CLDs are interdependent for centromeric localization and are physically associated. (A) CLD depletions cause CID and CENP-C mislocalization. Control cells showed strong centromeric localization of CID (red) and CENP-C (green; merged panel across top; DAPI shown in gray). Depletion of CID, CENP-C, CAL1, CYCA, and RCA1 all resulted in absent or reduced centromeric staining of CID and CENP-C. Note that CAL1 depletion resulted in diffuse mislocalization of CENP-C in the nucleus, which is in contrast to the elimination of CENP-C observed after CID depletion. In addition, residual centromeric CID staining was observed after CENP-C depletion. (B) CID or CLD depletion causes loss of centromeric CAL1. Control cells showed strong colocalization between CID (red) and GFP-CAL1 (green), whereas cells depleted for CID, CENP-C, CYCA, or RCA1 displayed severely reduced centromeric CAL1. (C) Centromeric enrichment of CYCA (green) was visible in control cells and was lost in cells depleted for CID (red) after CID, CENP-C, or CAL1 RNAi (DNA is shown in gray). RCA1 depletion resulted in a general decrease in CYCA staining. (D) Coimmunoprecipitation of CLDs. GFP-CLD fusions were immunoprecipitated from stable cell lines expressing GFP-CID, GFP–CENP-C, or GFP-CAL1 or from the parent Kc167 cell line (control). Immunoprecipitates were Western blotted with antibodies against CID (top), CENP-C (middle), and CAL1 (bottom). The position of the GFP fusion and the endogenous protein is labeled on the right. (E) Summary of epistatic and physical relationships. CID, CAL1, and CENP-C physically interact and are interdependent for centromere localization. CID, CAL1, and CENP-C require RCA1 and CYCA for centromere localization, and CYCA requires all three for centromere enrichment. Bars, 5 μm.