Fig. 9. Domain analysis of MIR1. (A) BHK cells were transfected with YFP fusions with different domains of MIR1 (Fig. 6A). The cells were analyzed for YFP fluorescence and for tubulin. The arrow indicates concentration of the C-terminal domains at the centrosome. (B) BHK cells were transfected with CFP fusions with N-terminal fragments of MIR1 (tN and tNP; Fig. 6A), as well as with YFP fusions with C-terminal fragments (tPC and tC). The cells were analyzed by CFP and YFP fluorescence and by staining for tubulin. The arrow points to cells in which the wild-type phenotype of microtubule bundling is restored by the simultaneous expression of N- and C-terminal domains that both contain the P-stretch. (C) BHK cells were transfected with a CFP fusion to a MIR1 fragment lacking the coiled-coil domain (delCC; Fig. 6A). The cells were analyzed for CFP fluorescence and for -tubulin staining with mAb GTU-88. CFP-delCC is visible in filaments and bundled structures (arrow heads), as well as at the centrosome (arrows). Colocolization with -tubulin is evident in the enlarged field. (D) MIR1 was expressed in a baculovirus system, purified, and subjected to sucrose gradient centrifugation. Fractions were analyzed by immunoblotting with MIR1 antibodies. Molecular weight standards were run in paralle. (E) Full-length MIR1 (wt) or a C-terminal truncation (tC) were synthesized in an in vitro translation system in the presence of ³⁵S-methionine. The labeled proteins were combined with an excess of microtubules polymerized from purified tubulin (0.5 mg/ml), and binding was assessed in a microtubule co-sedimentation assay.

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