁺ under the control of the nmt1⁺ promoter. Fzo1p (Fzo1p) is produced in the absence of thiamine, whereas it is not expressed (no Fzo1p) in its presence. (B) Yeasts with deletion of msp1⁺ (Δmsp1⁺) expressing the mitochondrial protein Arg11p fused to the fluorescent mCherry protein (Arg11p-mCherry), for which the ectopic expression of msp1⁺ was abolished by addition of thiamine (no Msp1p), were cultured at 25°C in dextrose minimal liquid medium for 72 h with or without 50 μM hexestrol (Hex), and stained with DAPI before observation under a fluorescence microscope. Left column: representative pictures of Arg11p-mCherry and DAPI staining. Scale bar: 5 μm. High magnifications are shown in insets (×1.7). Right column: the number of mitochondrial nucleoids was counted in yeasts with deletion of msp1⁺ (Δmsp1⁺) cultured at 25°C in dextrose liquid medium for 72 h with thiamine (no Mps1p) and without (-) or with 50 μM hexestrol (Hex). Data represent the mean±s.d. of three independent experiments, with 60 cells per condition, and were statistically analyzed by a two-tailed unpaired Mann–Whitney test (***P<0.001). (C) A WT strain and a strain deleted for the dnm1⁺ gene (Δdnm1⁺), expressing the mitochondrial protein Arg11p fused to the fluorescent mCherry protein (Arg11p-mCherry) were cultured in dextrose liquid medium at 25°C with or without 15 μM hexestrol (Hex) for 18 h as indicated. Sodium azide (Azide) was then added to a final concentration of 0.02% w/v, the cultures were incubated for 30 min, and the cells were fixed and observed by fluorescence microscopy. Scale bar: 5 μm. High magnifications are shown in insets (×1.7). A, B and C are representative of three experiments.