Fig. 3. Katanin inhibition reduces the area occupied by -tubulin at prometaphase spindle poles. (A-D) CV-1 cells were transfected with plasmids encoding GFP fusions to the dominant katanin inhibitor, P loop K-A p60, the weak katanin inhibitor, N-P loop K-A p60, or GFP alone. Cells were fixed and stained with an anti--tubulin monoclonal antibody within 24 hours of transfection or within 12 hours of release from a thymidine block to ensure that mitotic cells were in their first mitosis after expression of the GFP-fusion. The fluorescence micrographs show anti--tubulin staining of representative prometaphase cells expressing GFP (A) or GFP-P loop K-A p60 (B). The histograms in C-E show the fraction of prometaphase cells with -tubulin areas falling within each range of values shown. P indicates the significance of the difference between control (GFP alone) and experimental populations as determined with the Mann-Whitney test. (E) Affinity-purified anti-p60 katanin antibodies or a control IgG were introduced into CV-1 cells by Chariot mediated protein transfection (see Materials and Methods). Cells were fixed 4 hours after initiation of the antibody treatment and prometaphase cells containing significant cytoplasmic IgG concentrations were identified by DAPI-stained chromosome morphology and anti-rabbit IgG staining. Areas of increased -tubulin staining intensity were analyzed as in C and D.