Table 1. Comparison of effects of acute optogenetic removal of PRC1 and long-term depletion by siRNA.
All values are given as mean ± s.e.m. The numbers in the brackets denote the number of measurements and cells, respectively; a single number is the number of cells. Symbols (arrows and equal signs) denote trend of change of parameters; equal sign means no change; two arrows mark stronger effect. Measurements include cells without and with SiR-tubulin. Signal intensities of microtubule-associated proteins were normalized to the mean value of the signal of corresponding control for each treatment. BAC denotes live-cell experiments on HeLa cells expressing a fluorescently tagged protein from a BAC, immuno denotes immunocytochemistry experiments. #Consistent with our previous studies (Kajtez et al., 2016; Polak et al., 2017).
| Parameter | Acute removal | Long-term depletion | |||||||
|---|---|---|---|---|---|---|---|---|---|
| 0 min | 20 min | p-value | Untreated | siRNA | p-value | ||||
| dEQ (µm) | 0.78 ± 0.04 (290, 17) |
0.94 ± 0.05 (240, 16) |
0.035 | ↑ | 0.69 ± 0.05 (107, 8) |
0.69 ± 0.03 (333, 17) |
0.64 | = | |
| dEQ > 2 µm (%) | 4.1 ± 1.2 (290, 17) |
8.3 ± 1.8 (240, 16) |
0.043 | ↑ | 3.3 ± 1.0 (107, 8) |
0.9 ± 0.9 (333, 17) |
0.33 | = | |
| dKC (µm) | 0.87 ± 0.01 (226, 18) |
0.79 ± 0.01 (186, 18) |
1*10−8 | ↓ | 0.85 ± 0.01 (75, 8)# |
0.780 ± 0.008 (202, 17)# |
4*10−6 | ↓ | |
| αKC (°) | 13.8 ± 0.7 (290, 17) |
18.4 ± 1.0 (240, 16) |
0.0013 | ↑ | 13.3 ± 1.0 (107, 8) |
10.3 ± 0.7 (333, 17) |
1*10−4 | ↓ | |
| αKC > 35° (%) | 6.2 ± 1.4 (290, 17) |
12.1 ± 2.1 (240, 16) |
0.018 | ↑ | 4.7 ± 2.0 (107, 8) |
3.6 ± 1.0 (333, 17) |
0.83 | = | |
| Lagging kinetochores (%) | 23 ± 7 (39) ctrl |
46 ± 9 (28) opto |
0.044 | ↑ | 5 ± 4 (37) |
40 ± 11 (20) |
0.0036 | ↑ | |
| EB3 comets (min−1) | 1.8 ± 0.1 (47, 5) ctrl |
2.0 ± 0.3 (26, 3) opto |
0.59 | = | 1.91 ± 0.09 (144, 6) |
1.5 ± 0.1 (54, 4) |
0.01 | ↓ | |
| EB3 comets velocity (µm/min) | 19 ± 1 (17, 9) ctrl |
19.6 ± 0.8 (23, 9) opto |
0.73 | = | 16.0 ± 0.6 (16, 6) |
14.7 ± 0.8 (14, 4) |
0.19 | = | |
| Half-overlap length (µm) | 1.8 ± 0.2 (17, 9) ctrl |
2.6 ± 0.2 (23, 9) opto |
0.009 | ↑ | 2.0 ± 0.2 (16, 6) |
1.8 ± 0.2 (14, 4) |
0.45 | = | |
| N (MTs) in the bridging fiber | 14 ± 2 (22, 13) |
5.6 ± 0.9 (19, 10) |
0.0038 | ↓ | 14 ± 2 (18, 15) |
7.5 ± 1.1 (26, 18) |
0.0028 | ↓ | |
| Curvature (µm−1) |
0.134 ± 0.004 (40, 10) |
0.081 ± 0.005 (39, 10) |
6*10−11 | ↓↓ | 0.137 ± 0.006 (20, 5) |
0.108 ± 0.004 (52, 13) |
0.0014 | ↓ | |
| θ (°) | 142 ± 3 (20, 10) |
125 ± 3 (20, 10) |
2*10−4 | ↓ | 145 ± 3 (10, 5) |
137 ± 2 (26, 13) |
0.051 | = | |
| Kif4A intensity in the bridging fiber | 1 ± 0.1 (54, 11) |
0.24 ± 0.05 (32, 6) |
1*10−4 | ↓↓ | immuno | 1 ± 0.03 (72, 17) |
0.48 ± 0.02 (58, 15) |
1*10−4 | ↓ |
| BAC | 1 ± 0.1 (43, 12) |
0.35 ± 0.04 (43, 11) |
1*10−4 | ↓ | |||||
| Kif18A intensity in the bridging fiber | 1 ± 0.1 (57, 15) |
0.57 ± 0.09 (28, 11) |
0.01 | ↓ | immuno | 1 ± 0.07 (121, 33) |
0.62 ± 0.05 (89, 40) |
1*10−4 | ↓ |
| BAC | 1 ± 0.1 (38, 14) |
0.53 ± 0.11 (16, 6) |
0.009 | ↓ | |||||
| MKLP1 intensity in the bridging fiber | 1 ± 0.19 (25, 6) |
0.13 ± 0.05 (20, 6) |
0.008 | ↓ | 1 ± 0.22 (29, 8) |
0.17 ± 0.10 (20, 6) |
0.008 | ↓ | |