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. Author manuscript; available in PMC: 2021 Mar 9.
Published in final edited form as: Nature. 2020 Sep 9;585(7825):440–446. doi: 10.1038/s41586-020-2710-1

Figure 2. TRIM37 prevents the formation of PLK4 condensate-based ectopic microtubule-organizing centers.

Figure 2.

(a) Immunofluorescence images localizing PLK4 in interphase RPE1 cells. PLK4 localizes to centrioles (cyan arrowheads) and to a single ectopic condensate (yellow arrows) in TRIM37Δ cells. (b) Immunoblot showing PLK4 protein levels are not altered in TRIM37Δ cells. (c) Immunofluorescence images of TRIM37Δ cells that lack centrioles due to treatment with centrinone showing centrosome components in an array of small condensates. (d) Schematic highlighting differences between the single large condensate in TRIM37Δ cells with centrioles (top) and the small condensates in cells that lack centrioles due to treatment with centrinone (bottom). (e) Images of centrinone-treated TRIM37Δ cells with in situ mNG-tagged CEP192 and transgene-expressed red fluorescent microtubule-binding domain. Times are minutes after NEBD. (f) Control or TRIM37Δ RPE1 cells with in situ-tagged CEP192 treated with centrinone to inhibit PLK4 activity (top) or after inducibly knocking out PLK4 (bottom). Immunofluorescence (left) and plots of relative proliferation (right) show that foci formation and improved proliferation of centrinone-treated TRIM37Δ cells require PLK4 protein. Error bars are SD (n=3). (g) Mitotic duration analysis of the conditions in (f). Error bars are 95% CI. (h) (left) Images showing localization to the centrosome of FLAG-tagged WT TRIM37 expressed in TRIM37Δ RPE1 cells. (middle) schematic summarizing the ligase and TRAF domain TRIM37 mutations. (right) Graph plotting percent of cells with condensates after expression in TRIM37Δ cells of FLAG-tagged WT or mutant TRIM37 proteins. (i) Interaction analysis of TRIM37 variants with PLK4 following co-expression and PLK4 immunoprecipitation. Low expression of wild-type TRIM37 prompted use of ligase-mutant TRIM37 in this analysis. (j) TRIM37 ligase activity-dependent ubiquitination of PLK4, observed following co-expression. α-tubulin serves as a loading control for the input in (i) and (j). Scale bars are 10 μm. For gel source data see Supplementary Figure 1.