In preparation for mitosis, the centrioles duplicate, creating two γ-tubulin–containing centrosomes that nucleate MTs and ultimately become the poles of the mitotic spindle. However, centrioles are not needed for most cell divisions in flies, as MT nucleation around chromatin suffices for bipolar spindle formation (13
). In our screen, depletion of proteins involved in centriole duplication would be expected to produce a mixture of anastral spindles (no γ-tubulin staining at the poles) and monastral bipolar spindles (only one pole with normal γ-tubulin staining), because centriole numbers only gradually diminished with dilution through successive cell cycles in a 4-day dsRNA treatment ( and fig. S3A
Fig. 2 Genes required for localizing γ-tubulin to the centrosome and the spindle. (A) (Left) Anastral as well as monastral bipolar spindles were observed after RNAi to Sak or three previously unknown Ana genes (the latter two shown in fig. S3). (Right) (more ...)
Our screen identified several known proteins (Sak kinase, DSas-4, and Sas-6), as well as three previously unknown genes [anastral spindle phenotype (Ana
)]. Consistent with their RNAi phenotype, GFP-Ana1 and -Ana2 colocalized during interphase and mitosis with the centriolar markers mRFP-Sas-6 ( and fig. S3C
) and Sak and DSas-4 (fig. S3D
). GFP-Ana1 was not detected at anastral spindle poles after Sak RNAi, and RNAi depletion of Ana1 resulted in a substantial decrease in GFP-Sas6 fluorescence from spindle poles, suggesting an important role in centriole formation (). Thus, Ana1 and Ana2 (and possibly Ana3) may be core components of the centriole that are necessary for centriole duplication.
RNAi of the known genes Spd-2, Polo, centrosomin, Dgrip84, and Dgrip91 [these Dgrips make up the stable core (γTuSC) of the γ-tubulin ring complex (γTuRC)] decreased γ-tubulin staining at the pole without interfering with centriole marker localization (fig. S3, F and G
). By examining the effects of RNAi of these genes on centrosomin and γ-tubulin localization, we suggest a molecular pathway leading to γ-tubulin localization at the spindle pole (). We also found two previously unknown genes, Dgt1 and Dgt2 (dim γ-tubulin), that decreased spindle pole γ-tubulin staining without affecting centrosomin [a known γ-tubulin localization factor (18
)]. RNAi of these genes also produced long spindles, a phenotype characteristic of γ-tubulin RNAi, thus further suggesting a role in γ-tubulin function.
In addition to centrosome localization, a subset of γ-tubulin localizes to the spindle (16
), where it might contribute to MT nucleation within the spindle (16
). Phosphorylation of a γTuRC subunit is required for spindle localization of γ-tubulin in mammalian cells (19
), but otherwise, little is known about this population of spindle-localized γ-tubulin. In our screen, we identified genes that are needed to localize γ-tubulin to the spindle but not the pole ( and fig. S4A
). Among these are components of the γTuRC (Dgrip71, Dgrip75, Dgrip128, and Dgrip163). RNAi of several previously unknown genes (Dgt3 to Dgt6) also diminished γ-tubulin selectively within the spindle compared with the pole. Consistent with a role in targeting γ-tubulin to the spindle, GFP-tagged Dgt4, Dgt5, and Dgt6 localized uniformly on spindle MTs, with no enrichment at the centrosome; the spindle staining was lost upon MT depolymerization and was cell cycle dependent (noMT localization in interphase) ( and fig. S4B
). High-throughput, automated imaging of living cells expressing mCherry-tubulin and H2B-GFP as well as high-resolution confocal imaging of MTs further revealed that RNAi of these Dgts reducedMT density inside the spindle, increased monopolar spindle formation, and caused chromosome/kinetochore misalignment and mitotic delay (; and movie S1
of Dgt5 RNAi). Recently, γTuRC was implicated in the spindle-assembly checkpoint (SAC) signaling through binding Cdc20 and BubR1 (20
), but our results suggest that the loss of proper kinetochore-MT interactions after removal of spindle-localized γTuRC may constitute the primary reason for failure to satisfy the SAC.
This study suggests two pathways for γ-tubulin localization in mitosis (). At the spindle pole, a core set of proteins build centrioles (Ana1, Ana2, Sak, DSas-4, and Sas-6), which provide scaffolds for Spd-2, polo kinase, and centrosomin to recruit γ-tubulin through the γTuSC subunits. However, this pathway is dispensable for cell division in S2 cells. A second pathway involving a new set of proteins (Dgt3–6) and the outer γTuRC subunits recruit γ-tubulin to spindle MTs. Surprisingly, this site of γ-tubulin function is more important than the centrosome in building a normal-length bipolar spindle with properly aligned chromosomes.