Figure 4.
CEP55 inhibits cilia formation through interacting with Aurora A kinase. (A) HEK293T cells were transfected with Flag-vector or Flag-CEP55 and then immunoprecipitated with anti-Flag M2 Affinity Gel. Protein levels of endogenous Aurora A, Flag-CEP55, and endogenous HDAC6 in immunoprecipitants and cell lysates were detected. (B) HEK293T cell lysates were immunoprecipitated with anti-CEP55 polyclonal antibody or normal rabbit IgG, and then the immunoprecipitants were analyzed by immunoblotting with the indicated antibodies. (C) Lysates prepared from primary MEFs were immunoprecipitated with IgG or anti-CEP55 antibody and then immunoblotted with the indicated antibodies. (D) Schematic representation of full-length (FL) CEP55 and the indicated CEP55 truncations. A summary of the ability of CEP55 or the indicated CEP55 truncations to bind to Aurora A. (E) Flag-vector (−) or full-length or truncations of CEP55 was respectively expressed in HEK293T cells and immunoprecipitated with anti-Flag M2 affinity gel. Endogenous interactions with Aurora A were determined by indicated antibodies. IB, immunoblotting. (F) RPE-1 cells transfected with indicated plasmids under serum starvation (− Serum) were stained with ciliary marker (ARL13B) and Flag antibody. Quantification of the ciliation in Flag-positive cells. Data are means ± SD of three independent experiments. A one-way ANOVA test was performed, with each mean compared with the Flag-CEP55 group, followed by Dunnett’s multiple comparisons. ***, P < 0.001. n, number of cells. (G) Schematic diagram of CEP55 C256T mutant in human MKS (c.256C>T, p.Arg86*). (H) The nonsense mutation C256T in CEP55 abolished its centrosome localization. RPE-1 cells transfected with mCherry-CEP55-WT or C256T mutant were stained with γ-tubulin (green) and DNA (blue). Insets show zoomed-in views of the boxed regions. Scale bars, 5 µm (main image) and 0.5 µm (magnified region). (I) HEK293T cells were cotransfected with HA–Aurora A together with mCherry-vector (−), mCherry-CEP55-WT, or C256T mutant. Lysates were immunoprecipitated with anti-HA Agarose antibody, and then immunoprecipitants and whole-cell lysates were analyzed by immunoblotting with indicated antibodies. (J) Increased ciliation caused by CEP55 depletion can be rescued by the expression of an RNAi-resistant mCherry-CEP55 WT, but not by C256T mutant. RPE-1 cells were transfected with the indicated siRNA and plasmids (− indicates mCherry-vector) and then stained with Ac-tubulin (green) and DNA (blue). Insets show zoomed-in views of the boxed regions. Scale bars, 5 µm (main image) and 1 µm (magnified region). (K) Quantification of the ciliation in mCherry-positive cells in G. Data are presented as means ± SD of three independent experiments. A one-way ANOVA test was performed followed by Bonferroni’s multiple comparisons. ***, P < 0.001. n, number of cells. EABR, ESCRT and ALIX-binding region.

CEP55 inhibits cilia formation through interacting with Aurora A kinase. (A) HEK293T cells were transfected with Flag-vector or Flag-CEP55 and then immunoprecipitated with anti-Flag M2 Affinity Gel. Protein levels of endogenous Aurora A, Flag-CEP55, and endogenous HDAC6 in immunoprecipitants and cell lysates were detected. (B) HEK293T cell lysates were immunoprecipitated with anti-CEP55 polyclonal antibody or normal rabbit IgG, and then the immunoprecipitants were analyzed by immunoblotting with the indicated antibodies. (C) Lysates prepared from primary MEFs were immunoprecipitated with IgG or anti-CEP55 antibody and then immunoblotted with the indicated antibodies. (D) Schematic representation of full-length (FL) CEP55 and the indicated CEP55 truncations. A summary of the ability of CEP55 or the indicated CEP55 truncations to bind to Aurora A. (E) Flag-vector (−) or full-length or truncations of CEP55 was respectively expressed in HEK293T cells and immunoprecipitated with anti-Flag M2 affinity gel. Endogenous interactions with Aurora A were determined by indicated antibodies. IB, immunoblotting. (F) RPE-1 cells transfected with indicated plasmids under serum starvation (− Serum) were stained with ciliary marker (ARL13B) and Flag antibody. Quantification of the ciliation in Flag-positive cells. Data are means ± SD of three independent experiments. A one-way ANOVA test was performed, with each mean compared with the Flag-CEP55 group, followed by Dunnett’s multiple comparisons. ***, P < 0.001. n, number of cells. (G) Schematic diagram of CEP55 C256T mutant in human MKS (c.256C>T, p.Arg86*). (H) The nonsense mutation C256T in CEP55 abolished its centrosome localization. RPE-1 cells transfected with mCherry-CEP55-WT or C256T mutant were stained with γ-tubulin (green) and DNA (blue). Insets show zoomed-in views of the boxed regions. Scale bars, 5 µm (main image) and 0.5 µm (magnified region). (I) HEK293T cells were cotransfected with HA–Aurora A together with mCherry-vector (−), mCherry-CEP55-WT, or C256T mutant. Lysates were immunoprecipitated with anti-HA Agarose antibody, and then immunoprecipitants and whole-cell lysates were analyzed by immunoblotting with indicated antibodies. (J) Increased ciliation caused by CEP55 depletion can be rescued by the expression of an RNAi-resistant mCherry-CEP55 WT, but not by C256T mutant. RPE-1 cells were transfected with the indicated siRNA and plasmids (− indicates mCherry-vector) and then stained with Ac-tubulin (green) and DNA (blue). Insets show zoomed-in views of the boxed regions. Scale bars, 5 µm (main image) and 1 µm (magnified region). (K) Quantification of the ciliation in mCherry-positive cells in G. Data are presented as means ± SD of three independent experiments. A one-way ANOVA test was performed followed by Bonferroni’s multiple comparisons. ***, P < 0.001. n, number of cells. EABR, ESCRT and ALIX-binding region.

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