Figure 8.
Figure 8. CAL1 targeting bypasses the requirement of CENP-C for CENP-A recruitment. (A) Metaphase chromosome showing that mCherry–LacI–CENP-C (LacI–CENP-C; red) recruits GFP-CAL1 (green) at the ectopic lacO site (100%; n = 34). CENP-A is in blue and DAPI in gray. (B) Western blot with the indicated antibodies of total cell extracts from control-treated and CENP-C RNAi cells expressing CAL1–GFP–LacI. Decreasing amounts of extracts from control cells (100%, 50%, 25%, 10%) were loaded to qualitatively assess the levels of CENP-C and CENP-A 5 d after RNAi treatment (100% loaded). (C) Western blot with the indicated antibodies of total cell extracts from control cells and cells treated with CENP-C double-stranded RNA for 4, 6, and 8 d. (D) Metaphase chromosomes from control and CENP-C RNAi cells expressing CAL1–GFP–LacI (green) showing the recruitment of CENP-A (red) in the control but not in the RNAi. CENP-C is in blue and DAPI in gray. (E) Quantification of the CENP-A recruitment efficiency by CAL1–GFP–LacI (**, P = 0.0009). n = 35 (number of GFP-positive/lacO array chromosomes) for both control and RNAi. One (of two total) representative experiment is shown. (F) Western blots with the indicated antibodies of total cell extracts from control and CAL1 RNAi in cells expressing GFP–LacI–CENP-C. Decreasing amounts of extracts from control cells (100%, 50%, 25%, 10%) were loaded to qualitatively assess the levels of CAL1 and CENP-A after 5 d of RNAi (100% loaded). Anti-tubulin Western blot is a loading control in B, C, and F. (G) Metaphase chromosomes from control, CAL1, or CAL1/PPA RNAi performed in lacO cells expressing GFP–LacI–CENP-C (green). CENP-A is in red, CENP-C in blue, and DAPI in gray. (H) Quantification of the CENP-A recruitment efficiency by GFP–LacI–CENP-C. ***, P < 0.0001. ncontrol = 37, nRNAi = 36 (one of two experiments shown). Arrows indicate the position of the endogenous (green) and the ectopic (white) centromeres in A, D, and G. Bars, 1 µm. (I) Cartoon summarizing the mechanism of centromere specification in Drosophila. Free CAL1 binds directly to free CENP-A (blue half-circle) and histone H4 via its N-terminal domain. The C terminus of CAL1 interacts with the C terminus of centromere-bound CENP-C resulting in the recruitment of the CAL1–CENP-A–H4 complex to the centromere. CENP-C interacts with chromatin-associated CENP-A (gray half-circle) and CAL1 (not depicted; Erhardt et al., 2008) and binds to the KMN (KNL-1/Mis12 complex/Ndc80) complex through its N terminus during mitosis (Przewloka et al., 2011).

CAL1 targeting bypasses the requirement of CENP-C for CENP-A recruitment. (A) Metaphase chromosome showing that mCherry–LacI–CENP-C (LacI–CENP-C; red) recruits GFP-CAL1 (green) at the ectopic lacO site (100%; n = 34). CENP-A is in blue and DAPI in gray. (B) Western blot with the indicated antibodies of total cell extracts from control-treated and CENP-C RNAi cells expressing CAL1–GFP–LacI. Decreasing amounts of extracts from control cells (100%, 50%, 25%, 10%) were loaded to qualitatively assess the levels of CENP-C and CENP-A 5 d after RNAi treatment (100% loaded). (C) Western blot with the indicated antibodies of total cell extracts from control cells and cells treated with CENP-C double-stranded RNA for 4, 6, and 8 d. (D) Metaphase chromosomes from control and CENP-C RNAi cells expressing CAL1–GFP–LacI (green) showing the recruitment of CENP-A (red) in the control but not in the RNAi. CENP-C is in blue and DAPI in gray. (E) Quantification of the CENP-A recruitment efficiency by CAL1–GFP–LacI (**, P = 0.0009). n = 35 (number of GFP-positive/lacO array chromosomes) for both control and RNAi. One (of two total) representative experiment is shown. (F) Western blots with the indicated antibodies of total cell extracts from control and CAL1 RNAi in cells expressing GFP–LacI–CENP-C. Decreasing amounts of extracts from control cells (100%, 50%, 25%, 10%) were loaded to qualitatively assess the levels of CAL1 and CENP-A after 5 d of RNAi (100% loaded). Anti-tubulin Western blot is a loading control in B, C, and F. (G) Metaphase chromosomes from control, CAL1, or CAL1/PPA RNAi performed in lacO cells expressing GFP–LacI–CENP-C (green). CENP-A is in red, CENP-C in blue, and DAPI in gray. (H) Quantification of the CENP-A recruitment efficiency by GFP–LacI–CENP-C. ***, P < 0.0001. ncontrol = 37, nRNAi = 36 (one of two experiments shown). Arrows indicate the position of the endogenous (green) and the ectopic (white) centromeres in A, D, and G. Bars, 1 µm. (I) Cartoon summarizing the mechanism of centromere specification in Drosophila. Free CAL1 binds directly to free CENP-A (blue half-circle) and histone H4 via its N-terminal domain. The C terminus of CAL1 interacts with the C terminus of centromere-bound CENP-C resulting in the recruitment of the CAL1–CENP-A–H4 complex to the centromere. CENP-C interacts with chromatin-associated CENP-A (gray half-circle) and CAL1 (not depicted; Erhardt et al., 2008) and binds to the KMN (KNL-1/Mis12 complex/Ndc80) complex through its N terminus during mitosis (Przewloka et al., 2011).

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