Figure 6.
The Cin8 N-terminal extension interacts with the β-CTT. (A) Computational isoelectric point analysis of kinesin motor domains categorized by secondary structure regions from the amino-terminal through the neck linker for Cin8, Kip1, KIF11 (human kinesin-5), or KIF5B (human kinesin-1). (B) Image of a heterodimer (Tub1 and Tub2) demonstrating the electrostatic surface potential for β-tubulin, including the β-CTT (left, bracket marks acidic patch), or depicting a feasible interaction with the intrinsically disordered Cin8 N-terminal region from a side view (middle). A top view looking down on the heterodimer is shown on the right. Protein surfaces are colored by electrostatic potential calculated by Poisson-Boltzmann solution. Arrow generally marks Cin8 N-term, and arrowhead marks β-CTT. (C) Images demonstrating feasible interactions of the intrinsically disordered kinesin N-terminal regions and the β-CTTs. For kinesin motor domains and β-tubulin, protein surfaces are colored by electrostatic potential calculated by Poisson-Boltzmann solution. Arrows generally mark kinesin N-termini and arrowheads mark β-CTT. For Kip1, Tub1 (α-tubulin) and Tub2 (β-tubulin) are modeled. For KIF11 and KIF5B, TUBA1A (α-tubulin) and TUBB5 (β-tubulin) are modeled. (D) Example images of pre-anaphase cells expressing Cin8-3GFP, cin8K1NT-3GFP, or cin8K1L8-3GFP. Poles are labeled with Spc110-tdTomato. Scale bars = 1 µm. (E) Quantification of pre-anaphase spindle fluorescence of cells expressing wild-type Cin8-3GFP (n = 61 cells), cin8K1NT-3GFP (n = 88 cells, P < 0.001), or cin8K1L8-3GFP (n = 84, P = 0.037). Statistics are Student’s t test of replicate medians compared to wild-type Cin8. * indicates P < 0.05; ** indicates P < 0.01. Error bars mean ± 95% CI. (F) Quantification of pre-anaphase spindle fluorescence of cells expressing wild-type Cin8-3GFP (n = 68 cells), cin8K1NT-3GFP (n = 62 cells, P = 0.005), or cin8K1NT-3GFP and tub2-Δ438Q (n = 65 cells, P = 0.039). For E and F, values are normalized to the median wild-type value. Bolded, outlined points represent the median for each replicate. Statistics are Student’s t test of replicate medians compared to wild-type Cin8. Error bars mean ± 95% CI.

The Cin8 N-terminal extension interacts with the β-CTT. (A) Computational isoelectric point analysis of kinesin motor domains categorized by secondary structure regions from the amino-terminal through the neck linker for Cin8, Kip1, KIF11 (human kinesin-5), or KIF5B (human kinesin-1). (B) Image of a heterodimer (Tub1 and Tub2) demonstrating the electrostatic surface potential for β-tubulin, including the β-CTT (left, bracket marks acidic patch), or depicting a feasible interaction with the intrinsically disordered Cin8 N-terminal region from a side view (middle). A top view looking down on the heterodimer is shown on the right. Protein surfaces are colored by electrostatic potential calculated by Poisson-Boltzmann solution. Arrow generally marks Cin8 N-term, and arrowhead marks β-CTT. (C) Images demonstrating feasible interactions of the intrinsically disordered kinesin N-terminal regions and the β-CTTs. For kinesin motor domains and β-tubulin, protein surfaces are colored by electrostatic potential calculated by Poisson-Boltzmann solution. Arrows generally mark kinesin N-termini and arrowheads mark β-CTT. For Kip1, Tub1 (α-tubulin) and Tub2 (β-tubulin) are modeled. For KIF11 and KIF5B, TUBA1A (α-tubulin) and TUBB5 (β-tubulin) are modeled. (D) Example images of pre-anaphase cells expressing Cin8-3GFP, cin8K1NT-3GFP, or cin8K1L8-3GFP. Poles are labeled with Spc110-tdTomato. Scale bars = 1 µm. (E) Quantification of pre-anaphase spindle fluorescence of cells expressing wild-type Cin8-3GFP (n = 61 cells), cin8K1NT-3GFP (n = 88 cells, P < 0.001), or cin8K1L8-3GFP (n = 84, P = 0.037). Statistics are Student’s t test of replicate medians compared to wild-type Cin8. * indicates P < 0.05; ** indicates P < 0.01. Error bars mean ± 95% CI. (F) Quantification of pre-anaphase spindle fluorescence of cells expressing wild-type Cin8-3GFP (n = 68 cells), cin8K1NT-3GFP (n = 62 cells, P = 0.005), or cin8K1NT-3GFP and tub2-Δ438Q (n = 65 cells, P = 0.039). For E and F, values are normalized to the median wild-type value. Bolded, outlined points represent the median for each replicate. Statistics are Student’s t test of replicate medians compared to wild-type Cin8. Error bars mean ± 95% CI.

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