Figure S2.
The KI motifs and LLPS regions do not overlap in human KNL1; KNL1/Bub1 may be an RZZ receptor in human cells; and a model for how multimerization contributes to Spc105 function. (A) Schematic of human (Hs) KNL1 highlighting the N-terminal MT-(orange) and PP1-binding (magenta) motifs, the KI motifs (shades of green), and the six regions across Spc105 with catGRANULE LLPS propensity scores above 0 (blue) overlaid onto a corresponding disorder plot of KNL1. (B) The BubR1 fluorescence quantifications from Figs. 3 and 5 shown together on the same plots (WT; n = 140 kinetochores from 25 cells and two replicates, Δ267-383; n = 135 kinetochores from 24 cells and two replicates; WT; n = 150 kinetochores from 30 cells and three replicates, HsKI; n = 145 kinetochores from 29 cells and three replicates; WT; n = 150 kinetochores from 30 cells and three replicates, FUS-HsKI; n = 150 kinetochores from 30 cells and three replicates). (C) The Rod fluorescence quantifications from Figs. 3 and 5 shown together on the same plots (WT; n = 142 kinetochores from 25 cells and two replicates, Δ267–383; n = 140 kinetochores from 25 cells and two replicates; WT; n = 150 kinetochores from 30 cells and three replicates, HsKI; n = 150 kinetochores from 30 cells and three replicates; WT; n = 155 kinetochores from 31 cells and three replicates, FUS-HsKI; n = 160 kinetochores from 32 cells and three replicates). Each replicate was done with a corresponding WT control and normalized to the control mean fluorescence intensity ratios for that experiment. (D) Model of how the pathway characterized using D. melanogaster cells in this study could function in human cells where Bub1, rather than BubR1, may be an RZZ receptor. (E) Model for how higher-order multimerization via sticky IDRs contributes to Spc105 function. Spc105 is too dilute in the cytoplasm to multimerize; however, the concentration of Spc105 upon kinetochores assembly stimulates multimerization thereby increasing its avidity for the RZZ complex. Bar plots report mean ± SEM. The P values were generated by a randomization method (PlotsOfDifferences): **P < 0.001.

The KI motifs and LLPS regions do not overlap in human KNL1; KNL1/Bub1 may be an RZZ receptor in human cells; and a model for how multimerization contributes to Spc105 function. (A) Schematic of human (Hs) KNL1 highlighting the N-terminal MT-(orange) and PP1-binding (magenta) motifs, the KI motifs (shades of green), and the six regions across Spc105 with catGRANULE LLPS propensity scores above 0 (blue) overlaid onto a corresponding disorder plot of KNL1. (B) The BubR1 fluorescence quantifications from Figs. 3 and 5 shown together on the same plots (WT; n = 140 kinetochores from 25 cells and two replicates, Δ267-383; n = 135 kinetochores from 24 cells and two replicates; WT; n = 150 kinetochores from 30 cells and three replicates, HsKI; n = 145 kinetochores from 29 cells and three replicates; WT; n = 150 kinetochores from 30 cells and three replicates, FUS-HsKI; n = 150 kinetochores from 30 cells and three replicates). (C) The Rod fluorescence quantifications from Figs. 3 and 5 shown together on the same plots (WT; n = 142 kinetochores from 25 cells and two replicates, Δ267–383; n = 140 kinetochores from 25 cells and two replicates; WT; n = 150 kinetochores from 30 cells and three replicates, HsKI; n = 150 kinetochores from 30 cells and three replicates; WT; n = 155 kinetochores from 31 cells and three replicates, FUS-HsKI; n = 160 kinetochores from 32 cells and three replicates). Each replicate was done with a corresponding WT control and normalized to the control mean fluorescence intensity ratios for that experiment. (D) Model of how the pathway characterized using D. melanogaster cells in this study could function in human cells where Bub1, rather than BubR1, may be an RZZ receptor. (E) Model for how higher-order multimerization via sticky IDRs contributes to Spc105 function. Spc105 is too dilute in the cytoplasm to multimerize; however, the concentration of Spc105 upon kinetochores assembly stimulates multimerization thereby increasing its avidity for the RZZ complex. Bar plots report mean ± SEM. The P values were generated by a randomization method (PlotsOfDifferences): **P < 0.001.

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