Global cellular response to chemical perturbation of PLK4 activity and abnormal centrosome number
Centrosomes act as the main microtubule organizing center (MTOC) in metazoans. Centrosome number is tightly regulated by limiting centriole duplication to a single round per cell cycle. This control is achieved by multiple mechanisms, including the regulation of the protein kinase PLK4, the most upstream facilitator of centriole duplication. Altered centrosome numbers in mouse and human cells cause p53-dependent growth arrest through poorly defined mechanisms. Recent work has shown that the E3 ligase TRIM37 is required for cell cycle arrest in acentrosomal cells. To gain additional insights into this process, we undertook a series of genome-wide CRISPR/Cas9 screens to identify factors important for growth arrest triggered by treatment with centrinone B, a selective PLK4 inhibitor. We found that TRIM37 is a key mediator of growth arrest after partial or full PLK4 inhibition. Interestingly, PLK4 cellular mobility decreased in a dose-dependent manner after centrinone B treatment. In contrast to recent work, we found that growth arrest after PLK4 inhibition correlated better with PLK4 activity than with mitotic length or centrosome number. These data provide insights into the global response to changes in centrosome number and PLK4 activity and extend the role for TRIM37 in regulating the abundance, localization, and function of centrosome proteins.
This study analyses the molecular pathways that lead to growth arrest in human cells with altered centriole number, caused by inhibition of PLK4, the master regulator of centriole duplication. The authors identify the ubiquitin E3 ligase TRIM37 as a key mediator of this growth arrest, but, in contrast to previous work, they find that growth arrest correlates better with PLK4 activity than with the duration of mitosis or centrosome number. The work extends the role for TRIM37 in regulating centrosome protein levels, distribution, and function, and will be of interest to researchers interested in centrosome and growth regulation.https://doi.org/10.7554/eLife.73944.sa0
The centrosome is a multi-protein complex that is the major microtubule organizing center (MTOC) of metazoan cells influencing microtubule-based processes such as cell division, ciliogenesis, signalling, and cell motility (Conduit et al., 2015). Each centrosome consists of two microtubule-based centrioles surrounded by pericentriolar material (PCM) (Conduit et al., 2015). Each cell inherits a single centrosome from the previous cell cycle and subsequent centriole duplication is restricted to a single round of replication (Gönczy and Hatzopoulos, 2019). Centriole duplication initiates at the G1/S transition and is largely a stepwise pathway dependent on the upstream kinase PLK4 (Gönczy and Hatzopoulos, 2019). PLK4 is a low-abundance protein (Fode et al., 1996) that is recruited around the mother centriole (Sonnen et al., 2013; Kim et al., 2013). PLK4 kinase activity is regulated by interaction with STIL and CEP85 resulting in the recruitment of SASS6 to form the cartwheel of the nascent daughter centriole followed by the recruitment of centriole elongation factors (Moyer et al., 2015; Ohta et al., 2014; Liu et al., 2018; Schmidt et al., 2009; Kohlmaier et al., 2009; Tang et al., 2009; Comartin et al., 2013; Azimzadeh et al., 2009). PLK4 functions as a homodimer and autophosphorylates itself in trans to generate a phosphodegron sequence that limits its abundance. This sequence is recognized by SCFβ-TrCP and targets PLK4 for ubiquitin-mediated proteolysis (Rogers et al., 2009; Cunha-Ferreira et al., 2009; Guderian et al., 2010).
PLK4 misregulation is often associated with pathological states. Centrosome amplification is a hallmark of tumor cells (Chan, 2011) and may play a role in generating chromosome instability (Ganem et al., 2009; Silkworth et al., 2009) and promoting cell invasiveness (Arnandis et al., 2018). In cell culture and mouse models, overexpression, inhibition, or deletion of PLK4 results in p53-dependent arrest (Holland et al., 2012; Lambrus et al., 2016; Wong et al., 2015; Fong et al., 2016; Marthiens et al., 2013; Coelho et al., 2015; Vitre et al., 2015; Serçin et al., 2016; Hudson et al., 2001). A series of CRISPR/Cas9 screens identified the p53 pathway members, p53, p21(CDKN1A), 53BP1 and USP28 (Lambrus et al., 2016; Fong et al., 2016; Meitinger et al., 2016), and the E3 ligase TRIM37 as component of this response (Fong et al., 2016; Meitinger et al., 2016). A recent screen for mediators of supernumerary centrosome-induced arrest identified PIDDosome/p53 and placed the distal appendage protein ANKRD26 within this pathway (Evans et al., 2021; Burigotto et al., 2021).
