Role of dynein in kinetochore microtubule stability and kinetochore orientation. LLCPK1 cells transfected with the dynein tail fragment were incubated at 4°C for 10 min and then examined by confocal microscopy. (A) Dynein tail–expressing cell stained with anti-tubulin, DAPI, and anti-myc (inset) showing few remaining kinetochore microtubule bundles. (B) A control cell stained as in A showing numerous cold-stable kinetochore microtubule bundles. (C) Fraction of cells exhibiting decrease in the number of cold-stable microtubule bundles in tail-expressing versus control untransfected cells after cold treatment (n = 90). P = 0.012; two-tailed t test. (D–I) Analysis of kinetochore orientation in tail-expressing LLCPK1 cells. (D–F and D′–F′) Cells in late prometaphase/metaphase (D, D′, E, and E′, pretreated with MG132 as indicated) or prometaphase (F and F′) transfected with the tail were stained with anti-tubulin, CREST, and anti-myc antibodies (D′–F′) as indicated versus DAPI (not depicted). (G and H) Control LLCPK1 cells expressing GFP. The numbered boxes indicate images magnified at right. Insets show GFP staining. (I) Fraction of aligned kinetochore pairs exhibiting different attachment patterns. P = 0.0025; two-tailed t test. Error bars indicate SD from mean from three independent experiments. Bars, 5 μM.