Figure 1. Kinetochore-microtubule... | Rockefeller University Press

Figure 1.

$Figure 1. Kinetochore-microtubule occupancy scales linearly with the number of functional Hec1 subunits. (A) Schematic depicting experimental design and expected outcomes. After deleting endogenous Hec1, strong (Hec1-1D, blue) and weak (Hec1-9D, pink) microtubule-binding mutants are expressed. Cells randomly receive different fractions of functional binders and therefore have different microtubule occupancies. Depending on whether Hec1 subunits bind microtubules cooperatively or independently, microtubule attachment may change rapidly or gradually. Hec1-9D kinetochores are depicted without attached microtubules for simplicity but may have low-affinity attachments. (B) Immunofluorescence imaging (maximum-intensity projection) of microtubule attachments (tubulin), Hec1-1D intensity (anti-EGFP), and Hec1-9D intensity (anti-mKate, binds to FusionRed) in Hec1 knockout cells expressing Hec1-1D-EGFP and Hec1-9D-FusionRed. Cells were treated with 5 µM MG132 to accumulate them at a metaphase spindle steady state. The two highlighted examples were taken from the same coverslip, where the top has a high Hec1-1D to -9D ratio and the bottom a low ratio. Scale bars = 3 µm (large) and 1 µm (zoom). (C and D) Mean of cellular EGFP fraction for each cell versus mean cellular K-K distance (C) and mean cellular kinetochore microtubule intensity (D) from Hec1 knockout cells in B with mixed kinetochores (n = 345 pairs, 690 kinetochores, 23 cells; green) and cells with control Hec1-1D alone (n = 270, 540, 18; blue) and Hec1-9D alone coverslips (n = 300, 600, 20; pink; D). The relationship between microtubule attachment and amount of strong binders fits a linear relationship (r2 = 0.57) better than an exponential one (r2 = 0.51). Error bars = SEM. All data displayed were acquired at the same time for all conditions and with the data in Fig. 2.$

Kinetochore-microtubule occupancy scales linearly with the number of functional Hec1 subunits. (A) Schematic depicting experimental design and expected outcomes. After deleting endogenous Hec1, strong (Hec1-1D, blue) and weak (Hec1-9D, pink) microtubule-binding mutants are expressed. Cells randomly receive different fractions of functional binders and therefore have different microtubule occupancies. Depending on whether Hec1 subunits bind microtubules cooperatively or independently, microtubule attachment may change rapidly or gradually. Hec1-9D kinetochores are depicted without attached microtubules for simplicity but may have low-affinity attachments. (B) Immunofluorescence imaging (maximum-intensity projection) of microtubule attachments (tubulin), Hec1-1D intensity (anti-EGFP), and Hec1-9D intensity (anti-mKate, binds to FusionRed) in Hec1 knockout cells expressing Hec1-1D-EGFP and Hec1-9D-FusionRed. Cells were treated with 5 µM MG132 to accumulate them at a metaphase spindle steady state. The two highlighted examples were taken from the same coverslip, where the top has a high Hec1-1D to -9D ratio and the bottom a low ratio. Scale bars = 3 µm (large) and 1 µm (zoom). (C and D) Mean of cellular EGFP fraction for each cell versus mean cellular K-K distance (C) and mean cellular kinetochore microtubule intensity (D) from Hec1 knockout cells in B with mixed kinetochores (n = 345 pairs, 690 kinetochores, 23 cells; green) and cells with control Hec1-1D alone (n = 270, 540, 18; blue) and Hec1-9D alone coverslips (n = 300, 600, 20; pink; D). The relationship between microtubule attachment and amount of strong binders fits a linear relationship (r² = 0.57) better than an exponential one (r² = 0.51). Error bars = SEM. All data displayed were acquired at the same time for all conditions and with the data in Fig. 2.