TRIM37 is an E3 ligase that has been associated with a myriad of cellular functions including gene expression (Bhatnagar et al., 2014), peroxisome maturation (Wang et al., 2017), various signalling pathways (Zhu et al., 2020; Fu et al., 2021; Chen et al., 2020; Wang et al., 2018), and centriole biology (Balestra et al., 2013; Balestra et al., 2021; Meitinger et al., 2021; Yeow et al., 2020). There is no consensus on how TRIM37 mediates these functions since its activities have been linked to mono-ubiquitination (Bhatnagar et al., 2014; Wang et al., 2017), poly-ubiquitination (Zhu et al., 2020; Chen et al., 2020; Yeow et al., 2020; Meitinger et al., 2020), and E3-independent functions (Wang et al., 2018) that result in changes in protein activity (Bhatnagar et al., 2014; Wang et al., 2018), localization (Meitinger et al., 2016; Balestra et al., 2021; Meitinger et al., 2021), and abundance (Wang et al., 2017; Zhu et al., 2020; Chen et al., 2020; Yeow et al., 2020; Meitinger et al., 2020). A number of centrosome-related TRIM37 functions have been described. In the absence of TRIM37, a collection of centriole proteins such as CNTROB, PLK4, CETN1/2, CP110 accumulate to form aberrant assemblies referred to as condensates or ‘Cenpas’ (centriolar protein assemblies) (Balestra et al., 2021; Meitinger et al., 2021). These structures are dependent on the presence of CNTROB, but it is unclear why TRIM37 might suppress their formation in normal cells. TRIM37 is part of the 17q23 amplicon present in approximately 18% of breast cancer tumors (Kallioniemi et al., 1994) and overexpression of TRIM37 in these lines renders them sensitive to the PLK4 inhibitor centrinone (Yeow et al., 2020; Meitinger et al., 2020). TRIM37 interacts with PLK4 and CEP192 (Meitinger et al., 2020). Although TRIM37 can promote ubiquitination of PLK4, it is distinct from SCFβ−TRCP modification since it does not result in changes to PLK4 abundance (Meitinger et al., 2020). In contrast, transiently overexpressed TRIM37 leads to CEP192 ubiquitination and its subsequent degradation (Yeow et al., 2020; Meitinger et al., 2020). In this model of TRIM37 function, PLK4-nucleated condensates consisting of PCM components facilitate mitosis in the absence of TRIM37 and the overexpression of TRIM37 decreases the cellular levels of CEP192 rendering cells sensitive to the loss of centrioles.
Here, we sought to determine how growth arrest is initiated in response to alterations in PLK4 activity and centrosome number. Using the specific PLK4 inhibitor, centrinone B, we modulated PLK4 activity to generate supernumerary centrosomes or centrosome loss and performed genome-wide chemical genetic screens in RPE-1 and A375 cells. Our screens identified distinct pathways mediating the response to partial and full PLK4 inhibition. Intriguingly, TRIM37 was required for growth when PLK4 was partially or fully inhibited but was dispensable for arrest triggered by PLK4 overexpression. Moreover, TRIM37 growth arrest activity was partially independent of its E3 ligase activity. These results highlight the complex role of TRIM37 and regulators of its function in the control of centrosome number homeostasis.
