Supplemental material

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1 Supplemental material THE JOURNAL OF CELL BIOLOGY Civelekoglu-Scholey et al., Figure S1. Spindle dynamics in Drosophila embryos and in vitro gliding assays with Ncd and KLP61F. (A) Time series of a prometaphase spindle in living transgenic Drosophila embryos expressing GFP-tubulin (green) and RFP-histone (red) taken at the indicated time points (vertical arrows) on the plot of pole pole spacing in the right panel. A stack of four planes was acquired and projected to capture the positions of the chromosomes that lie outside the plane containing the centrosomes during early prometaphase. (B) Histogram of observed instantaneous MT gliding velocities. (left) At the balance point Ncd mole fraction. (right) At all Ncd mole fractions assayed, excluding the balance point mole fraction. Note the Gaussian distribution with a single peak at all Ncd mole fractions, showing that the MTs move at slow intermediate instantaneous velocities, i.e., mean rates do not arise from mixtures of MTs moving at unloaded (free) KLP61F and Ncd velocities. (C) Distances traveled by MTs at the balance point mole fraction of Ncd in competitive gliding assays with Ncd and FL-KLP61F. (left) Plots show the typical behavior of 10 MTs. (right) Behavior of an MT exhibiting atypical, persistent unidirectional movement. Zero in the vertical axis was arbitrarily chosen as the initial position of the MT. Prometaphase spindle length maintenance Civelekoglu-Scholey et al. S1

2 Figure S2. Protein friction model results for competitive gliding assays with Ncd and HL-KLP61F and F V relations. (A) Computed MT velocity at mole fractions of Ncd ranging between [0 1], based on the protein friction model, for increasing values of the protein drag coefficient for HL-KLP61F, increasing from 20 to 200 pns/µm in steps of 20 pns/µm are shown in cold to warm colors, respectively. (B, left) Qualitative representation of the piecewise linear continuous F V relations for KLP61F and Ncd considered in the stochastic opposing power stroke model for the KLP61F and Ncd motors. Increasing superstall forces of the motors in the backward velocity regime are shown in dotted, solid, and dashed blue and red lines for KLP61F and Ncd, respectively. (right) A discontinuous F V relation, as proposed in the protein friction model of Tao et al. (2006), for Ncd and KLP61F. Note that the motor s drag coefficient,, is the slope of the F V relation in the backward velocity/superstall regime (dashed red and blue lines for KLP61F and Ncd, respectively). S2

3 Figure S3. Quantitative test of the role of motors F V relations in superstall regime in competitive gliding assays. (A) Computed MT gliding velocities (red) superimposed on experimental data (black) in competitive gliding assay for motors exerting superstall forces in the backward velocity regime (Fig. S2 B, left, dashed red and blue lines) when transiently bound to MTs gliding in the opposing direction. All other parameters are the same as in Fig. 2 A. (B) Quantitative test of the role of Ncd s F V relation in superstall regime in competitive gliding assays. Computed MT gliding velocities (red) superimposed ncd on experimental data (black) in competitive gliding assay for only Ncd exerting superstall forces in the backward velocity regime, V back = 0.02 µm/s (Fig. S2 B, left, dashed red line), i.e., when transiently bound to MTs gliding in the KLP61F direction. All other parameters are the same as in Fig. 2 A. (A and B) Error bars show SD. Prometaphase spindle length maintenance Civelekoglu-Scholey et al. S3

4 Figure S4. Quantitative test of the role of MT dynamics on prometaphase steady-state s sensitivity. Effect of MT dynamics on prometaphase phase I: varying MT catastrophe rate. (A, left) Mean pole pole separation rate (mean of instantaneous velocities in 10 virtual spindles) computed for various values of f cat. All other parameters in the model are as in Fig. 4 D. Note that the steady-state pole pole separation is lost for low f cat values (large SD at the balance point Ncd mole fraction, 0 velocity), and the steady-state becomes less sensitive to variations in the Ncd mole fraction for high f cat values. Error bars show SD. (right) The mean and SD of pole pole separation rate for indicated values of f cat at the mole fraction that gives rise to a steady-state pole separation. Note that the SD is >0.06 µm/s for low f cat values. This indicates that, even though the mean pole pole separation rate is near zero at this mole fraction, the variance is too large, and a steady-state pole pole separation cannot be maintained. (B, left) Pole pole distance over time for the indicated values of f cat at the Ncd mole fraction that gives rise to a mean zero pole pole separation rate. (right) The corresponding mean AP MT overlap over time for the spindles shown in the left panel. All other parameters in the model are as in Fig. 4 D. (C, from left to right) Mean number of engaged motors over time for the spindles in which the MT dynamics are altered (values of f cat are indicated in the plots) and whose pole pole dynamics and AP overlap is shown in B. All other parameters in the model are as in Fig. 4 D. S4

