A tropomyosin point mutant recapitulates the <i>rng8/9</i> deletion phenotype.

<p>(A) Cross of wildtype <i>h+ myo52-tdTomato</i> with <i>h-</i> wildtype or <i>rng8</i>Δ cells expressing nmt41-Cdc8-GFP. (B) Measurements of Cdc8-GFP accumulation at fusion site (N = 15). (C) Measurements of Cdc8-GFP zone width at the cell-cell contact site (N = 10); (***) P < 10⁻⁷, t-test. (D) Fusion efficiency of <i>h90</i> wildtype, <i>rng8</i>Δ, <i>cdc8-382</i>, <i>cdc8</i>^<i>R121A</i> and <i>cdc8</i>^<i>E104A</i> mutants on pads (N>600). Note that all strains were incubated at 30°C except for <i>cdc8-382</i>, which was incubated at the semi-restrictive temperature of 33°C. (E) Localization of Myo52-tdTomato and Myo51-3YFP in <i>h90 cdc8</i>^<i>R121A</i> and <i>cdc8</i>^<i>E104A</i> mutants. (F) Quantifications of the number of Myo52-tdTomato dots observed in <i>cdc8</i>^<i>E104A</i> <i>and cdc8</i>^<i>E104A</i> <i>myo51</i>Δ mutants in comparison to wildtype (N>100 cells). The quantification of Myo52-tdTomato dots in <i>myo51Δ</i> mutants is identical to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006721#pgen.1006721.g004" target="_blank">Fig 4F</a> and shown for comparison. (G) Kymographs of Myo52-tdTomato showing multiple stable dots over >60s in <i>cdc8</i>^<i>E104A</i> mutant. (H) Myo51-3YFP fluorescence at the fusion site is decreased in <i>cdc8</i>^<i>E104A</i> mutant (N = 15); (***) P < 3 × 10⁻⁵, t-test. (I) Localization of Rng8-mEGFP in <i>h+ cdc8</i>^<i>E104A</i> and <i>myo51</i>Δ <i>cdc8</i>^<i>E104A</i> during vegetative growth and when crossed to <i>h-</i> Myo52-tdTomato wildtype cells during mating. (J) Measurements of Rng8-mEGFP fluorescence at the division and the fusion sites show a significant reduction in the <i>cdc8</i>^<i>E104A</i> and <i>myo51</i>Δ <i>cdc8</i>^<i>E104A</i> mutant allele (N = 15); (***) P < 1.7 × 10⁻⁸, t-test. (K) Fusion efficiency of wildtype, <i>rng8</i>Δ <i>cdc8</i>^<i>E104A</i>, <i>myo51</i>Δ <i>cdc8</i>^<i>E104A</i>, <i>rng8</i>Δ <i>myo51</i>Δ <i>cdc8</i>^<i>E104A</i> and <i>rng8</i>Δ <i>myo51</i>Δ (N>150); (***) P < 6 × 10⁻⁵, t-test. Bars, 2 μm.