Figure 3. A full-length p150 glued that... | Rockefeller University Press

Figure 3.

$Figure 3. A full-length p150glued that can no longer be phosphorylated by aurora A binds more strongly to microtubules but cannot rescue glued spindle assembly defect. (A) Scheme of the GFP fusion proteins expressed in S2 cell lines under the control of the metallothionein promoter (inducible by Cu2+). The constructs lack the glued gene 3′UTR (top) targeted by RNAi. (B) p150glued (top) or actin (bottom) Western blot showing p150-GFP (left) and p150-SA8-GFP (right) protein levels after 0 and 18 h induction on the corresponding cell line. (C) p150glued, p150-GFP, p150-SA8-GFP, and actin protein levels after glued 3′UTR RNAi (lanes 2 and 3). Note the knockdown of endogenous p150glued, whereas p150-GFP protein level remains stable. (D) p150glued Western blot analysis of the different sucrose fractions after the sedimentation assay (from 5 to 40%) of S2 cell extracts expressing either p150-GFP (top) or p150-SA8-GFP (bottom). (E, left) Fixed control or p150glued-depleted metaphase cells are stained for DNA in blue, microtubules in red (monochrome in bottom), and centrosomes in green. The arrows point to disconnected centrosomes. (E, right) Quantification of the centrosome disconnection phenotype in control or glued 3′UTR dsRNA-treated S2-cultured cells. Note the rescue obtained after induced expression of p150-GFP protein but not p150-SA8-GFP. (F) Spindle morphology in p150glued cells after p150-GFP (top) or p150-SA8-GFP (bottom) expression. DNA (blue), microtubules (red; monochrome in the third column), centrosomes (green), and GFP (green in merge; monochrome on the right) are displayed. p150-SA8-GFP interacts more strongly with spindle microtubules than its wild-type counterpart. The arrows point out the disconnected centrosomes. Error bars indicate mean ± SD. Bars, 10 µm.$

A full-length p150^glued that can no longer be phosphorylated by aurora A binds more strongly to microtubules but cannot rescue glued spindle assembly defect. (A) Scheme of the GFP fusion proteins expressed in S2 cell lines under the control of the metallothionein promoter (inducible by Cu²⁺). The constructs lack the glued gene 3′UTR (top) targeted by RNAi. (B) p150^glued (top) or actin (bottom) Western blot showing p150-GFP (left) and p150-SA8-GFP (right) protein levels after 0 and 18 h induction on the corresponding cell line. (C) p150^glued, p150-GFP, p150-SA8-GFP, and actin protein levels after glued 3′UTR RNAi (lanes 2 and 3). Note the knockdown of endogenous p150^glued, whereas p150-GFP protein level remains stable. (D) p150^glued Western blot analysis of the different sucrose fractions after the sedimentation assay (from 5 to 40%) of S2 cell extracts expressing either p150-GFP (top) or p150-SA8-GFP (bottom). (E, left) Fixed control or p150^glued-depleted metaphase cells are stained for DNA in blue, microtubules in red (monochrome in bottom), and centrosomes in green. The arrows point to disconnected centrosomes. (E, right) Quantification of the centrosome disconnection phenotype in control or glued 3′UTR dsRNA-treated S2-cultured cells. Note the rescue obtained after induced expression of p150-GFP protein but not p150-SA8-GFP. (F) Spindle morphology in p150^glued cells after p150-GFP (top) or p150-SA8-GFP (bottom) expression. DNA (blue), microtubules (red; monochrome in the third column), centrosomes (green), and GFP (green in merge; monochrome on the right) are displayed. p150-SA8-GFP interacts more strongly with spindle microtubules than its wild-type counterpart. The arrows point out the disconnected centrosomes. Error bars indicate mean ± SD. Bars, 10 µm.