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Mesoscale regulation of microtubule-organizing centers by the E3 ligase TRIM37

Nature Structural & Molecular Biology volume 32, pages 1787–1799 (2025)Cite this article

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Abstract

Centrosomes ensure accurate chromosome segregation during cell division. Although the regulation of centrosome number is well established, less is known about the suppression of noncentrosomal microtubule-organizing centers (ncMTOCs). The E3 ligase TRIM37, implicated in Mulibrey nanism and 17q23-amplified cancers, has emerged as a key regulator of both centrosomes and ncMTOCs. Yet, the mechanism by which TRIM37 achieves enzymatic activation to target these mesoscale structures had thus far remained unknown. Here we elucidate the activation process of TRIM37, unveiling a process that initiates with TRAF domain-directed substrate recognition followed by B-box domain-mediated oligomerization and culminates in RING domain dimerization. Using optogenetics, we demonstrate that the E3 activity of TRIM37 is directly coupled to the assembly state of its substrates, being activated only when centrosomal proteins cluster into higher-order assemblies resembling MTOCs. This regulatory framework provides a mechanistic basis for understanding TRIM37-driven pathologies and echoes the restriction of the human immunodeficiency virus capsid by TRIM5, thus unveiling a conserved activation blueprint among TRIM proteins to control turnover of complexes assembled at the mesoscale level.

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Fig. 1: Domain-specific impact of MUL TRIM37 mutations on MTOC regulation.
Fig. 2: Proximity-dependent BioID identifies TRAF domain interactors of TRIM37.
Fig. 3: Chimeric TRIM5 bearing the TRIM37 TRAF domain regulates MTOCs.
Fig. 4: TRAF-directed higher-order assembly of TRIM37 at the centrosome.
Fig. 5: Endogenous TRIM37 localization at the centrosome is revealed by E3 ligase inactivation.
Fig. 6: Mutations in the B-box domain impair TRIM37 higher-order assembly.
Fig. 7: Optogenetic clustering of centrosomal substrates triggers recognition and activation of TRIM37.
Fig. 8: TRIM37 regulates MTOC function through substrate-templated higher-order assembly.

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Data availability

The BioID MS proteomics data were deposited to the ProteomeXchange Consortium through the PRIDE partner repository under accession number PXD061083. Source data are provided with this paper.

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Acknowledgements

This work was supported by research grants R01GM114119, R01GM133897 and R01CA266199 (to A.J.H) from the National Institutes of Health. M.v.B. was supported by the Queen Mary University of London and by the Biotechnology and Biological Sciences Research Council (BB/X013030/1). We thank M. R. Leung and N. K. Sinha for providing valuable experimental insights.

Author information

Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Zhong Y. Yeow, Sonia Sarju, Fang-Chi Chang, Lance Y. Xu & Andrew J. Holland

  2. School of Biological and Behavioural Sciences, Queen Mary University of London, London, UK

    Mark van Breugel

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  1. Zhong Y. Yeow
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  3. Fang-Chi Chang
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Contributions

Z.Y.Y. designed, performed and analyzed the majority of the experiments and prepared the figures. S.S., F.-C.C. and L.Y.X. assisted with the cloning and immunofluorescence analyses. M.v.B. performed the experiments to identify TRIM37-binding regions within Centrobin. Z.Y.Y. and A.J.H. conceptualized the study. A.J.H. supervised the study. Z.Y.Y. and A.J.H. cowrote the paper.

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Correspondence to Zhong Y. Yeow or Andrew J. Holland.

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Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Dimitris Typas, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Effect of TRIM37 mutations on the regulation of the centrosome and Centrobin assemblies.

