Figure 4. Quantitative stochastic... | Rockefeller University Press

Figure 4.

$Figure 4. Quantitative stochastic force-balance model of the prometaphase phase I spindle: spindle MT and motor properties reveal a finely tuned steady-state. (A, from left to right) Spindle length, total AP overlap, and number of engaged Ncd and KLP61F motors on the AP overlaps during Phase I. Mole fraction of available Ncd is 0.7 (i.e., ∼2 Ncd per KLP61F). (B, top) Experimental (left) and computed (right) tubulin kymograph starting at NEB (t = 0) during prometaphase. (bottom) The corresponding pole–pole (PP) dynamics, experimental (black) and computed (blue), are shown. (C) Computed normalized spindle length dynamics in representative individual spindles: wild type (blue), Ncd null (orange), and KLP61F antibody injected into wild-type (black) or Ncd-null background (red). (D, left) Mean prometaphase phase I spindle dynamics for varying mole fractions of Ncd, increasing from 0 to 1 in 31 equally spaced steps. At t = 0 (NEB) MTs (not depicted) begin invading the spindle region. (right) Mean pole–pole separation rate (mean of instantaneous velocities during the last 40 s of prometaphase in 10 virtual spindles) plotted for varying Ncd mole fractions. The steady-state separation (mean velocity is ∼0) is confined to a narrow range of Ncd mole fractions. Error bars show SD. (E) Measurement of the molar ratio of endogenous KLP61F to Ncd. (left) Western blots showing (a) polypeptides from a serial dilution of recombinant protein (standard) purified from baculovirus expression system on the left and (b) corresponding polypeptides in high speed supernatants (HSS) from Drosophila embryo lysates; KLP61F was detected by anti-KLP61F antibody (top), and Ncd was detected by anti-Ncd antibody (bottom). (right) Corresponding standard curves showing polypeptide density versus standard protein’s amount from the left panels (KLP61F standards are shown as black dots, and Ncd standards are shown as gray dots). The amount of standard protein loaded was adjusted so that the densities of the endogenous protein (labeled as high speed supernatant; two different loading volumes for each protein) fell within the linear range, i.e., the middle of the standard curve. The amount of endogenous KLP61F and Ncd were calculated from linear fits to the standard curves. AU, arbitrary unit.$

Quantitative stochastic force-balance model of the prometaphase phase I spindle: spindle MT and motor properties reveal a finely tuned steady-state. (A, from left to right) Spindle length, total AP overlap, and number of engaged Ncd and KLP61F motors on the AP overlaps during Phase I. Mole fraction of available Ncd is 0.7 (i.e., ∼2 Ncd per KLP61F). (B, top) Experimental (left) and computed (right) tubulin kymograph starting at NEB (t = 0) during prometaphase. (bottom) The corresponding pole–pole (PP) dynamics, experimental (black) and computed (blue), are shown. (C) Computed normalized spindle length dynamics in representative individual spindles: wild type (blue), Ncd null (orange), and KLP61F antibody injected into wild-type (black) or Ncd-null background (red). (D, left) Mean prometaphase phase I spindle dynamics for varying mole fractions of Ncd, increasing from 0 to 1 in 31 equally spaced steps. At t = 0 (NEB) MTs (not depicted) begin invading the spindle region. (right) Mean pole–pole separation rate (mean of instantaneous velocities during the last 40 s of prometaphase in 10 virtual spindles) plotted for varying Ncd mole fractions. The steady-state separation (mean velocity is ∼0) is confined to a narrow range of Ncd mole fractions. Error bars show SD. (E) Measurement of the molar ratio of endogenous KLP61F to Ncd. (left) Western blots showing (a) polypeptides from a serial dilution of recombinant protein (standard) purified from baculovirus expression system on the left and (b) corresponding polypeptides in high speed supernatants (HSS) from Drosophila embryo lysates; KLP61F was detected by anti-KLP61F antibody (top), and Ncd was detected by anti-Ncd antibody (bottom). (right) Corresponding standard curves showing polypeptide density versus standard protein’s amount from the left panels (KLP61F standards are shown as black dots, and Ncd standards are shown as gray dots). The amount of standard protein loaded was adjusted so that the densities of the endogenous protein (labeled as high speed supernatant; two different loading volumes for each protein) fell within the linear range, i.e., the middle of the standard curve. The amount of endogenous KLP61F and Ncd were calculated from linear fits to the standard curves. AU, arbitrary unit.