Figure 6.
The chaperonin CCT complex is required for cilia disassembly by stabilizing Aurora A, and CEP55 promotes Aurora A binding to the chaperonin CCT complex. (A) HEK293T cells were serum starved for 48 h and then stimulated with serum for 18 h. Endogenous proteins immunoprecipitated with anti-CEP55 or anti-Aurora A antibody from these cells lysates were analyzed by liquid chromatography-tandem mass spectrometry. A list of top-20 proteins identified both in anti-CEP55 and anti-Aurora A immunoprecipitants were shown. (B) HEK293T cell lysates during cilia disassembly were immunoprecipitated with anti-CEP55, anti-Aurora A, anti-CCT5 antibody or normal rabbit IgG, and then the immunoprecipitants were analyzed by immunoblotting with the indicated antibodies. (C) RPE-1 cells were transfected with the indicated siRNAs individually, and cell lysates were detected with the indicated antibodies. The amounts of Aurora A, CEP55, and CCT proteins were quantified in ImageJ software and then normalized to respective GAPDH levels, which is shown at the bottom of each immunoblot. (D) RPE-1 cells were transfected with control, CCT1, CCT2, CCT5, or CCT8 siRNAs and serum starved for 48 h. Next, ciliated RPE-1 cells were stimulated with serum for the indicated times and then quantified. Ciliation in the siCCT1, siCCT2, siCCT5, or siCCT8 group is compared with their respective time-point controls in the siControl group. Data are presented as means ± SD of three independent experiments. A two-way ANOVA test was performed followed by Dunnett’s multiple comparisons. *, P < 0.05; ***, P < 0.001. n, number of cells. (E and F) Increased ciliation in RPE-1 cells induced by CCT5 knockdown were rescued by expressing GFP-Aurora A. GFP-vector (−) or GFP-Aurora A plasmids were transfected in RPE-1 cells after 24 h of siRNA transfection. 24 h later, cell lysates were detected with the indicated antibodies (E). IB, immunoblotting. (F) Quantification of the ciliation in GFP-positive cells in E. Data are presented as means ± SD of three independent experiments. A two-way ANOVA test was performed followed by Bonferroni's multiple comparisons. ***, P < 0.001. n, number of cells. (G) CCT5 binds to Aurora A in the presence of CEP55 in vitro. Increasing amounts of full-length (FL) or 1–355 truncation of Flag-CEP55 proteins were purified from sonicated HEK293T cell lysates by anti-Flag M2 affinity gel, eluted with 3×Flag peptide, and then respectively incubated with HA–Aurora A–bound Agarose (pulled down from sonicated HEK293T cell lysates by anti-HA Agarose antibody). HA-vector–bound Agarose was incubated with eluted Flag-vector sample as control (− indicates the corresponding vector). An equal amount of translated CCT5 protein was added to all groups. Proteins retained on anti-HA Agarose were then analyzed by immunoblotting.

The chaperonin CCT complex is required for cilia disassembly by stabilizing Aurora A, and CEP55 promotes Aurora A binding to the chaperonin CCT complex. (A) HEK293T cells were serum starved for 48 h and then stimulated with serum for 18 h. Endogenous proteins immunoprecipitated with anti-CEP55 or anti-Aurora A antibody from these cells lysates were analyzed by liquid chromatography-tandem mass spectrometry. A list of top-20 proteins identified both in anti-CEP55 and anti-Aurora A immunoprecipitants were shown. (B) HEK293T cell lysates during cilia disassembly were immunoprecipitated with anti-CEP55, anti-Aurora A, anti-CCT5 antibody or normal rabbit IgG, and then the immunoprecipitants were analyzed by immunoblotting with the indicated antibodies. (C) RPE-1 cells were transfected with the indicated siRNAs individually, and cell lysates were detected with the indicated antibodies. The amounts of Aurora A, CEP55, and CCT proteins were quantified in ImageJ software and then normalized to respective GAPDH levels, which is shown at the bottom of each immunoblot. (D) RPE-1 cells were transfected with control, CCT1, CCT2, CCT5, or CCT8 siRNAs and serum starved for 48 h. Next, ciliated RPE-1 cells were stimulated with serum for the indicated times and then quantified. Ciliation in the siCCT1, siCCT2, siCCT5, or siCCT8 group is compared with their respective time-point controls in the siControl group. Data are presented as means ± SD of three independent experiments. A two-way ANOVA test was performed followed by Dunnett’s multiple comparisons. *, P < 0.05; ***, P < 0.001. n, number of cells. (E and F) Increased ciliation in RPE-1 cells induced by CCT5 knockdown were rescued by expressing GFP-Aurora A. GFP-vector (−) or GFP-Aurora A plasmids were transfected in RPE-1 cells after 24 h of siRNA transfection. 24 h later, cell lysates were detected with the indicated antibodies (E). IB, immunoblotting. (F) Quantification of the ciliation in GFP-positive cells in E. Data are presented as means ± SD of three independent experiments. A two-way ANOVA test was performed followed by Bonferroni's multiple comparisons. ***, P < 0.001. n, number of cells. (G) CCT5 binds to Aurora A in the presence of CEP55 in vitro. Increasing amounts of full-length (FL) or 1–355 truncation of Flag-CEP55 proteins were purified from sonicated HEK293T cell lysates by anti-Flag M2 affinity gel, eluted with 3×Flag peptide, and then respectively incubated with HA–Aurora A–bound Agarose (pulled down from sonicated HEK293T cell lysates by anti-HA Agarose antibody). HA-vector–bound Agarose was incubated with eluted Flag-vector sample as control (− indicates the corresponding vector). An equal amount of translated CCT5 protein was added to all groups. Proteins retained on anti-HA Agarose were then analyzed by immunoblotting.

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