-Tubulin Is Required for Proper Recruitment and Assembly of Kar9–Bim1 Complexes in Budding Yeast

Supplemental Material

This article contains the following supporting material:

• Movie 1 - Dynamic Kar9-GFP localization in the bud of a wild-type cell.
  Time-lapse of a small budded wild-type cell expressing Kar9-GFP (green) and Spc42-CFP (red) fusion proteins. In wild-type cells, Kar9-GFP foci exhibit dynamic movements towards and away from the SPB. Kar9-GFP foci probe the bud neck/cortex and then retract back to the SPB to facilitate spindle placement at the bud neck. 0.5μm optical sections for Kar9-GFP and Spc42-CFP were acquired through a 4.0 μm stack at 10 sec intervals for a total time of 5 minutes.

• Movie 2 - Kar9-GFP dwelling in the bud of a tub4-Δdsyl cell.
  Time-lapse of a small budded tub4-Δdsyl cell expressing Kar9-GFP (green) and Spc42-CFP (red) fusion proteins. In tub4-Δdsyl cells, Kar9-GFP foci dwell within the bud cortex and do not exhibit dynamic movements between the bud and the SPB. As a result, the spindle is often mis-positioned and localized >2μm from the bud neck. 0.5μm optical sections for Kar9-GFP and Spc42-CFP were acquired through a 4.0 μm stack at 10 sec intervals for a total time of 5 minutes.

• Movie 3 - Kar9-GFP dwelling in the bud of a tub4-Δdsyl cell (vector control).
  Time-lapse of a small budded tub4-Δdsyl cell containing the control vector pRS423 and expressing Kar9-GFP (green) and Spc42-CFP (red) fusion proteins. tub4-Δdsyl pRS423 cells behave similar to tub4-Δdsyl cells in that Kar9-GFP foci remain statically localized near the bud cortex and rarely retract back to the SPB. Notice that the spindle is pushed away from the bud neck and out of the plane of focus as a result of the Kar9 dwelling. 0.5μm optical sections for Kar9-GFP and Spc42-CFP were acquired through a 4.0 μm stack at 10 sec intervals for a total time of 5 minutes.

• Movie 4 - Kar9-GFP movements in the bud of a tub4-Δdsyl cell over-producing Bim1.
  Time-lapse of a small budded tub4-Δdsyl cell over-producing Bim1 (pBIM1) and expressing Kar9-GFP (green) and Spc42-CFP (red) fusion protein. Over production of Bim1 was found to restore Kar9-GFP dynamic movements/localization in tub4-Δdsyl cells. Kar9-GFP foci dynamically probed the bud neck/cortex and retracted back to the SPB in a manner similar to wild-type cells. 0.5μm optical sections for Kar9-GFP and Spc42-CFP were acquired through a 4.0 μm stack at 10 sec intervals for a total time of 5 minutes.