Consequences of TRIM37 loss in interphase RPE1 cells and mulibrey nanism patient-derived fibroblasts. (A) Schematic of TRIM37, which has a RING–B-box–coiled-coil (RBCC) ubiquitin ligase domain. Mutation of catalytic Cys18 to Arg (Lig^mut TRIM37) disrupts ligase activity. (B) Expansion microscopy of centrioles colabeled for acetylated tubulin and the distal centriole component CEP290. Normal centrioles at different cell cycle stages and rare aberrant centriolar configurations are shown in TRIM37Δ RPE1 cells. Scale bar, 2 µm. (C) Quantification of mother centriole length from expansion microscopy of the indicated conditions. Error bars represent standard deviation. n, number of centrioles; ns, not significant, based on a t test. (D) Quantification of centriole number for the indicated cell lines following expansion microscopy as in B. Normal G1, S, G2, and M centriolar configurations were counted as “2 (single or duplicated)”. For B–D, cells were expanded twice with similar results; images and quantification of centriole length are from one experiment, and the centriole number data is pooled from both experiments. (E) Analysis of ciliogenesis, assessed by ARL13B labeling, in control RPE1 and TRIM37Δ RPE1 cells. Scale bar, 10 µm. The experiment was performed twice, once in triplicate and once in duplicate, with similar results; ciliation frequency from one experiment is shown here and from the second experiment in Fig. S1 A. (F) Immunostaining of TRIM37Δ and control USP28Δ RPE1 cells for centrobin and PLK4. Scale bars, 5 µm and 1 µm (insets). (G) Quantification of immunofluorescence signals at centrosomes in control USP28Δ cells and at centrosomes and condensates in TRIM37Δ cells. The signal for both centrosomes in a cell was measured together. Values were normalized relative to the mean centrosomal signal in USP28Δ cells; inset in the centrobin graph shows centrosome signals plotted on a different y-axis scale. Mean and 95% confidence interval (CI) are plotted on top of individual values; P value is from an unpaired t test. The condensate signal values plotted here are the same as those shown in Fig. S4 B (−Dox). Experiments in F and G were repeated twice; images and quantification are from one experiment. (H) Immunostaining of control and mulibrey nanism patient-derived fibroblasts. Scale bars, 5 µm and 1 µm (insets). The experiment was performed once; data for each cell line were pooled from three replicate wells imaged in parallel. (I) Localization of mNG-fused TRIM7 (WT or Lig^mut) expressed in TRIM37Δ cells. TRIM37 activity reduces its own levels; hence, the signal for WT TRIM37-mNG was enhanced to show centrosomal localization. Scale bars, 5 and 1 µm (insets). The experiment was performed once; an equivalent experiment with similar results was performed using an HA tag shown in (Meitinger et al., 2020). (J) Top: Schematic of condensate observed in TRIM37Δ RPE1 cells and mulibrey nanism patient fibroblasts. Bottom: Summary of the localization of centrosomal components in TRIM37Δ cells; components shown in brackets localize to centriolar satellites. Analysis of CPAP, CEP63, CEP135, PCM1, KIAA0753, and CCDC14 is from Meitinger et al. (2020) and was conducted with PLK4 colabeling. In F, H, and I, centrosomes (white arrowheads) and the condensate (yellow arrows) are indicated, and numbers below images indicate percentage of cells with a condensate.