Figure 3.
Mitotic delay increases Kip1 levels. (A) Cartoon depicting possible non-mutually exclusive models to explain the opposite response of Cin8 and Kip1 to the loss of the β-CTT. (B) Quantification of background-subtracted Cin8-mNeonGreen spindle fluorescence in wild-type KIP1 or kip1Δ cells. Values are normalized to the median wild-type value. Bolded, outlined points represent the median for each replicate. Error bars are the mean ± 95% CI for the replicate medians. KIP1 n = 95 cells, kip1Δ n = 97 cells. P = 0.03; * indicates P < 0.05. Statistics are Student’s t test for the replicate medians. (C) Quantification of background-subtracted Kip1-mNeonGreen spindle fluorescence in wild-type CIN8 or cin8Δ cells. Values are normalized to the median wild-type value. Bolded, outlined points represent the median for each replicate. Error bars are the mean ± 95% CI for the replicate medians. CIN8 n = 93 cells, cin8Δ n = 89 cells. P = 0.002; ** indicates P < 0.01. Statistics are Student’s t test for the replicate medians. (D) Doubling time in minutes for wild-type (WT), tub2-Δ430 (P < 0.0001, t test with Welch’s correction), kip1Δ (P = 0.46, t test), or cin8Δ (P = 0.0001, t test with Welch’s correction) cells. Error bars are the mean ± 95% CI. Statistics are compared with wild type. (E) Quantification of background-subtracted Cin8-3GFP spindle fluorescence in asynchronous TUB2 cells or 2-h treatment with hydroxyurea (HU) to arrest TUB2 or tub2-Δ430 cells. Values are normalized to the median asynchronous wild-type value. Bolded, outlined points represent the median for each replicate. Error bars are the mean ± 95% CI for the replicate medians. Asynchronous TUB2 n = 75 cells, HU-treated TUB2 n = 89 cells, HU-treated tub2-Δ430 n = 78 cells. Compared with asynchronous TUB2, HU-treated TUB2 P = 0.02, HU-treated tub2-Δ430 P = 0.04; HU-treated TUB2 compared with HU-treated Δ430 P = 0.0014; Student’s t test with Welch’s correction. (F) Quantification of background-subtracted Kip1-mNeonGreen spindle fluorescence in asynchronous TUB2 cells or 2-h treatment with hydroxyurea (HU) to arrest TUB2 or tub2-Δ430 cells. Values are normalized to the median asynchronous wild-type value. Bolded, outlined points represent the median for each replicate. Error bars are the mean ± 95% CI for the replicate medians. Asynchronous TUB2 n = 88 cells, HU-treated TUB2 n = 93 cells, HU-treated tub2-Δ430 n = 80 cells. Compared to asynchronous TUB2, HU-treated TUB2 P = 0.0005, HU-treated tub2-Δ430 P = 0.004; HU-treated TUB2 compared to HU-treated Δ430 P = 0.47; Student’s t test with Welch’s correction. (G) Proposed model for how the β-CTT regulates Cin8 and Kip1 with letters indicating which panel supports that conclusion. The β-CTT directly promotes Cin8 function, which in turn promotes efficient mitotic timing and inhibits excessive Kip1 spindle localization. The grey dotted lines that represent our data do not rule out the possibility that the β-CTT promotes efficient mitotic timing through other factors such as microtubule dynamics. Cin8 and Kip1 may also inhibit each other’s spindle localization by competing for binding sites on the spindle.

Mitotic delay increases Kip1 levels. (A) Cartoon depicting possible non-mutually exclusive models to explain the opposite response of Cin8 and Kip1 to the loss of the β-CTT. (B) Quantification of background-subtracted Cin8-mNeonGreen spindle fluorescence in wild-type KIP1 or kip1Δ cells. Values are normalized to the median wild-type value. Bolded, outlined points represent the median for each replicate. Error bars are the mean ± 95% CI for the replicate medians. KIP1 n = 95 cells, kip1Δ n = 97 cells. P = 0.03; * indicates P < 0.05. Statistics are Student’s t test for the replicate medians. (C) Quantification of background-subtracted Kip1-mNeonGreen spindle fluorescence in wild-type CIN8 or cin8Δ cells. Values are normalized to the median wild-type value. Bolded, outlined points represent the median for each replicate. Error bars are the mean ± 95% CI for the replicate medians. CIN8 n = 93 cells, cin8Δ n = 89 cells. P = 0.002; ** indicates P < 0.01. Statistics are Student’s t test for the replicate medians. (D) Doubling time in minutes for wild-type (WT), tub2-Δ430 (P < 0.0001, t test with Welch’s correction), kip1Δ (P = 0.46, t test), or cin8Δ (P = 0.0001, t test with Welch’s correction) cells. Error bars are the mean ± 95% CI. Statistics are compared with wild type. (E) Quantification of background-subtracted Cin8-3GFP spindle fluorescence in asynchronous TUB2 cells or 2-h treatment with hydroxyurea (HU) to arrest TUB2 or tub2-Δ430 cells. Values are normalized to the median asynchronous wild-type value. Bolded, outlined points represent the median for each replicate. Error bars are the mean ± 95% CI for the replicate medians. Asynchronous TUB2 n = 75 cells, HU-treated TUB2 n = 89 cells, HU-treated tub2-Δ430 n = 78 cells. Compared with asynchronous TUB2, HU-treated TUB2 P = 0.02, HU-treated tub2-Δ430 P = 0.04; HU-treated TUB2 compared with HU-treated Δ430 P = 0.0014; Student’s t test with Welch’s correction. (F) Quantification of background-subtracted Kip1-mNeonGreen spindle fluorescence in asynchronous TUB2 cells or 2-h treatment with hydroxyurea (HU) to arrest TUB2 or tub2-Δ430 cells. Values are normalized to the median asynchronous wild-type value. Bolded, outlined points represent the median for each replicate. Error bars are the mean ± 95% CI for the replicate medians. Asynchronous TUB2 n = 88 cells, HU-treated TUB2 n = 93 cells, HU-treated tub2-Δ430 n = 80 cells. Compared to asynchronous TUB2, HU-treated TUB2 P = 0.0005, HU-treated tub2-Δ430 P = 0.004; HU-treated TUB2 compared to HU-treated Δ430 P = 0.47; Student’s t test with Welch’s correction. (G) Proposed model for how the β-CTT regulates Cin8 and Kip1 with letters indicating which panel supports that conclusion. The β-CTT directly promotes Cin8 function, which in turn promotes efficient mitotic timing and inhibits excessive Kip1 spindle localization. The grey dotted lines that represent our data do not rule out the possibility that the β-CTT promotes efficient mitotic timing through other factors such as microtubule dynamics. Cin8 and Kip1 may also inhibit each other’s spindle localization by competing for binding sites on the spindle.

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