Related to Fig. 1. (a) Immunoblot showing TRIM37 protein levels in parental and CRISPR–Cas9 edited RPE-1 TRIM37−/− cells. Ponceau-stained blot indicates loading. Representative data; n = 3 biological replicates. (b) Left, Tracking of Indels by Decomposition (TIDE) analysis histogram reveals a one base pair insertion ( + 1 bp) adjacent to the predicted cut site in the RPE-1 TRIM37−/− cell line. Right, representative Sanger sequencing traces used for TIDE analysis, highlighting the +1 bp insertion. (c) Representative images of RPE-1 TRIM37−/− cells and those expressing the indicated HA-tagged TRIM37 variants. Inset #1 denotes the centrosome, marked by CEP192, and inset #2 denotes the Centrobin assembly, identified by intense Centrobin staining that is non-centrosome localized. Representative data; n = 3 biological replicates. Scale bars, 5 μm. (d) Schematic representation of TRIM37 HA-tagged domain-specific deletion constructs compared to full-length (FL) protein. (e) Immunoblot showing expression levels of FL TRIM37 and the respective deletion mutants in RPE-1 tet-on TRIM37 cells. Ponceau-stained blot indicates loading. Representative data; n = 3 biological replicates.

Source data

Extended Data Fig. 2 Characterization of higher molecular weight (HMW) TRIM37 species.

Related to Fig. 4. (a) Immunoblot showing expression of WT TRIM37 and indicated mutants in RPE-1 tet-on TRIM37 cells. HMW TRIM37 species are prominently formed in the C18R mutant and indicated with an arrow. β-Actin, loading control. Representative data; n = 3 biological replicates. (b) Same as in (a) but with MG132 (10 μM) treatment to achieve proteasomal inhibition and stabilization of WT TRIM37. β-Actin, loading control. Representative data; n = 3 biological replicates. (c) Top, immunoblot showing detection of HMW TRIM37 species with increasing concentrations of DSS crosslinker. Vinculin is the loading and oligomerization control. Dotted lines indicate separate cropped sections of the same immunoblot. Representative data; n = 3 biological replicates. Bottom, Densitometric analysis of normalized HMW TRIM37 intensity upon increasing DSS concentrations relative to DMSO control ( − DSS). Mean ± s.e.m. (d) Sanger sequencing traces of the TRIM37 locus in parental and CRISPR–Cas9 edited RPE-1 TRIM37C18R cells, highlighting the mutation (TGT > CGT) responsible for the biallelic C18R residue substitution, denoted by an asterisk. (e) Top, immunoblot showing endogenous TRIM37 protein levels across the indicated cellular fractions. Validation markers include CEP192, Centrobin, and SAS6 for centrosomal proteins, and Lamin A/C for the nuclear fraction. Ponceau-stained blot indicates loading. Representative data; n = 3 biological replicates. WCE, whole-cell extract; exp, exposure. Bottom, Densitometric analysis of endogenous TRIM37 enrichment in indicated fractions relative to WCE. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate enrichment of TRIM37 in each cellular fraction relative to WCE. Mean ± s.e.m. (f) Top, immunoblot showing detection of various HMW species of endogenous TRIM37 upon treatment with increasing concentrations of DSG crosslinker. Vinculin is the loading and oligomerization control. Representative data; n = 3 biological replicates. Bottom, Densitometric analysis of normalized HMW TRIM37 intensity upon increasing DSG concentrations relative to DMSO control ( − DSG). Mean ± s.e.m.

Source data

Extended Data Fig. 3 Defining the minimal TRIM37 domain architecture required for centrosome regulation.

a) Schematic of the miniTRIM37 (RBCC-TRAF) construct compared to full-length TRIM37. (b) Representative images of the localization and effect of indicated HA-tagged TRIM37 constructs on centrosomal CEP192 levels in RPE-1 tet-on TRIM37 cells. Arrows indicate the positions of centrosomes. Representative data; n = 3 biological replicates. Scale bars, 5 μm. (c) Quantification of centrosomal CEP192 signal upon doxycycline-induced expression of indicated HA-tagged TRIM37 constructs in RPE-1 tet-on TRIM37 cells from (b). n = 3 biological replicates, each with >90 cells. P values, one-way ANOVA with post hoc Tukey’s multiple comparisons test. Mean ± s.e.m. (d) Immunoblot showing total protein levels of indicated HA-tagged TRIM37 constructs in RPE-1 tet-on TRIM37 cells from (b-c). GAPDH, loading control. Representative data; n = 3 biological replicates. (e) Immunoblot showing detection of various higher molecular weight (HMW) species of miniTRIM37 upon treatment with increasing concentrations of DSG crosslinker. Vinculin is used as a loading and oligomerization control. Representative data; n = 3 biological replicates.