Centrinone B induces concentration-dependent changes in centrosome number
Centrinone is a PLK4-specific inhibitor that is often used to study the cellular response to centrosome loss (Wong et al., 2015). Another PLK4 inhibitor, CFI-400945, induces both centrosome amplification, or loss, in a concentration-dependent manner (Mason et al., 2014). Since CFI-400945 also inhibits other mitotic kinases such as AURKB (Suri et al., 2019) it is unclear if its reported phenotypes are due to effects on PLK4, mitosis, or both. Centrinone and centrinone B are both potent inhibitors of PLK4 that show selectivity over the Aurora-family kinases (Wong et al., 2015). We used centrinone B for our experiments since it shows an even greater selectivity over the Aurora kinases compared to centrinone (Wong et al., 2015; Suri et al., 2019). We treated RPE-1 cells with 200 and 500 nM centrinone B and found that cell growth was greatly inhibited at both centrinone B concentrations (Figure 1A). As expected, cell growth arrest was correlated with induction of p53 and p21 (Figure 1B, C, Figure 1—figure supplement 1A, B), resulting in the accumulation of cells with 1 N DNA content (Figure 1—figure supplement 1C). We next imaged cells treated with centrinone B for 3 days to determine how centrosome number was affected as a function of centrinone B concentration. As expected, 500 nM centrinone B resulted in cells containing either a single or no centrosome, but cells treated with 200 nM failed to lose centrosomes and instead accumulated supernumerary centrosomes in approximately 50% of cells (Figure 1C and D, Figure 1—figure supplement 1B, D). Staining for a panel of centriole and centrosome markers revealed that these extra structures contained CEP135, CEP120, CETN2, glutamylated tubulin, and could accumulate PCNT and CEP192 indicating that treatment with 200 nM centrinone B can induce amplification of bona fide centrosomes (Figure 1—figure supplement 1E). Together, these data indicate that centrosomes can be amplified or lost using 200 or 500 nM centrinone B, respectively, and that both phenotypes result in p53-dependent G1 arrest.
Global cellular responses to abnormal centrosome number
To understand the mechanisms of centrinone-induced cell cycle arrest, we performed genome-wide CRISPR screens in the presence of 200 and 500 nM centrinone B (Figure 2A; Hart et al., 2015). We reasoned that cell fitness at each centrinone B condition would require distinct sets of genes. Since loss of components in the p53 pathway itself would also increase fitness, we performed a parallel screen in the presence of Nutlin-3a, a small molecule that prevents the MDM2-mediated inactivation of p53 (Vassilev et al., 2004), allowing us to filter out core p53 pathway components. RPE-1 or A375 cells stably expressing Cas9 were infected with the TKOv1 lentiviral sgRNA library (Hart et al., 2015), selected, and subsequently treated with the indicated drug concentrations (Figure 2A). After growth for 21 days, cells were harvested and subjected to next-generation sequencing (NGS) and model-based analysis of genome-wide CRISPR-Cas9 knockout (MAGeCK) analysis (Li et al., 2014). Genes with a false discovery rate (FDR) of <0.05 were considered hits for subsequent analyses. The combined screens to identify regulators of growth arrest using Nutlin-3a, 200 and 500 nM centrinone B yielded 91, 136, and 56 high-confidence hits that positively or negatively affected cell growth, respectively (Supplementary file 1).
We created a network diagram to visualize the hits on a global scale (Figure 2B). Each unique cell line and condition act as the hubs (i.e., RPE-1, 200 nM centrinone B) while the hits from each condition are the remaining nodes. Each screen identified overlapping and distinct sets of genes, supporting our hypothesis that cells respond differently to each of the conditions tested (Figure 2B, Figure 2—figure supplement 2A-D, Supplementary file 2). The Nutlin-3a dataset contained core p53 pathway genes TP53 and CDKN1A and genes coding for p53 regulators TP53BP1 and USP28, consistent with their role in promoting p53 transcriptional activity (Cuella-Martin et al., 2016). This dataset likely contains other mediators of the p53 pathway. The disruption of both BAG6 and EP300 increased fitness in Nutlin-3a. EP300 is an acetyltransferase that binds to and affects the acetylation of p53 while BAG6 modulates this acetylation event by EP300 (Sebti et al., 2014; Liu et al., 1999). Likewise, inactivation of AHR and ARNT, which form a transcriptional complex activated by exogenous ligands, promoted growth when p53 is activated in RPE-1 cells, but not A375. ARNT was previously identified as a FRET interactor with p53 (Li et al., 2017) and interacts with EP300 (Tohkin et al., 2000; Tong et al., 2016). Deletion of TSC1/2 that integrates p53 signalling with the mTOR pathway (Armstrong et al., 2017; Lee et al., 2007) caused decreased fitness after p53 activation. We used Genemania (Franz et al., 2018) to further probe the pairwise physical interactions among the hits from the RPE-1 Nutlin-3a screen and generated a significantly enriched network (~20-fold enrichment, p=4.1 × 10–31). In this network, 27 of the 57 hits formed physical interactions with eight proteins forming complexes with p53 itself (Figure 2—figure supplement 1E). Our high-confidence hits from the Nutlin-3a screen identified known p53 pathway members and likely contains unknown regulators of this pathway that will warrant further characterization.
