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Files in this Data Supplement:
Fig. S1. ENTH2 overexpression cell division phenotype. (A) Cells overexpressing ENTH2 for 16 hours were stained with calcofluor white (CFW) or with DAPI as described in Materials and Methods. CFW staining was used to analyze the morphological defects of ENTH2-overexpressing cells. We observed at least four major types of abnormalities: cells with very elongated buds (1), branched bud chains emerging from a common neck (2), linear chains of buds (3) and cells with multiple and branched ramifications (4). DAPI staining revealed different degrees of multinucleation associated with each morphological category. Scale bar, 5 µm. (B) Phenotype penetrance. We estimated the percentage of failed cell division attempts as described in Materials and Methods by counting more than 200 cells per data point. Quantifications were repeated between six (empty vector) and nine (ENTH1 or ENTH2) times. The median for each group of quantifications is indicated by a horizontal line. Statistical significance of the cell division failure induced by ENTH2 with respect to ENTH1-overexpressing cells was assessed by the Wilcoxon test (**P<0.05). (C) The contribution of the different morphological categories described in A to the population of cells transformed with empty vector or overexpressing ENTH domains is shown.
Fig. S2. Cell division phenotype depends on ENTH2 overexpression but not on the promoter or growth conditions used. (A) ENTH2 was overexpressed for 16 hours from MET25 or GAL1 promoters in high-copy (2µ) vectors with either URA3 or TRP1 auxotrophic markers. Cells were stained with calcofluor white and observed by epifluorescence microscopy using UV optics. Scale bar, 5 µm. (B) W303 cells expressing ENTH2 from the methionine-repressible MET25 promoter were grown overnight in media containing different methionine concentrations to generate a gradient of ENTH2 intracellular dosages (Rönicke et al., 1997). The percentage of failed cell division attempts was estimated as described in Material and Methods and expressed as the mean ± s.d.
Fig. S3. Growth and morphological distribution of cell overexpressing ENTH2 mutants. W303 cells overexpressing ENTH1 or ENTH2 (WT or mutant) or transformed with empty vector were grown overnight in selective media lacking uracil and methionine. (A) Cell population distribution according to the morphological categories described in Fig. S1A was determined. (B) Optical density of triplicate cultures was measured at 600 nm.
Fig. S4. Chs2 dynamics in empty vector transformants and ENTH1 overexpressors. Chs2-GFP dynamics was analyzed by live-cell imaging in empty vector transformants and ENTH1-overexpressing cells. Individual frames represent time progression. Chs2-GFP structures are pseudo-colored black on a white background. Right panels shows average fluorescence intensity (FI) over a 12 minute interval to highlight the transient nature of Chs2-GFP localization. Scale bar: 5 µm.
Fig. S5. Overexpression of ENTH2 but not ENTH1, leads to mislocalization of the actomyosin ring protein Myo1. Cells expressing Myo1-GFP and overexpressing ENTH1 or ENTH2 were grown in selective media lacking methionine for up to 4 hours and processed for fluorescence and DIC microscopy as described in the Materials and Methods. GFP-DIC merged images are shown. Scale bar: 5 µm.
Fig. S6. Intracellular localization of proteins involved in septin assembly in cells overexpressing ENTH1 or transformed with empty vector. W303 cells expressing the indicated GFP fusion protein and transformed with empty vector or pMET25::ENTH1 were cultured as described in Fig. 5 and observed by epifluorescence microscopy using a GFP filter. Scale bar: 5 µm.
Fig. S7. Overexpression of the Bem3Δ1-114 mutant induces cell division defects. (A) W303 cells overexpressing Bem3Δ1-114 were observed by DIC microscopy. (B) Localization of Bem3Δ1-114-GFP. (C) W303 cells bearing integrated Cdc3-mCherry and overexpressing Bem3Δ1-114 were observed using appropriate optics. Boxes indicate location of magnified areas, dotted lines highlights cell boundaries. Scale bar,:5 µm.
