Fig. 1

Aip5 accelerates bulk actin assembly and Bni1-mediated actin polymerization in the polarisome. a Representative maximum Z-projection images of Aip5-GFP at the presumptive bud site in wild-type and polarisome yeast mutants. S small-budded cell, M medium size-budded cell, BN bud neck. Scale bar represents 3 µm. b Domain schematic of the polarisome scaffold protein Spa2. Three classes of Aip5 localization that are quantitatively determined by the ratio of fluorescence signal intensity (R) of Aip5-GFP at presumptive bud site and cytoplasm (Aip5-GFP, n = 41 bud sites; spa2∆ Aip5-GFP, n = 23 bud sites; bni1∆ Aip5-GFP, n = 29 bud sites; bud6∆ Aip5-GFP, n = 59 bud sites). c The SDS-PAGE gel of purified Aip5 protein from budding yeast. d Pyrene-actin polymerization reaction with an increased concentration of full-length Aip5. e Pyrene-actin polymerization reaction of Aip5 in the presence of Bni1FH1COOH. f Representative total internal reflection fluorescence microscope (TIRFM) images of actin filaments formed at 5 and 10 min in the field of 48 µm³. Actin filaments were assembled using 0.5 µM actin (10% Oregon green 488 labeled and 0.5% biotin–actin), with or without 5 nM Bni1FH1COOH and 20 nM Aip5. The scale bar represents 5 µm. g Quantification of the number of nucleated-actin seeds at 5 min (n = 60 ROIs for each sample, ROI = 64 µm³). The box plot covers data from minimal to maximal with the central line indicating the mean value. h Representative kymograph of elongating actin filament generated from movies over 5 min with 5-s interval between each frame. The scale bar represents 3 µm. i Quantification of actin filament barbed-end elongation speed of the indicated protein combinations as shown in f (n = 30 actin filament for each sample). j Yeast spotting assay of the indicated strains that were grown on YPD plate at 25 °C for 36 h before imaging. k Yeast growth curves of the liquid cultures at 25 °C in the presence of 1 µM LatA (n = 4). p Values were determined by one-way ANOVA, ns not significant, ****p < 0.0001. All the bar graph represents mean value; error bar, S.D. Source data are provided as a Source Data file