The 200 nM centrinone B screens (i.e., condition that produces supernumerary centrosomes) revealed a core set of 23 genes that suppressed the growth arrest in both cell lines (Supplementary file 1, Figure 2—figure supplement 2C). Notably, we identified the ANKRD26/CASP2/PIDD1/CRADD (PIDDosome) complex recently implicated in the response to supernumerary centrosomes (Evans et al., 2021; Burigotto et al., 2021). This set also included p53 pathway genes (TP53, CDKN1A, TP53BP1, and USP28), centriole duplication factors (CEP85, CENPJ, STIL, USP9X), centriolar satellite proteins (C2CD3, CEP350, KIAA0753, PIBF1), and TRIM37. The A375 screen identified additional centriole components namely CP110, CEP97, CEP135, SASS6, CEP76, and PLK4. Twenty-two of the hits from both cell lines combined also overlapped with the high-confidence hits from a similar screen that induced centrosome amplification by overexpressing PLK4 (Evans et al., 2021). The overexpression screen also yielded the ANKRD26/PIDDOsome and some, but not all the centriole-associated genes, nor TRIM37 (Figure 2—figure supplement 2F).
We identified a total of 37 suppressors probing the response to centriole depletion (500 nM centrinone B), with five scoring in both cell lines (TP53, CDKN1A, TP53BP1, USP28, and TRIM37). Our results are similar to previous screens aimed at identifying suppressors of growth arrest due to centrosome loss from PLK4 inhibition that identified 31, 41, and 27 genes, respectively (Lambrus et al., 2016; Fong et al., 2016; Meitinger et al., 2016). Four of the five common hits (TP53, TP53BP1, USP28, CDKN1A) correspond to core p53 pathway components and were the only genes identified by all screens performed to date (Figure 2—figure supplement 2G). CHD8 and FBXO42 were previously identified in the response to centrosome loss (Fong et al., 2016) and also scored in our 200 and 500 nM centrinone B screens, respectively; however they also appeared in our Nutlin-3a hits suggesting that these genes might not be specific to centrosome biology. Indeed, both FBXO42 and CHD8 are known to negatively regulate p53 activity (Sun et al., 2009; Nishiyama et al., 2009; Lü, 2022). Two of the previous centriole loss screens also identified TRIM37 (Lambrus et al., 2016; Meitinger et al., 2016) which was unique among all the other hits since it was the only gene outside the p53 pathway that scored in both RPE-1 and A375 cells in both centrinone B concentrations. We therefore chose to study TRIM37 further.
TRIM37 localizes near the centrosomes but is not required for centriole duplication
Our combined screens indicated that TRIM37 was required for growth arrest in response to PLK4 inhibition that results in either centrosome overduplication or loss. Since TRIM37 has been implicated in centriole duplication (Balestra et al., 2013), we determined if loss of TRIM37 affected centrosome number after treatment with centrinone B. RPE-1 cells were infected with viruses expressing two independent sgRNAs targeting TRIM37 (Figure 3—figure supplement 1) and selected cell pools were treated with 200 and 500 nM centrinone B and assessed for centriole number (Figure 3A). As previously observed (Balestra et al., 2013), we noted a small number of TRIM37-disrupted cells harbored extra centrioles in untreated cells. However, centriole amplification or loss after centrinone B treatment was not greatly affected (Figure 3A). To further characterize TRIM37, we created a TRIM37-disrutped RPE-1 clonal cell line using CRISPR/Cas9 (Figure 3—figure supplement 1). After treatment of cells with centrinone B, p53 and p21 failed to accumulate in TRIM37-/- cells at 200 or 500 nM centrinone B (Figure 3B). In the presence of supernumerary centrosomes, but not in the absence of centrosomes, MDM2 is cleaved via the ANKRD26/PIDDosome pathway that relieves its p53 inhibitory function and promotes p53 transcriptional activity (Evans et al., 2021; Burigotto et al., 2021; Fava et al., 2017). We also noted a small amount of cleaved MDM2 (Fava et al., 2017) in untreated TRIM37-/- cells with no additional increase after 200 nM centrinone B. This cleavage product was lost after treatment with 500 nM centrinone B. Thus, we find that TRIM37 does not affect gain or loss of centrosomes after centrinone B treatment but is required for the induction of both p53 and p21 in response to these treatments as previously observed (Meitinger et al., 2016), and is required for MDM2 cleavage.