Source data

Extended Data Fig. 4 Impairment of TRIM37 oligomerization attenuates synthetic lethality with PLK4 inhibition in 17q23-amplified cells.

(a) Immunoblot showing TRIM37 protein levels in TP53−/− MCF-7 cells. TRIM37 wild-type (WT), TRIM37 knockdown (KD) via shRNA, and cells harboring the C109S mutation in approximately half of the TRIM37 alleles present (TRIM37C109S) were used. Vinculin, loading control. Representative data; n = 3 biological replicates. (b) Left, Representative data of a 10-d clonogenic survival of indicated MCF-7 cell lines from (a) treated with DMSO (control) or PLK4 inhibitor (PLK4i) (250 nM). Right, Quantification of relative growth in the presence PLK4i relative to DMSO. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate differences between each experimental condition (KD and C109S) and WT. Mean ± s.e.m. (c) Quantification of mitotic CEP192 foci in PLK4i-treated TP53−/− MCF-7 cells that lack centrosomes. n = 3, biological replicates, each comprising >30 cells. P values, one-way ANOVA with post hoc Dunnett’s multiple comparisons test to evaluate differences between each experimental condition (KD and C109S) and WT. Mean ± s.e.m. (d) Representative images for (c). Scale bars, 5 μm. (e) Representative Sanger sequencing traces for the TRIM37 locus in parental TP53−/− MCF-7 cells subjected to TRIM37 knockdown (KD) via shRNA, and CRISPR–Cas9 edited TRIM37C109S KI cells. The mutation (TGT > TCT) leading to the C109S residue substitution is denoted by an asterisk. Silent mutations introduced to prevent re-editing are highlighted.

Source data

Extended Data Fig. 5 Substrate-independent clustering is sufficient to activate TRIM37.

Related to Fig. 7. (a) Top, schematic of the TRIM37G322V-mNeonGreen-CRY2 optogenetic fusion construct. The star denotes the TRAF domain mutation (G322V). Bottom, illustration of the blue light (BL)- activated optogenetic system enabling TRIM37 clustering independent of binding to a centrosome substrate. (b) Representative time-lapse images of RPE-1 cells expressing the optogenetic construct detailed in (a) incubated in the presence or absence of MG132. Timestamps indicate minutes post blue light exposure. Scale bar = 10 µm. (c) Quantification of mNeonGreen fluorescence intensity from (b), with each condition comprising >30 cells. Mean ± s.d. (d) RPE-1 cells expressing optogenetic constructs detailed in (a) were incubated with or without doxycycline (Dox) and MG132 (10 μM) in the absence or presence blue light for 3 h before immunoblotting for the indicated proteins. Higher molecular weight (HMW) TRIM37 species were prominently formed only in MG132 and BL-stimulated conditions and are indicated with an arrow. Ponceau-stained blot indicates loading. Representative data; n = 3 biological replicates. exp, exposure.

Source data

Supplementary information

Reporting Summary

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Supplementary Video 1 (related to Extended Data Fig. 5)

Time-lapse of an RPE-1 cell expressing the optogenetic TRIM37 G322V–mNG–CRY2 construct, with blue-light pulses (470-nm filter) applied during imaging intervals to induce and sustain CRY2 clustering and to visualize TRIM37–mNG dynamics. Timestamps indicate minutes from the initial blue-light exposure.

Supplementary Video 2 (related to Extended Data Fig. 5)

Time-lapse of an RPE-1 cell expressing the optogenetic TRIM37 G322V–mNG–CRY2 construct, with blue-light pulses (470-nm filter) applied during imaging intervals to induce and sustain CRY2 clustering and to visualize TRIM37–mNG dynamics. Cells were incubated with MG132 1 h before blue-light exposure. Timestamps indicate minutes from the initial blue-light exposure.

Supplementary Video 3 (related to Fig. 7)

Time-lapse of an RPE-1 TRIM37−/− cell expressing the optogenetic mCherry–CRY2 construct, incubated with doxycycline but in the absence of blue light. Timestamps indicate minutes from the first imaged frame. mCherry–CRY2 is displayed in grayscale.

