Figure 9.

GRASP65 depletion perturbs γ-tubulin recruitment to the centrosome. (A) A nonsynchronous population of scrambled control or GRASP65-specific siRNA-transfected HeLa cells was processed for immunofluorescence microscopy 48 h after siRNA transfection using antibodies to the centrosomal marker protein Centrin2 or the pericentriolar protein γ-tubulin to monitor the composition of the aberrant spindle poles (2 cells are shown for each phenotype). These cells were positive for the mitotic marker phospho-histone H3 (unpublished data). (B) A HeLa cell line stably expressing Centrin2-GFP was generated, transfected with scrambled control or GRASP65-specific siRNA and processed for immunofluorescence microscopy to monitor Centrin2-GFP and α-tubulin in the same mitotic cell. The α-tubulin staining reveals a number of disorganized spindle asters of which only two contained Centrin2-GFP (arrows).