Table 1.
Spindle phenotype and γ-tubulin localization analysis after RNAi
| Targeted gene product |
Relative mitotic index |
Spindle phenotype |
% abnormal spindles (in %), (n) |
Spindles with localized γ-tubulin (in %), (n) |
Knockdown confirmation |
|---|---|---|---|---|---|
| None | 1 | 24±9 (496)† | 100±0 (114) | ||
| γ-tubulin (23C) | 3.3±0.7* | Monopolar/anastral | 98±2 (279) | 3±2 (118) | |
| Dgrip84 | 2.8±0.9* | Monopolar/anastral | 98±3 (303) | 14±5 (146) | + |
| Dgrip91 | 2.9±0.6* | Monopolar/anastral | 95±3 (371) | 11±5 (133) | + |
| Dgrip128 | 2.6±0.3* | Monopolar/anastral | 84±4 (285) | 94±3 (125) | − |
| Dgrip163 | 2.0±0.7* | Monopolar/anastral | 76±5 (301) | 89±7 (166) | − |
| Dgrip71 | 2.5±0.3* | Monopolar/anastral | 85±7 (385) | 96±1 (82) | − |
| Dgrip75 | 2.3±0.3* | Monopolar/anastral | 82±1 (331) | 91±5 (129) | − |
| Msps | 3.2±0.4* | Multiple defects | 97±2 (292) | 99±1 (135) | + |
| Msps and Asp | 2.0±0.6* | Bipolar/anastral spindles | 88±5 (134) | 95±4 (111) | − |
| Centrosomin | 1.0±0.2 | Anastral | 93±4 (246) | 10±1 (108) | + |
| Asp | 1.2±0.7 | Detached/unfocused spindles poles | 91±6 (222) | 98±3 (109) | − |
| Dmob4 | 1.1±0.4 | Monopolar/monastral bipolar | 73±7 (342) | 96±6 (83) | − |
| Mps1 | 0.5±0.2* | Monastral bipolar/other | 51±2 (122) | 99±2 (63) | − |
| γ-tubulin (37C) | 0.9±0.5 | Normal | 33±11 (235) | 99±2 (85) | − |
| Dgrip223○ | 1.2±0.1 | Normal | 26±8 (152) | 100 (39) | ○ |
| Dgrip79○ | 0.7±0.6 | Normal | 29±4 (176) | 100±0 (84) | ○ |
| D-TACC | 1.2±0.7 | Normal | 25±7 (241) | 100±0 (101) | + |
| Centrin 1 | 0.6±0.1 | Normal | 15±6 (190) | 99±2 (105) | − |
| Centrin 2○ | 0.6±0.1 | Normal | 26±1 (174) | 100±0 (77) | ○ |
| Niki/Nek2○ | 1.1±0.1 | Normal | 30±5 (168) | 100±0 (89) | ○ |
| CP60 | 0.9±0.3 | Normal | 36±3 (260) | 94±4 (116) | + |
| CP190 | 0.9±0.2 | Normal | 24±6 (241) | 99±2 (77) | + |
| Dmob1 | 0.8±2.4 | Normal | 25±2 (247) | 100± (93) | − |
| Dmob2 | 1.0±0.2 | Normal | 24±10 (286) | 100±0 (19) | − |
| Dmob3 | 1.3±0.4 | Normal | 24±3 (484) | 95±1 (148) | − |
| Pericentrin | 0.8±0.3 | Normal | 24±12 (206) | 100±0 (99) | − |
For complete gene information, including CG-identification numbers and RNAi primer sequences, see supplementary material Table S1. Cells were analyzed on day 4 or 5 of RNAi treatment. Values of mean ± s.d. are from three individual experiments unless noted otherwise. n=number of spindles observed.
Dmob1, Dmob2, Dmob3, Dmob4; Drosophila Mob1 (CG13852), Mob2 (CG11711), Mob3 (CG4946), Mob4 (CG3403), respectively. They have been identified as Drosophila Mob proteins based on homology to the Saccharomyces cerevisiae Mob1p (He et al., 2005).
Msps, minispindles protein; Asp, abnormal spindle protein.
γ-tubulin (23C) and γ-tubulin (37C): two isotypes of the γ-tubulin gene are present in Drosphila. The 23C isotype is expressed ubiquitously, whereas the 37C isotype is primarily expressed in the ovaries and in embryos.
Relative mitotic index: Percentage of nuclei that stained positive for phsphorylated histone H3 (a marker of mitotic cells), primarily scored using an automated microscope (see Materials and Methods). Numbers give the average fold increase in mitotic index for at least three separate RNAi experiments (mean ± s.d.) relative to the average mitotic index of untreated control cells (1.41±0.47, derived from untreated cells in three independent experiments). The average mitotic index of untreated cells from each experiment was determined from measurements of 90 separate wells.
P<0.005 (t-test), fold increase in mitotic index is statistically different from untreated cells.
As reported previously (Goshima and Vale, 2003), S2 cells often show aberrant mitotic spindle phenotypes. Some S2 cells form monopolar spindles that subsequently can be converted to a bipolar spindle by a rescue process in which microtubules become organized to form a second, acentrosomal pole (~10%). These monastral bipolar spindles are capable of entering anaphase and segregating chromosomes.
Spindles with localized γ-tubulin: Values are the mean ± s.d. of three experiments, except that for Dgrip223, which was obtained from a single experiment. n, number of mitotic cells.
+, protein reduction after RNAi confirmed using specific antibodies; −, not tested because antibodies were unavailable;
○, expression of these genes was at background levels in the S2 cell line by Affimatrix DNA microarray analysis (J. Hollien and J.Weissman, personal communication), suggesting that they are not expressed in this cell line at significant levels.
Data for Drosophila pericentrin are from RNAi experiments targeting Computed Gene (CG) 6735; however, RNAi targeting of alternative segments of the Drosophila pericentrin gene contained in CG18648 and CG13459 produced similar results.