Fig. S8. Yeast two-hybrid interaction between ENTH domains with Cdc42 GAPs Rga2 and Bem3. The yeast two-hybrid strain AH109 was co-transformed with plasmids encoding Gal4BD-ENTH2 (WT and mutants, as indicated) and Gal4AD-Bem3/-Rga2 fusions. Transformants were grown on liquid media containing (+His) or lacking (−His) histidine and supplemented with 20 mM 3AT. The growth in liquid media was measured as the optical density at 600 nm, in duplicate. Results were expressed as the −His/+His ratio of the OD600nm, mean ± (SD−His+SD+His).
Fig. S9. Localization of Ent2-GFP and Bem3-GFP in bem3Δ and ent2Δ cells respectively. bem3Δ cells expressing Ent2-GFP or ENTH2-GFP and ent2Δ cells expressing Bem3-GFP were analyzed by epifluorescence microscopy with a FITC filter. Scale bar: 5 µm.
Movie 1. Growth pattern of ENTH1/2-overexpressing cells. W303 yeast cells transformed with pMET25::ENTH1 or pMET25::ENTH2 were arrested with nocodazole as described in Materials and Methods, released from arrest while expression was induced for 4-6 hours and then spotted on media-embedded agarose beds and imaged with DIC optics using a 63× objective. Interval between frames is 10 minutes. Movie playback rate: 5 frames/second.
Movie 2. Clathrin-GFP dynamics in empty vector-transformed and ENTH-overexpressing cells. Clathrin-GFP-expressing cells transformed with empty vector or pMET25::ENTH1/2 were treated as indicated in Movie 1, spotted on media-embedded agarose beds and imaged with GFP filter using a 100× objective. Interval between frames is 2 seconds. Movie playback rate: 10 frames/second. Scale bar: 5 µm.
Movie 3. Chs2-GFP dynamics in ENTH1-overexpressing cells. W303 yeast cells transformed with pMET25::ENTH1 and expressing Chs2-GFP were treated as indicated for Movie 1, and then spotted on media-embedded agarose beds and imaged by epifluorescence with a GFP filter at 63× magnification. Movie playback rate: 5 frames/second. Elapsed time is indicated. Scale bar: 5 µm.
Movie 4. Chs2-GFP dynamics in empty vector-transformed cells. W303 yeast cells transformed with empty vector and expressing Chs2-GFP were treated as indicated in Movie 1, and then spotted on media-embedded agarose beds and imaged by epifluorescence with a GFP filter at 100× magnification. Elapsed time is indicated. Scale bar: 5 µm.
Movie 5. Chs2-GFP dynamics in ENTH2-overexpressing cells. W303 yeast cells transformed with pMET25::ENTH2 and expressing Chs2-GFP were treated as indicated in Movie 1, and then spotted on media-embedded agarose beds and imaged by epifluorescence with a GFP filter at 63× magnification. Movie playback rate: 5 frames/second. Elapsed time is indicated. Scale bar: 5 µm.
Movie 6. Cdc3-GFP dynamics in ENTH2-overexpressing cells. W303 yeast cells transformed with pMET25::ENTH2 and expressing Cdc3-GFP were treated as indicated in Movie 1, and then spotted on media-embedded agarose beds and imaged by epifluorescence with a GFP filter at 100× magnification. Movie playback rate: 5 frames/second. Arrows point to growing septin filaments and ectopic deposition. Elapsed time is indicated. Movie loops three times to allow visualization. Scale bar: 5 µm.
Movie 7. Ent1-GFP and Ent2-GFP dynamics. Yeast cells expressing Ent1- or Ent2-GFP (as indicated) were spotted on media-embedded agarose beds and imaged by epifluorescence with a GFP filter at 100× magnification. Movie playback rate: 10 frames/second. Arrow indicates bud tip recruitment of Ent2-GFP. Scale bar: 5 µm.
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