Table I.

Parameters of dynamic instability

ControlCLASP knockdown
1-μm region at the cell edgeCell interior1-μm region at the cell edgeCell interior
Rate of growth, μm/min
Direct MT observation9.9 ± 3.511.7 ± 4.811.1 ± 1.412.6 ± 5.1
Subtraction analysis14.4 ± 3.918.2 ± 5.8
EB1 tracks15.9 ± 6.022.8 ± 7.3
Rate of shortening, μm/min
Direct MT observation      −12.0 ± 6.2−20.7 ± 11.6      −12.6 ± 6.5−17.8 ± 11.3
Subtraction analysis−35.6 ± 11.2−28.2 ± 9.8
Transition frequencies
Growth–Shortening, s−10.0780.0350.0300.045
Growth–Pause, s−10.4020.2380.3600.250
Shortening–Growth, s−10.0660.0850.0190.072
Shortening–Pause, s−10.2750.2130.2170.210
Pause–Growth, s−10.0160.0530.0320.046
Pause–Shortening, s−10.0260.0380.0380.040
Time in growth, %6.016.713.314.5
Time in pauses, %88.969.380.170.7
Time in shortening, %5.114.06.614.8
  • HeLa cells, stably expressing GFP-α-tubulin or EB1-GFP, were imaged with a 2-s interval 72 h after transfection with the control or CLASP1+2#B siRNAs. Life history plots of 65 MTs in five control cells and 75 MTs in five CLASP knockdown cells were analyzed as described by Shelden and Wadsworth (1993). In addition, to obtain more accurate values of growth and shortening rates in the internal cytoplasm, subtraction analysis (Vorobjev et al., 1999) and measurements of displacements of EB1-GFP comets were used. The differences in rates, obtained by different methods, can be explained by a higher contribution of the pausing state to the growth/shortening episodes at the cell periphery, which can be detected by direct observation, as compared with those inside the cells, which can be identified by subtraction analysis. EB1 associates predominantly with growing MTs (Mimori-Kiyosue et al., 2000) and, therefore, MT pauses hardly contribute to the growth episodes, which are detected by this method. The data are presented in format: mean ± SD. Rates are instantaneous (measured within one interval between successive frames).