Figure 6.
Tub1 preferentially directs Dynein pathway proteins to astral microtubule plus ends.(a) Schematic of Dyn1-3GFP localization (green) in WT cells. (b) Dyn1-3GFP localization to microtubule plus ends. Left: Representative wide-field and DIC images showing Dyn1-3GFP localization to SPB and astral microtubule plus end. Right: Dyn1-3GFP signal at microtubule plus ends. (c) Percentage of cells with visible Dyn1-3GFP foci at astral microtubule plus ends. (d) Dyn1-3GFP localization to mother cell cortex. Left: Cell images showing Dyn1-3GFP localization at mother cortex. Right: Dyn1-3GFP signal at mother cortex foci. (e) Bik1 localization to microtubule plus ends. Left: Cell images showing Bik1-3GFP localization to the spindle, SPBs and astral microtubule plus ends. Right: Bik1-3GFP signal at microtubule plus ends. (f) Bik1 localization along astral microtubules. Left: Cell images showing Bik1-3GFP localization along astral microtubules. Right: Number of Bik1-3GFP foci along bud-directed astral microtubules. For Tub1 only versus Tub3 only, P = 0.002, 0.04, and 0.004 for one, two, and three or more foci; for Tub3 only versus WT-Tub3, P = 0.012 and 0.024 for one and two foci, respectively. (g) Kip2 localization to microtubule plus ends. Left: Cell images showing Kip2-3YFP localization along and at plus ends of astral microtubules. Right: Kip2-3YFP signal at microtubule plus ends. For panels b–g, microtubules are visualized by mRUBY3-Tub1 or mRUBY3-Tub3; merge shows microtubules in red and GFP-tagged proteins in green. Graphs show mean ± SEM from three trials. In panel b, for Tub1 only, n = 47, 58, and 60; for WT-Tub1, n = 54, 57, and 70; for WT-Tub3, n = 54, 68, and 52; for Tub3 only, n = 61, 60, and 51. In panel c, for Tub1 only, n = 69, 59, and 58; for WT-Tub1, n = 82, 55, and 61; for WT-Tub3, n = 63, 60, and 87; for Tub3 only, n = 53, 63, and 85. In panel d, for Tub1 only, n = 26, 48, and 42; for WT-Tub1, n = 38, 25, and 21; for WT-Tub3, n = 40, 54, and 54; for Tub3 only, n = 55, 42, and 25. In panel e, for Tub1 only, n = 49, 58, and 57; for WT-Tub1, n = 56, 50, and 60; for WT-Tub3, n = 56, 62, and 60; for Tub3 only, n = 50, 61, and 55. In panel f, for Tub1 only, n = 65, 62, and 63; for WT-Tub1, n = 76, 73, and 61; for WT-Tub3, n = 67, 63, and 70; for Tub3 only, n = 68, 63, and 81. In panel g, for Tub1 only, n = 59, 60, and 60; for WT-Tub1, n = 60, 60, and 67; for WT-Tub3, n = 60, 60, and 60; for Tub3 only, n = 63, 60, and 60. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 by unpaired, two-tailed Student’s t test. Scale bars = 2 µm. MT, microtubule.

Tub1 preferentially directs Dynein pathway proteins to astral microtubule plus ends. (a) Schematic of Dyn1-3GFP localization (green) in WT cells. (b) Dyn1-3GFP localization to microtubule plus ends. Left: Representative wide-field and DIC images showing Dyn1-3GFP localization to SPB and astral microtubule plus end. Right: Dyn1-3GFP signal at microtubule plus ends. (c) Percentage of cells with visible Dyn1-3GFP foci at astral microtubule plus ends. (d) Dyn1-3GFP localization to mother cell cortex. Left: Cell images showing Dyn1-3GFP localization at mother cortex. Right: Dyn1-3GFP signal at mother cortex foci. (e) Bik1 localization to microtubule plus ends. Left: Cell images showing Bik1-3GFP localization to the spindle, SPBs and astral microtubule plus ends. Right: Bik1-3GFP signal at microtubule plus ends. (f) Bik1 localization along astral microtubules. Left: Cell images showing Bik1-3GFP localization along astral microtubules. Right: Number of Bik1-3GFP foci along bud-directed astral microtubules. For Tub1 only versus Tub3 only, P = 0.002, 0.04, and 0.004 for one, two, and three or more foci; for Tub3 only versus WT-Tub3, P = 0.012 and 0.024 for one and two foci, respectively. (g) Kip2 localization to microtubule plus ends. Left: Cell images showing Kip2-3YFP localization along and at plus ends of astral microtubules. Right: Kip2-3YFP signal at microtubule plus ends. For panels b–g, microtubules are visualized by mRUBY3-Tub1 or mRUBY3-Tub3; merge shows microtubules in red and GFP-tagged proteins in green. Graphs show mean ± SEM from three trials. In panel b, for Tub1 only, n = 47, 58, and 60; for WT-Tub1, n = 54, 57, and 70; for WT-Tub3, n = 54, 68, and 52; for Tub3 only, n = 61, 60, and 51. In panel c, for Tub1 only, n = 69, 59, and 58; for WT-Tub1, n = 82, 55, and 61; for WT-Tub3, n = 63, 60, and 87; for Tub3 only, n = 53, 63, and 85. In panel d, for Tub1 only, n = 26, 48, and 42; for WT-Tub1, n = 38, 25, and 21; for WT-Tub3, n = 40, 54, and 54; for Tub3 only, n = 55, 42, and 25. In panel e, for Tub1 only, n = 49, 58, and 57; for WT-Tub1, n = 56, 50, and 60; for WT-Tub3, n = 56, 62, and 60; for Tub3 only, n = 50, 61, and 55. In panel f, for Tub1 only, n = 65, 62, and 63; for WT-Tub1, n = 76, 73, and 61; for WT-Tub3, n = 67, 63, and 70; for Tub3 only, n = 68, 63, and 81. In panel g, for Tub1 only, n = 59, 60, and 60; for WT-Tub1, n = 60, 60, and 67; for WT-Tub3, n = 60, 60, and 60; for Tub3 only, n = 63, 60, and 60. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 by unpaired, two-tailed Student’s t test. Scale bars = 2 µm. MT, microtubule.

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