A. Wild-type cells labeled with dis1p-GFP and mCherry-Tubulin. Fluorescence intensity profiles of selected IMAs showing that Dis1p-GFP does not localize to the whole overlap regions of mCherry-tubulin (regions of the red line above the turquoise shade). B. Wild-type cells labeled with dis1p-GFP and ase1p-mCherry and intensity profiles of selected IMAs showing that dis1-GFP and ase1-mCherry have both co-localized and segregated signals in different IMAs. C. ase1Δ cells labeled with dis1p-GFP and mCherry-Tubulin and intensity profiles of selected IMAs where Dis1p-GFP localizes to the overlap regions of mCherry-tubulin. D. Kymograph showing the dynamics of dis1p-GFP along an IMA (left) where dotted lines indicate different velocities movements. Speckles of dis1p that appear and disappear are visible (arrowheads). The velocities distribution is represented in the box-plot graph. Kymograph of ase1-GFP (center right) along time. Several events of incorporation of newly created overlaps are visible (arrowheads). Graph (right) displaying several events of ase1-GFP overlaps. *p<0.05; ***p<0.001. E. Ase1p-GFP cells over-expressing dis1p. Several regions of ase1p-GFP are visible along the same IMA (dotted line) as well as faint long regions of ase1p-GFP along an IMA (arrowhead). The average number of ase1p-GFP regions per cell is depicted in the bar graph. F. Kymograph showing highly dynamic ase1p-GFP regions in cells over-expressing dis1p. Compare this kymograph with the ase1-GFP wild-type in panel D. Cell images are maximum projections. Bars: 5 µm in A to C; 2 µm in D, E and F. Vertical bars: 30 seconds.

A. Models obtained from selected cells that are representative of each mutant cytoskeleton organization. In the triple mutant the inset shows the MT bundle of the dotted square isolated and rotated for better view. MT bundles are associated or non-associated with the SPB (yellow volume). See also Figure S1B and Movies S4–S6. B. Selected longitudinal tomographic sections of two adjacent MTs (asterisks) (1 and 2) showing electron-dense bridges (arrows). Cross-section of 1 and 2, respectively, in the plane of red and blue arrows. C. Nda analysis show a small decrease in the inter-MT distance for dis1Δ cells, while ase1Δ dis1Δ cells have a significant reduction of the inter-MT distance (arrowheads indicate the mean of the peak). D. Gallery of bundles showing inter-MT associations (100 nm stretches of MT that are at (or closer than) the higher peak values of the Nda analysis to an adjacent MT). MT minus end structure is represented by a red sphere. Percentage of anti-parallel (blue) and parallel (red) MT associations. Bars: 500 nm in A; 25 nm in B; 100 nm in D.

A. Maximum projection of GFP-tubulin labeled cells where brighter fluorescence regions (arrowheads) are visible in wild-type and the different mutants. B. Number of IMAs per cell for wild-type and the different mutants. *p<0.05; **p<0.001 (mean ± SEM). Black asterisk are in relation to wild-type and red asterisks to ase1Δ. C. Models obtained from selected cells that are representative of wild-type and each mutant cytoskeleton organization. All the volumes show an inset of the dotted square where the MT bundle is isolated and rotated for better view. MT bundles are associated or non-associated with the SPB (yellow volume). MT ends structures are represented in colored spheres: red (capped structure), blue (blunt structure), cyan (open structures) and white (undetermined structure). Central volume represents the nucleus and surrounding volume represents the plasma membrane (equal pattern to all model images). See also Figure S1A and Movies S1–S3. D. Selected longitudinal tomographic sections of two adjacent MTs (asterisks) (1 and 2) showing electron-dense bridges (arrows). Cross-section of 1 and 2, respectively, in the plane of red and blue arrows. (E) Nda analysis shows that ase1Δ mutants have significantly smaller inter-MT distances than wild-type while klp2Δ cells only show a slight spacing reduction (arrowheads indicate the peak mean). Bars: 5 µm in A; 500 nm in C and 25 nm in D.

A. Number of IMAs per cell for wild-type and the different mutants. * p<0.05; ** p<0.001 (mean ± SEM). Black asterisk are in relation to wild-type and red asterisks to ase1Δ. B. GFP-tubulin labeled cells show that the triple mutant ase1Δdis1Δklp2Δ has more IMAs than wild-type or any other mutant. Regions of higher fluorescence intensity indicative of MT overlaps (arrowheads) are also visible. Over-expression of dis1p-GFP leads to the rescue of the ase1Δ phenotype. C. dis1Δ cells do not affect de novo nucleation or MT dynamics with the exception of the increase in shrinkage velocity (mean ± SEM). Bar: 3 µm in B.