Supplementary Video 4 (related to Fig. 7)

Time-lapse of an RPE-1 TRIM37−/− cell expressing the optogenetic mCherry–CRY2 construct, subjected to blue-light pulses at each imaging interval, but incubated without doxycycline. Timestamps indicate minutes from the initial blue-light exposure. mCherry–CRY2 is displayed in grayscale.

Supplementary Video 5 (related to Fig. 7)

Time-lapse of an RPE-1 TRIM37−/− cell expressing the optogenetic mCherry–CRY2 construct, incubated with doxycycline and subjected to blue-light pulses at each imaging interval. Timestamps indicate minutes from the initial blue-light exposure. mCherry–CRY2 is displayed in grayscale.

Supplementary Video 6 (related to Fig. 7)

Time-lapse of an RPE-1 TRIM37−/− cell expressing the optogenetic mCherry–CRY2–Centrobin567–836 construct, incubated with doxycycline but in the absence blue light. Timestamps indicate minutes from the first imaged frame. mCherry–CRY2–Centrobin567–837 is displayed in grayscale.

Supplementary Video 7 (related to Fig. 7)

Time-lapse of an RPE-1 TRIM37−/− cell expressing the optogenetic mCherry–CRY2–Centrobin567–836 construct, subjected to blue-light pulses at each imaging interval but incubated without doxycycline. Timestamps indicate minutes after initial blue-light exposure. mCherry–CRY2–Centrobin567–836 is displayed in grayscale.

Supplementary Video 8 (related to Fig. 7)

Time-lapse of an RPE-1 TRIM37−/− cell expressing the optogenetic mCherry–CRY2–Centrobin567–836 construct, incubated with doxycycline and subjected to blue-light pulses at each imaging interval. Timestamps indicate minutes from the initial blue-light exposure. mCherry–CRY2–Centrobin567–836 is displayed in grayscale.

Supplementary Video 9 (related to Fig. 7)

Time-lapse of an RPE-1 TRIM37−/− cell expressing the optogenetic mCherry–CRY2–Centrobin567–836 construct and TRIM37–mNG WT subjected to blue-light pulses at each imaging interval. Timestamps indicate minutes from the initial blue-light exposure. mCherry–CRY2–Centrobin567–836 is displayed in magenta and TRIM37–mNG is displayed in green.

Supplementary Video 10 (related to Fig. 7)

Time-lapse of an RPE-1 TRIM37−/− cell expressing the optogenetic mCherry–CRY2–Centrobin567–836 construct and TRIM37–mNG WT subjected to blue-light pulses at each imaging interval. Cells were incubated with MG132 1 h before blue-light exposure. Timestamps indicate minutes from the initial blue-light exposure. mCherry–CRY2–Centrobin567–836 is displayed in magenta and TRIM37–mNG is displayed in green.

Supplementary Video 11 (related to Fig. 7)

Time-lapse of an RPE-1 TRIM37−/− cell expressing the optogenetic mCherry–CRY2–Centrobin567–836 construct and TRIM37–mNG C18R subjected to blue-light pulses at each imaging interval. Timestamps indicate minutes from the initial blue-light exposure. mCherry–CRY2–Centrobin567–836 is displayed in magenta and TRIM37–mNG is displayed in green.

Supplementary Video 12 (related to Fig. 7)

Time-lapse of an RPE-1 TRIM37−/− cell expressing the optogenetic mCherry–CRY2–Centrobin567–836 construct and TRIM37–mNG G322V subjected to blue-light pulses at each imaging interval. Timestamps indicate minutes from the initial blue-light exposure. mCherry–CRY2–Centrobin567–836 is displayed in magenta and TRIM37–mNG is displayed in green.

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Yeow, Z.Y., Sarju, S., Chang, FC. et al. Mesoscale regulation of microtubule-organizing centers by the E3 ligase TRIM37. Nat Struct Mol Biol 32, 1787–1799 (2025). https://doi.org/10.1038/s41594-025-01540-6

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