Figure 4.
The β-CTT acidic patch promotes Cin8 spindle localization and recruits Cin8 to the midzone. (A) Table of wild-type Tub2 and mutant tub2 β-CTT amino acids. The bold, underlined residues represent the “acidic patch,” E431 through E438. (B) Example pre-anaphase spindle images of Cin8-3GFP and Spc110-tdTomato (poles) in wild-type TUB2 or mutant tub2 cells. Scale bars = 1 µm. (C) Quantification of background-subtracted Cin8-3GFP pre-anaphase spindle fluorescence in wild-type TUB2 or mutant tub2 cells. Values are normalized to the median wild-type value. Bolded, outlined points represent the median for each replicate. Error bars are mean ± 95% CI for the replicate medians. TUB2: n = 113 cells; tub2-Δ430: n = 113 cells, P < 0.0001; tub2-Δ438: n = 112 cells, P = 0.66; tub2-Δ438Q: n = 117 cells, P = 0.005. Statistics are Student’s t test compared with wild-type TUB2. (D) Example anaphase spindle images of Cin8-3GFP and Spc110-tdTomato (poles) in wild-type TUB2 or mutant tub2 cells. Scale bars = 1 µm. (E) Cartoon model of methods to quantify anaphase spindle localization. A 3-pixel-wide line scan was drawn along the spindle as determined by Cin8-3GFP and Spc110-tdTomato fluorescence, and a box in the cytoplasm was used to determine the cellular background for subtraction. The length of the spindle was divided into five equally sized bins and the background-subtracted intensity within each bin along the line scan was calculated. (F) Quantification of background-subtracted Cin8-3GFP anaphase spindle fluorescence along the total length of the spindle in wild-type TUB2 or mutant tub2 cells. Values are normalized to the median wild-type value. Bolded, outlined points represent the median for each replicate. Error bars are mean ± 95% CI for the replicate medians. TUB2: n = 76 cells; tub2-Δ430: n = 69 cells, P < 0.0001; tub2-Δ438: n = 71 cells, P = 0.39; tub2-Δ438Q: n = 75 cells, P = 0.04. Statistics are Student’s t test compared with wild-type TUB2. (G) Quantification of the proportion of background-subtracted Cin8-3GFP anaphase spindle fluorescence in the middle 20% of the spindle in the same cells from G. Values are normalized to the median wild-type value. Bolded, outline pointed represent the median for each replicate. Error bars are mean ± 95% CI for the replicate medians. TUB2: n = 76 cells; tub2-Δ430: n = 69 cells, P < 0.0001; tub2-Δ438: n = 71 cells, P = 0.12; tub2-Δ438Q: n = 75 cells, P < 0.0001. Statistics are Student’s t test compared to wild-type TUB2. * indicates P < 0.05; ** indicates P < 0.01.

The β-CTT acidic patch promotes Cin8 spindle localization and recruits Cin8 to the midzone. (A) Table of wild-type Tub2 and mutant tub2 β-CTT amino acids. The bold, underlined residues represent the “acidic patch,” E431 through E438. (B) Example pre-anaphase spindle images of Cin8-3GFP and Spc110-tdTomato (poles) in wild-type TUB2 or mutant tub2 cells. Scale bars = 1 µm. (C) Quantification of background-subtracted Cin8-3GFP pre-anaphase spindle fluorescence in wild-type TUB2 or mutant tub2 cells. Values are normalized to the median wild-type value. Bolded, outlined points represent the median for each replicate. Error bars are mean ± 95% CI for the replicate medians. TUB2: n = 113 cells; tub2-Δ430: n = 113 cells, P < 0.0001; tub2-Δ438: n = 112 cells, P = 0.66; tub2-Δ438Q: n = 117 cells, P = 0.005. Statistics are Student’s t test compared with wild-type TUB2. (D) Example anaphase spindle images of Cin8-3GFP and Spc110-tdTomato (poles) in wild-type TUB2 or mutant tub2 cells. Scale bars = 1 µm. (E) Cartoon model of methods to quantify anaphase spindle localization. A 3-pixel-wide line scan was drawn along the spindle as determined by Cin8-3GFP and Spc110-tdTomato fluorescence, and a box in the cytoplasm was used to determine the cellular background for subtraction. The length of the spindle was divided into five equally sized bins and the background-subtracted intensity within each bin along the line scan was calculated. (F) Quantification of background-subtracted Cin8-3GFP anaphase spindle fluorescence along the total length of the spindle in wild-type TUB2 or mutant tub2 cells. Values are normalized to the median wild-type value. Bolded, outlined points represent the median for each replicate. Error bars are mean ± 95% CI for the replicate medians. TUB2: n = 76 cells; tub2-Δ430: n = 69 cells, P < 0.0001; tub2-Δ438: n = 71 cells, P = 0.39; tub2-Δ438Q: n = 75 cells, P = 0.04. Statistics are Student’s t test compared with wild-type TUB2. (G) Quantification of the proportion of background-subtracted Cin8-3GFP anaphase spindle fluorescence in the middle 20% of the spindle in the same cells from G. Values are normalized to the median wild-type value. Bolded, outline pointed represent the median for each replicate. Error bars are mean ± 95% CI for the replicate medians. TUB2: n = 76 cells; tub2-Δ430: n = 69 cells, P < 0.0001; tub2-Δ438: n = 71 cells, P = 0.12; tub2-Δ438Q: n = 75 cells, P < 0.0001. Statistics are Student’s t test compared to wild-type TUB2. * indicates P < 0.05; ** indicates P < 0.01.

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