Effects of dynein tail expression on mitotic stage and kinetochore composition during metaphase. (A) Dynein tail–expressing COS7 cells with bipolar spindles were evaluated for chromosome distribution, revealing an increase in late prometaphase cells versus untransfected controls (n = 300). Error bars indicate SD from mean from three independent experiments. (B) Example of cells used for A stained with anti-myc, anti-tubulin, and CREST (not depicted) antibodies versus DAPI (inset). Numbered boxes indicate images magnified below. (C–E) Control COS7 cell expressing a myc vector stained with anti-p150Glued, anti-Mad1, and anti-BubR1 antibodies in each case versus anti-tubulin and anti-myc (not depicted) versus DAPI (inset), showing loss of these proteins from kinetochores at metaphase. (C′–E′) COS7 cells expressing the tail construct and stained as in C–E. The numbered boxes indicate images magnified at right. Dynein tail, p150Glued, Mad1, and BubR1 are all observed to associate with both kinetochores of aligned chromatid pairs, including those kinetochores associated with microtubules. Note examples of misoriented kinetochore pairs in numbered insets (and see Fig. 4). Note that these proteins are absent at spindle poles in tail-expressing cells, in contrast to controls. (F–H and F′–H′) Analysis of Hec1, MCAK, and Kif2b immunoreactivity at aligned kinetochores. Control and tail-expressing COS7 cells were stained using anti-myc (insets) and anti-Hec1, anti-MCAK, and anti-Kif2b antibodies, respectively versus DAPI (n = 150). Mean kinetochore spacing in Hec1-stained cells was decreased relative to controls (see text), which is consistent with loss of tension at kinetochores. MCAK and Kif2b localize to prometaphase kinetochores (G and H) and were normally redistributed to spindle poles in dynein-inhibited cells (G′ and H′). Bars, 5 μM.
