Fig. 1. Two of three mutations in the centrosomin motif 1 (CM1) region of Mto1 abolish cytoplasmic MT nucleation. (A) Schematic of Mto1 protein, showing CM1 and Mto2-binding region in grey. Predicted coiled-coils (PAIRCOIL score >0.5) are indicated in red (Berger et al., 1995). Amino acid residue numbers are indicated below. (B) Alignment of Mto1 CM1 region with selected related proteins in fission yeast, Neurospora, Drosophila, zebrafish and human, with the amino acids mutated to alanine in the mto1-9A1, mto1-9A2 and mto1-9A3 mutants indicated above. The mto1-9A3 mutation lies at the beginning of the first predicted coiled-coil of Mto1. (C) Wild-type and mto1 mutant cell morphology (strains KS515, KS1017, KS2007, KS2010, KS2013) after growth to stationary phase and refeeding with fresh medium. (D) Anti-tubulin immunofluorescence time-course of MT regrowth after cold-induced depolymerization in wild-type and mutant strains (strains KS515, KS1017, KS2007, KS2010, KS2013). (E) Stills from movies of strains expressing GFP-tubulin and the SPB component Sad1-dsRed (strains KS2863, KS3609, KS3604, KS3605, KS3607; see supplementary material Movies 1-5). Interphase cells are shown on the left of each pair (40 second intervals) and mitotic cells on the right (100 second intervals). White arrowheads indicate examples of interphase MT nucleation. Yellow arrowheads indicate examples of mitotic astral MTs. Blue arrowheads indicate example of PAA MTs. Asterisks indicate bend-breakage events in which a single MT or MT bundle breaks into two. Values underneath images indicate average number of interphase nucleation events per cell per minute (see Materials and Methods). Bars, 10 µm.