Figure 6.

LIC is required for dynein to move off the kinetochore but not to bind the kinetochore. (A) Immunolocalization of dynein (DHC; red) and CID, a kinetochore marker (green), in metaphase S2 cells. In untreated control cells, dynein is present along the spindle and throughout the cytoplasm but is distinctly decreased in the region of the metaphase plate. After Dlic RNAi, dynein is enriched at kinetochores. Bar, 5 μm. (B) The intensity of the dynein HC signal at metaphase kinetochores was determined after Dlic RNAi (see Materials and Methods). Error bars indicate ± SEM. See also Supplemental Table 2. (C) Cells were treated with colcemid to remove kinetochore microtubules, thereby preventing dynein from exiting the kinetochore. Under these conditions, cells depleted of LIC still show some dynein accumulation at kinetochores. In contrast, in the absence of IC, binding of dynein HC to kinetohcores is largely suppressed. Bar, 2 μm. (D) Quantitation of experiments illustrated in C. Bar graph shows mean relative intensity of DHC, ± SEM. For each condition, n = 31–39 kinetochores. See also Supplemental Table 3.