Since TRIM37 was a prey for multiple centrosome baits in our previous BioID survey of centrosomal proteins (Gupta et al., 2015), we sought to determine if TRIM37 localized to centrosomes. Immunofluorescence staining of endogenous TRIM37 indicated that the protein was associated with the centrosome in most cells although it did not strictly co-localize with either γ-tubulin or CEP120 (Figure 3C). We verified that the anti-TRIM37 antibody used reliably detected endogenous TRIM37. The overall TRIM37 signal was greatly reduced in TRIM37-/- cells (Figure 3—figure supplement 1) and TRIM37 detected at the centrosome in interphase cells was largely diminished (Figure 3—figure supplement 1). We noted that a minor TRIM37 signal at the centrosome remained in TRIM37-/- cells but could not be further reduced using siRNA directed against TRIM37 (Figure 3—figure supplement 1) suggesting the antibody displays weak cross reactivity with another centrosomal component. Since the intensity difference in mitotic cells was not as large that observed in interphase cells after disruption and/or knockdown, we restricted our analysis to interphase cells (Figure 3—figure supplement 1). To determine if TRIM37 localization was cell cycle-dependent, we co-stained TRIM37 with PCNA and CEP120 (Figure 3D) and detected TRIM37 in all cell cycle stages we could discern. We further determined if TRIM37 preferentially localized to mother or daughter centrioles by staining with CETN2 to detect all centrioles and CEP120 that preferentially localizes to daughter centrioles (Figure 3F and G). TRIM37 localized to both mother and daughter centrioles in most cells and localized exclusively to the mother or daughter in only a small percentage of cells. Interestingly, in cells with two centrosomes, TRIM37 appeared to favor one centrosome over the other (Figure 3E, arrowhead) and we observed a minor preference for exclusive association with mother or daughter centrioles in G1 cells (Figure 3G). Given that the fluorescence signal from detecting endogenous protein was too low for super-resolution imaging, we performed 3D-SIM on RPE-1 Cas9 TRIM37-/- cells stably expressing FLAG-BirA-TRIM37 (FB-TRIM37) (Figure 3H, Figure 3—figure supplement 2). FB-TRIM37 formed partial ring structures preferentially surrounding one of the γ-tubulin foci. Moreover, the FB-TRIM37 signal was discontinuous with a dot-like distribution around the ring.
As a member of the TRIM family of E3 ligases, TRIM37 contains an N-terminal RING domain followed by a B-box and a coiled-coil region CCR (Hatakeyama, 2017). The E3 ligase activity of TRIM37 has been implicated in its centrosomal-related functions in mitotic length (Meitinger et al., 2020), PCM stability (Yeow et al., 2020; Meitinger et al., 2020), and PLK4 localization (Meitinger et al., 2020). We created two TRIM37 E3 ligase mutants, one containing a C18R point mutation (Bhatnagar et al., 2014) and another deleting the RING domain entirely (ΔRING). After stable expression in RPE-1 TRIM37-/- cells, the steady-state abundance of both ligase mutants was greater than the wild type (WT) protein, consistent with TRIM37 auto-regulating its stability (Meitinger et al., 2020; Figure 3—figure supplement 2). Correspondingly, immunofluoresence for the tagged proteins confirmed their relative abundances and demonstrated that the proteins were expressed in all cells (Figure 3—figure supplement 2). The proteins were found primarily in the cytoplasm, sometimes in punctate structures possibly representing their peroxisomal localization (Wang et al., 2017). Further, we quantified the centrosomal localization of these proteins and found that both E3 mutants localized to the centrosome, albeit at slightly lower levels than the WT protein (Figure 3I and J). Thus, TRIM37 localizes to an area near the centrosomes proper, surrounding the PCM in a manner that is partially dependent on its E3 ligase activity.
An E3-independent TRIM37 activity mediates growth arrest after PLK4 inhibition
To determine if TRIM37 E3 ligase activity promotes growth arrest after centrinone B treatment, we performed clonogenic survival assays with the E3 mutant rescue lines in the presence of DMSO, 200 or 500 nM centrinone B (Figure 4A). Expression of FB-TRIM37 fully restored the growth arrest triggered by centrinone B as did the expression of either C18R or ΔRING (Figure 4A). These data suggest that TRIM37 promotes growth arrest in response to centrinone B in an E3-independent manner.
