Extended Data Figure 8. Les1 truncations and genetic interactions.

a. Schematic indicating Les1 truncation constructs, with numbers representing amino acid positions starting from 1 at the N-terminus. Lower panels, cells expressing Les1 (1-291)-mNeonGreen, replacing Les1 at the endogenous locus, as well as Cut11-mCherry. See Methods for details on the truncation constructs (all at endogenous locus, replacing endogenous copy). Note the absence of detectable stalks and the delocalisation of Cut11 in the bridge. Representative of >10 cells across 2 biological repeats. Scale bar = 5 μm. b. Confocal maximum intensity projections of cells expressing Cmp7-mNeonGreen and Les1-mScarlet. Arrows indicate Cmp7 foci at the tips of retracting stalks. Representative of >20 cells across 4 biological repeats. Scale bar = 2 μm. c. Single iSIM slice of a dividing cell expressing Cmp7-mNeonGreen and Les1-mScarlet, representative of >5 cells drawn from 2 technical repeats. Scale bar = 2 μm. d. Tetrad dissection assay for les1Δ crossed with either lem2Δ or cmp7Δ, showing colonies grown from individual spores. See Methods for details. e. Single image of double deletion strains, derived from the tetrad assay clones shown in (d), expressing a synthetic NLS-GFP construct. Scale bar = 10 μm. Representative of >200 cells drawn from 2 biological repeats. f. Averaged line traces (darker lines = mean, lighter bands = standard deviation) of Nup60-mCherry intensities in les1Δ cells (gray: control; blue: treated with 10 μM Cerulenin) at bridge length 3 μm (n=45 cells for cerulenin-treated; n=27 cells for control). g. Confocal maximum intensity projections of les1Δ lem2Δ cells expressing Cut11-mCherry, either in the presence (lower panel) or absence (upper panel) of 5 μM Latrunculin A. Representative of >20 cells across 3 technical repeats. Scale bar = 2 μm.