Figure 7.
Effect of dynactin subunit depletion on kinetochore behaviour. (A) Representative images of monopolar spindles generated by monastrol treatment of asynchronous Cos7 cells stained with DAPI plus Abs to tubulin and ACA. Structures were observed using deconvolution microscopy. These images are maximal projections of three z-planes but kinetochore–microtubule associations were assessed by examining the entire deconvolved image. The insets provide images of selected kinetochore pairs at higher magnification to illustrate the different interactions seen. Bar=5 μm. (B) Images as in (A) were scored for the types of kinetochore–microtubule interaction observed (end-on=mono-oriented k-fibres; lateral=lateral k-fibres; mean±s.d., n>100 kinetochore pairs per condition). (C) The distances between sister kinetochores on bi-oriented chromosomes were measured in asynchronous Cos7 cells stained with DAPI plus Abs to tubulin and ACA (mean±s.e.m.; *P<0.01 compared to control; N=number of kinetochores). Taxol treatment (1 μM, 12 min) was used to provide a control for the amount of tension expected at unattached kinetochores. (D, E) Analysis of kinetochore Mad1 in cells treated with dynactin subunit siRNAs. (D) Representative images of Mad1 localization at prometaphase kinetochores in asynchronous cell populations. Bar=5 μm. (E) Quantitation of Mad1 levels at kinetochores of unaligned (i.e., prometaphase) and aligned (i.e., metaphase) chromosomes (judged by DAPI staining). Mad1 fluorescence intensity was normalized to ACA pixel values (mean±s.e.m., n>600 kinetochores, 15 cells) and expressed as per cent of the intensity at kinetochores of unaligned chromosomes in control cells. (F, G) As in (D) and (E), but in cells subjected to nocodazole treatment. Bar=5 μm.
Source data for this figure is available on the online supplementary information page.
