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. 2021 Jul 9;220(9):e202011133. doi: 10.1083/jcb.202011133

Figure 10.

Figure 10.

MNR recruits CEP90, which recruits CEP83 to build distal appendages. (a) 3D-SIM imaging of WT, CEP90−/−, and MNR−/− serum-starved RPE1 cells immunostained for CEP90 (yellow), γ-tubulin (cyan), and TubulinAc (magenta). Scale bar = 1 µm. (b) Quantification of CEP90 fluorescence intensity at centrioles. Horizontal lines in scatter dot plots indicate means ± SEM. ***, P < 0.0001, one-way ANOVA. n = 31–37 measurements. CEP90 fails to localize to MNR−/− and CEP90−/− centrioles, although protein levels of CEP90 remained unchanged in MNR−/− RPE1 cells (Fig. S1 d). (c) 3D-SIM imaging of WT, CEP90−/−, and MNR−/− serum-starved RPE1 cells immunostained for MNR (yellow), γ-tubulin (cyan), and CEP164 (magenta). Scale bar = 1 µm. (d) Quantification of MNR fluorescence intensity at centrioles. Horizontal lines in scatter dot plots indicate means ± SEM. ***, P < 0.0001, one-way ANOVA. n = 73–122 measurements. MNR localization is reduced but present at CEP90−/− centrioles. (e) Quantification of Talpid3 diameter at mother (MC) and daughter centrioles (DC) in serum-starved WT and CEP90−/− cells. Horizontal lines in scatter dot plots indicate means ± SEM. ***, P < 0.05, one-way ANOVA. n = 11–16 measurements. The Talpid3 ring diameter is increased at WT mother centrioles, but not CEP90−/− mother centrioles. (f) Quantification of OFD1 diameter at mother (MC) and daughter centrioles (DC) in serum-starved WT and CEP90−/− cells. Horizontal lines in scatter dot plots indicate means ± SEM. Asterisks indicate P < 0.05 (*, P < 0.05; **, P < 0.005; ***, P < 0.0005), one-way ANOVA. n = 17–20 measurements. In the absence of CEP90, the mother centriole ring of OFD1 does not expand as in WT cells. (g) WT or RPE1 cells expressing CEP83-HA were lysed, immunoprecipitated with anti-HA antibody–bound beads, and immunoblotted with antibodies to HA and CEP90. (h) Model of the hierarchical recruitment of the DISCO complex. MNR recruitment of OFD1 restrains centriole elongation and MNR recruits CEP90, which, in turn, recruits CEP83, the base of the distal appendage.