Figure 2.

The interphase and mitotic phenotypes of the Q18→E mutation in the context of wild-type and pseudophosphorylated Op18. K562 cells were transfected with the pMEP derivative indicated (8 μg DNA mixed with 8 μg vector-Co DNA) and counterselected with hygromycin for 5 d. Cd²⁺ was added to induce ectopic expression from the hMTIIa-promoter, for the indicated time periods. (A) Immunoblots of total cellular lysates from Cd²⁺-induced cells (8 h) using the antibodies indicated for detection. Arrow indicates the migration of endogenous Op18. (B and C) The MT content of interphase cells was determined before or after 8 h of Cd²⁺-induced expression (B, 0 h, and C, 8 h, respectively). Data are expressed as percentage of total cellular tubulin that is polymerized. (D) After 24 h of induced expression, mitotic figures were inspected and categorized as bipolar, multipolar, or, if spindle MTs were completely absent, “no spindle.” Data are expressed as percentage of total cells (n = 300 mitotic cells) and are the average of duplicate determinations of coded samples. (E) Epifluorescence images of representative examples of a normal bipolar metaphase spindle, a multipolar spindle, or a mitotic cell lacking spindle MTs, termed no spindle. Fixed cells were stained with anti-α-tubulin (green) and propidium iodide (red). All spindles with more than three spindle poles were categorized as “multipolar.” Bar, 6 μm. (F) MT-specific fluorescence in gated mitotic populations was determined by flow cytometry as in Figure 1F. (G) Transfected cell lines were Cd²⁺-induced for 24 h in the presence of the mitosis-blocking drug paclitaxel (0.5 μM) and phospho-isomers of the indicated Flag epitope-tagged Op18 derivative were resolved by native PAGE and revealed using anti-Flag antibodies. The position of unphosphorylated Op18 (non-P) is defined by the migration of kinase target site-deficient Op18-tetraA, which is Ala-substituted at all four phosphorylation sites. Op18-Q18E migrates somewhat faster due to introduction of the negatively charged Glu residue. The data are representative of four independent transfection experiments.