Figure 5.
The β-CTT promotes Cin8 plus-end motility. (A) Example kymographs from timelapse fluorescence microscopy of Cin8-3GFP and Spc110-tdTomato (poles) in cells at 30°C. Arrowheads represent midzone-directed motility events. Asterisks mark spindle midzone. (B) Quantification of velocity of Cin8 midzone-directed motility events in cells expressing TUB2 (n = 20 events) or tub2-Δ438Q (n = 15 events). Error bars are mean ± 95% CI. P = 0.0046; student’s t test. (C) Cartoon model of in vitro Cin8-3GFP motility on yeast microtubules; arrows indicate steps in the protocol. First, a concentrated yeast lysate was used to grow yeast microtubules from rhodamine-labeled, porcine brain GMPCPP seeds. Next, proteins were removed from the grown microtubules with several wash steps. Microtubules were maintained with a high concentration of stabilizing epothilone A (Epo). Finally, a dilute yeast lysate was used to image motility. An example kymograph is shown on the right. (D) Quantification of Cin8 plus-end-directed velocity. TUB2: n = 111 events; tub2-Δ438Q: n = 113 events, P = 0.0056; tub2-Δ438: n = 149 events, P = 0.67. Statistics are Student’s t test of replicate medians compared with wild-type TUB2. Error bars mean ± 95% CI. (E) Quantification of Cin8 plus-end distance traveled during motility event from D with a start and stop during the imaging. TUB2: n = 89 events; tub2-Δ438Q: n = 84 events, P = 0.041; tub2-Δ438: n = 119 events, P = 0.81. Statistics are Student’s t test of replicate medians compared to wild-type TUB2. Error bars mean ± 95% CI. (F) Quantification of time spent moving during a plus-end-directed motility event from D with a start and stop during the imaging. TUB2: n = 89 events; tub2-Δ438Q: n = 84 events, P = 0.63; tub2-Δ438: n = 119 events, P = 0.81. Statistics are Student’s t test of replicate medians compared to wild-type TUB2. Error bars mean ± 95% CI. (G) Quantification of the number of foci per microtubule (MT) length. TUB2: n = 180 microtubules; tub2-Δ438Q: n = 195 microtubules, P = 0.023; tub2-Δ438: n = 196 microtubules, P = 0.61. Statistics are student’s t test of replicate medians compared to wild-type TUB2. Error bars mean ± 95% CI. * indicates P < 0.05; ** indicates P < 0.01.

The β-CTT promotes Cin8 plus-end motility. (A) Example kymographs from timelapse fluorescence microscopy of Cin8-3GFP and Spc110-tdTomato (poles) in cells at 30°C. Arrowheads represent midzone-directed motility events. Asterisks mark spindle midzone. (B) Quantification of velocity of Cin8 midzone-directed motility events in cells expressing TUB2 (n = 20 events) or tub2-Δ438Q (n = 15 events). Error bars are mean ± 95% CI. P = 0.0046; student’s t test. (C) Cartoon model of in vitro Cin8-3GFP motility on yeast microtubules; arrows indicate steps in the protocol. First, a concentrated yeast lysate was used to grow yeast microtubules from rhodamine-labeled, porcine brain GMPCPP seeds. Next, proteins were removed from the grown microtubules with several wash steps. Microtubules were maintained with a high concentration of stabilizing epothilone A (Epo). Finally, a dilute yeast lysate was used to image motility. An example kymograph is shown on the right. (D) Quantification of Cin8 plus-end-directed velocity. TUB2: n = 111 events; tub2-Δ438Q: n = 113 events, P = 0.0056; tub2-Δ438: n = 149 events, P = 0.67. Statistics are Student’s t test of replicate medians compared with wild-type TUB2. Error bars mean ± 95% CI. (E) Quantification of Cin8 plus-end distance traveled during motility event from D with a start and stop during the imaging. TUB2: n = 89 events; tub2-Δ438Q: n = 84 events, P = 0.041; tub2-Δ438: n = 119 events, P = 0.81. Statistics are Student’s t test of replicate medians compared to wild-type TUB2. Error bars mean ± 95% CI. (F) Quantification of time spent moving during a plus-end-directed motility event from D with a start and stop during the imaging. TUB2: n = 89 events; tub2-Δ438Q: n = 84 events, P = 0.63; tub2-Δ438: n = 119 events, P = 0.81. Statistics are Student’s t test of replicate medians compared to wild-type TUB2. Error bars mean ± 95% CI. (G) Quantification of the number of foci per microtubule (MT) length. TUB2: n = 180 microtubules; tub2-Δ438Q: n = 195 microtubules, P = 0.023; tub2-Δ438: n = 196 microtubules, P = 0.61. Statistics are student’s t test of replicate medians compared to wild-type TUB2. Error bars mean ± 95% CI. * indicates P < 0.05; ** indicates P < 0.01.

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