Fig 2: TRIM37 displays E3-dependent and -independent mitotic activities.(A) WT RPE-1 and TRIM37-/- cells were incubated with DMSO (0) or the indicated concentration of centrinone B for 3 days before live imaging for 24 hr. The time from nuclear envelope breakdown (NEBD) to telophase was determined. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3). Significant p-values (< 0.05) from pairwise t-tests comparing WT and TRIM37-/- samples are shown. (n=3, N=30). Table below indicates average mitotic length and p-value from a Dunnett post hoc test using ‘DMSO’ as control after one-way ANOVA. (B) RPE-1 TRIM37-/- cells were treated with DMSO or 500 nM centrinone B (Cent.B) in the absence or presence of doxycycline (DOX) for 3 days before live imaging for 24 hr. The time from NEBD to telophase was determined. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3). Significant p-values (< 0.05) from Dunnett post hoc test using ‘uninduced, DMSO treated’ cells as a control after a one-way ANOVA are shown (n=3, N=46). (C) RPE-1 TRIM37-/- cells expressing the indicated TRIM37 protein (WT or C18R) were treated with DMSO or 500 nM centrinone B (Cent.B) in the absence or presence of DOX for 3 days before fixing and staining for CEP192. Representative images shown. (D) The number of discernable microtubule organizing centers (MTOCs) characterized by the accumulation of CEP192 in cells from (C) was quantified. Means and standard deviation shown (n=3, N=29). For each TRIM37 construct, the number of cells incubated with centrinone B and with two MTOCs in uninduced and induced samples was compared using a pairwise t-test. Significant p-values (< 0.05) are shown. See Figure 5—source data 1. Figure 5—source data 1.Source data for Figure 5.

Fig 3: TRIM37-dependent PLK4 modification is observed in multiple cell lines.293T, HeLa and RPE-1 cells were transfected with plasmids to express Myc-PLK4 and one of eGFP, T7-TRIM37 or T7-TRIM37D505-709. Cells were incubated for 48 with 30 µm MG132 added for the final 7 h. Cells were harvested directly in SDS-PAGE sample buffer and prepared for Western blot to detect PLK4. Ponc.S indicates total protein. A representative experiment from 3 Western blots is shown.

Fig 4: TRIM37 negatively regulates centriole and centrosome proteins.(A) WT RPE-1 and TRIM37-/- cells were processed for Western blot and probed for the indicated proteins. Band intensity was quantified and expressed as expression compared to WT cells. Relative intensity from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3). Data was tested by pairwise t-test between WT and TRIM37-/-. No significant differences were observed. (B) TRIM37-/- RPE-1 cells stably expressing FLAG-BirA or the indicated FB-TRIM37 protein were processed for Western blot and probed for the indicated proteins. Band intensity was quantified and expressed as expression compared to cells expressing FLAG-BirA. Relative intensity from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3). Significant p-values (< 0.05) from Dunnett post hoc test using FLAG-BirA as a control after a one-way ANOVA are shown. The band intensity from FLAG-BirA cells was set to ‘1’ and is omitted from the plot for clarity. (C) WT RPE-1 cells expressing doxycline-inducible TRIM37-3xFLAG (WT) or TRIM37 C18R-3xFLAG (C18R) were induced with doxycycline for 0, 4, or 8 hr. At each time point, extracts were prepared and analyzed by Western blot for the indicated protein (right). Ponc.S indicates equal loading. CEP192 abundance was quantified and normalized to the intensity at time 0 hr (bottom). Mean and standard deviation shown (n=3). Significant p-values (< 0.05) from Dunnett post hoc test using time 0 hr as a control after a one-way ANOVA are shown. (D) Cells from (C) were also fixed and immunostained for the indicated proteins. The centrosomal intensity from mitotic cells was determined. Intensity values were normalized to 0 hr. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3, N=60). Significant p-values (< 0.05) from Dunnett post hoc test using time 0 hr as a control after a one-way ANOVA are shown. (E) RPE-1 TRIM37-/- cells were fixed and stained for CEP120 and the indicated proteins. (F) RPE-1 (WT) or TRIM37-/- cells stably expressing FLAG-BirA or the indicated FB-TRIM37 protein were fixed and stained for the indicated protein. Arrowhead indicates centrosome defined by CEP192. Caret mark indicates ectopic structure defined by CEP120. See Figure 6—source data 1. Figure 6—source data 1.Source data for Figure 6.

Fig 5: TRIM37 promotes PLK4 phosphorylation in an E3-dependent manner.(A) TRIM37 domain schematic. Constructs used for structure-function experiments indicated below. (B) RPE-1cells were transfected to express Myc-PLK4 and FLAG-BirA or the indicated FB-TRIM37 fusion protein (top). Cells were lysed and subjected to anti-FLAG immunoprecipitation. Input and immunoprecipitates were analyzed by immunoblotting for the FLAG-BirA fusions (FLAG) or for Myc-PLK4. Ponc.S indicates total protein. * indicates position of FLAG-Cas9. (C) WT RPE-1, TRIM37-/- and TRIM37-/- expressing FB or the indicated FB-TRIM37 construct were seeded for clonogenic assays and grown in DMSO or the indicated concentration of centrinone B for 14 days. Colony density was quantified and growth compared to that in DMSO determined. Means from each replicate are shown as open circles. Resulting mean and standard deviation shown (n=3). Significant p-values (< 0.05) from Dunnett post hoc test using ‘WT’ as a control after one-way ANOVA shown. Note that the results from this experiment and those in Figure 4B are from the same experiment; therefore ‘WT’, ‘TRIM37-/- none’, ‘TRIM37-/- FLAG-BirA’, and ‘TRIM37-/- WT’ are duplicated in these panels. (D) HEK293T cells transfected to express Myc-PLK4 and the indicated protein (top) were grown overnight and subsequently treated with DMSO or MLN4924 for 22 hr and MG132 for the final 4 hr. Cell extracts were prepared and probed by Western blot using the indicated antibodies. Ponc.S indicates total protein. (E) HEK293T cells were transfected to express Myc-PLK4, HA-Ub, and the indicated protein (top). Cells were harvested after 48 hr and subjected to immunoprecipitation using anti-Myc antibodies. Input and immunoprecipitates were analyzed by immunoblotting for PLK4 and HA-Ub. Ponc.S indicates total protein. (F) HEK293T cells were transfected to express Myc-PLK4 and eGFP or the indicated T7-TRIM37 protein (top) for 48 hr. MG132 was added for the final 4 hr. Lysates were mock treated (-?) or incubated with ?-phosphatase (+?) and subsequently subjected to immunoblot for PLK4. (CB) indicates total protein. (G) HEK293T cells were transfected to express Myc-PLK4 and T7-TRIM37 ?505–709 for 48 hr. Cells were treated with the indicated inhibitor (top) for 3 or 6 hr and analyzed by immunoblot for PLK4. Ponc.S indicates total protein. See Figure 7—source data 1. Figure 7—source data 1.Source data for Figure 7.