FIGURE 5:

Elevated Alp16^GCP6 expression increases γTuC levels at mitotic SPBs and spindle microtubule levels. (A) Increase in expression levels of Alp16^GCP6 increases γTuC levels at SPBs. Alp16^GCP6 was expressed at the lys1 locus in alp16Δ cells containing GFP-Alp4^GCP2 and mCh-Atb2 under the control of thiamine-repressible P81nmt, P41nmt, or P3nmt promoter in the presence (+T) and absence (−T) of 2 μM thiamine at 27ºC for 22–24 h. Alp16^GCP6 expression levels were the lowest under the P81nmt promoter with thiamine (P81-Alp16 (+T)) and increased in the following order: P81 (+T) < P41 (+T) < P3 (+T) < P81 (−T) < P41 (−T) < P3 (−T). The intensity of GFP-Alp4^GCP2 signals at SPBs increases along with the increase in expression levels of Alp16^GCP6 but decreases at the highest expression with P3nmt promoter in the absence of thiamine. Scale bar, 10 μm. (B) Typical images of spindles observed in wild-type, alp16Δ, and alp16Δ cells with P81nmt-Alp16 (−T). Note that spindles look thicker with higher levels of γTuC at SPBs. (C) Elevated Alp16^GCP6 expression increases γTuC levels at mitotic SPBs and spindle microtubule levels. Quantification of Alp4^GCP2 (γTuC) and microtubule levels for spindles <3.5 μm in length. The p values are from unpaired t test: ****p < 0.0001, *p < 0.05, and ns, p > 0.05. (D) The ratio of spindle microtubule to γTuC level increases when Alp16^GCP6 is expressed. Spindle microtubule level was plotted against Alp4^GCP2 level using the data used for C. The dotted line was drawn connecting alp16Δ and wild-type levels, assuming that the increase in Alp4^GCP2 level increases spindle microtubule level linearly (y = 0.69x + 0.31, where x = Alp4^GCP2 level and y = spindle microtubule level). Alp16^GCP6 expression increases spindle microtubule level to higher than the expected value from the equation. The ratio of spindle microtubule to Alp4^GCP2 level was calculated as a percentage of the expected value and plotted against the Alp4^GCP2 level. (E) Gfh1^GCP4 and Mod21^GCP5 localize to mitotic SPBs when Alp16^GCP6 is overexpressed in alp16Δ cells. Localization of GFP-Gfh1^GCP4 and GFP- Mod21^GCP5 was observed in alp16Δ cells with P3nmt-Alp16 (+T). Scale bar, 10 μm. (F) Gfh1^GCP4 and Mod21^GCP5 are not required for Alp16^GCP6-dependent γTuC recruitment and spindle microtubule assembly. Alp4^GCP2 (γTuC) and microtubule levels for spindles <3.5 μm in length were quantified in wild-type, alp16Δ, alp16Δ with P3-Alp16 (+T), alp16Δ gfh1Δ with P3-Alp16 (+T), and alp16Δ gfh1Δ mod21Δ cells with P3-Alp16 (+T). The p values are from unpaired t test: ****p < 0.0001, **p < 0.01, and ns, p > 0.05.