5 Figure S5. Quantitative test of the role of motors kinetics, KLP61F s ultrastructural organization, and motors F V relationships on prometaphase steadystate s sensitivity. (A C) The left panels show the spindle dynamics for varying mole fractions of Ncd (mean of 10 virtual spindles at each mole fraction), and the right panels show the mean pole pole separation rate (mean of instantaneous velocities in 10 virtual spindles) plotted for varying Ncd mole fractions (31 equally spaced steps between 0 and 1). Compare with the wild-type/control solution in Fig. 4 D. Error bars show SD. (A) Effect of motor processivity on prometaphase phase I. Here, the motors have a lower detachment rate (k off * = k off /4 for both Ncd and KLP61F) compared with the results shown in Fig. 4 D. All other model parameters are as in Fig. 4 D. (left) Spindle pole dynamics. (right) Mean pole pole separation rate in spindles with processive motors (k off * = k off /4; red) superimposed on the results shown in Fig. 4 D (black). Note that increased processivity of the motors shifts the balance point (mole fraction of Ncd for steady-state) toward higher Ncd concentrations. (B) Effect of KLP61F structure on prometaphase phase I. Here, the KLP61F is assumed to have a monopolar structure. All other model parameters are as in Fig. 4 D. (left) Spindle pole dynamics. (right) Mean pole pole separation rate in spindles with monopolar KLP61F motors (red) superimposed on the results shown in Fig. 4 D (black). Note that monopolar KLP61F shifts the balance point (mole fraction of Ncd for steady-state) toward lower Ncd concentrations and the steady-state becomes more sensitive to variations in the Ncd mole fraction. (C) Effect of motors F V relationships on prometaphase phase I. Here, both the KLP61F and Ncd motors exert superstall forces in the backward velocity regime (Fig. S2 B, left, dashed blue and red lines). All other model parameters are same as in Fig. 4 D. (left) Mean spindle pole dynamics for varying mole fractions of Ncd for motors exerting very high superstall forces while stepping backward slowly. (right) The corresponding mean pole pole separation rates (red) superimposed on the mean pole pole separation rates shown in Fig. 4 D (black) in which motors F V relations suggested from the gliding assay model fit were used. Prometaphase spindle length maintenance Civelekoglu-Scholey et al. S5

6 Video 1. Dynamics of spindle MTs and chromosomes during cycle 11 in a transgenic Drosophila embryo expressing GFPtubulin and histone-rfp. Tubulin and histone are pseudocolored in green and red, respectively. Only a single confocal plane containing the centrosomes was acquired during imaging. Images were acquired with exposure times of 1.8 s for GFP and 0.6 s for RFP, and total time shown is 432 s. The video is shown at 10 frames/s. Video 2. Dynamics of spindle MTs and chromosomes during cycle 11 in a transgenic Drosophila embryo expressing GFP-tubulin and histone-rfp. Tubulin and histone are pseudocolored in green and red, respectively. The projection of a stack of four confocal planes, to view all four pairs of chromosomes during early prometaphase, is seen in this video. Exposure time was 0.5 s for each channel, time between images is 5.94 s, and total time shown is 422 s. The video is shown at 10 frames/s. Reference Tao, L., A. Mogilner, G. Civelekoglu-Scholey, R. Wollman, J. Evans, H. Stahlberg, and J.M. Scholey A homotetrameric kinesin-5, KLP61F, bundles microtubules and antagonizes Ncd in motility assays. Curr. Biol. 16: doi: /j.cub S6