To corroborate these observations, we created TRIM37 knockout pools in RPE-1 and A375 cells using an sgRNA distinct from that used to make the clonal line (Figure 3—figure supplement 1). These pools were infected with virus to express TRIM37 and the indicated TRIM37 mutants (Figure 4—figure supplement 1 and B). In addition, we performed clonogenic assays using varying centrinone B concentrations to fully characterize the growth arrest activity promoted by TRIM37 (Figure 4B, Figure 4—figure supplement 1). Robust cell arrest of WT cells was observed after treatment with 125 nM centrinone B or greater. Correspondingly, in RPE-1 cells, we observed increases of p53 and p21 abundance with increasing centrinone B that was attenuated in TRIM37-/- cells (Figure 4—figure supplement 1). Interestingly, cells lacking TRIM37 arrested after treatment with very low doses of centrinone B (50–125 nM) but only partially after higher concentrations (≥150 nM). As observed with cell lines derived from the clonal TRIM37 disruption, both E3-defective mutants, C18R and ΔRING, promoted growth arrest activity. To examine PLK4 function more closely after inhibition by centrinone B, we monitored centrosome number and cellular PLK4 mobility. There was a dose-dependent increase in centrosome number up to 167 or 200 nM after which cells harbored one or no centrosomes (Figure 4C and Figure 4—figure supplement 1). Surprisingly, abnormal centrosome number did not correlate with robust growth arrest in WT cells; cell growth was almost completely inhibited at 125 nM centrinone B although we observed minor centrosome abnormalities at this concentration. Recently, PLK4 phosphorylation status near its phosphodegron sequence was linked to its cellular mobility where decreased phosphorylation at this site resulted in decreased mobility (Yamamoto and Kitagawa, 2019). We expressed a PLK4 reporter construct (GFP-PLK kin + L1) and monitored PLK4 mobility using FRAP after treatment with centrinone B (Figure 4D; Yamamoto and Kitagawa, 2019). PLK4 mobility decreased with increasing centrinone B concentrations that mirrored growth arrest activity. Based on RPE-1 cells, our data uncovered three phases in response to PLK4 inhibition. Phase I is TRIM37-independent and occurs at centrinone B concentrations where cells display minor centrosome number aberration (≤125 nM); a TRIM37-dependent phase II occurs at ≥150 nM centrinone B and can be further separated based on centrosome number; phase IIa cells harbor overduplicated centrosomes (150–200 nM) while phase IIb cells have lost one or both centrosomes (500 nM) (Figure 4E). Similar trends were observed using A375 cells, although the exact concentrations of centrinone B required differed between the cell lines (Figure 4—figure supplement 1).
We noted that our FB-TRIM37 construct was overexpressed compared to the endogenous protein (Figure 4—figure supplement 2A), so we created TRIM37-/- cells lines that expressed inducible TRIM37-3xFLAG or TRIM37C18R-3xFLAG. Compared to our stable cell lines, the inducible lines expressed TRIM37 closer to endogenous levels, although the C18R mutant was always more highly expressed, similar to previous studies (Figure 4—figure supplement 2E; Meitinger et al., 2020). While the total cellular amount of the stable FLAG-BirA constructs was ~20-fold higher than that of the DOX-inducible system (Figure 4—figure supplement 2), the centrosomal difference was only ~3-fold (Figure 4—figure supplement 2). We performed clonogenic growth assays in the absence or presence of DOX and 0, 167, or 500 nM centrinone B (Figure 4F). Expression of either WT or C18R TRIM37 resulted in growth arrest although the growth arrest phenotype caused by TRIM37 C18R expression was weaker than that of WT cells. These data suggest that TRIM37 can support growth arrest after PLK4 inhibition in the absence of E3 ligase activity.
To distinguish between PLK4 activity and centrosome number, we used orthologous methods tTo corroborate these observations, we creato control centrosome number. First, we disrupted the gene encoding SASS6, a protein required for centriole duplication (Leidel et al., 2005; Dammermann et al., 2004), to induce centrosome loss in TRIM37-/- cells (Figure 4—figure supplement 3A). We recovered cells lacking centrioles indicating that TRIM37 is also required for growth arrest in these conditions since SASS6 is essential in WT cells (Leidel et al., 2005). Interestingly, TRIM37-/-SASS6-/- cells were completely resistant to any dose of centrinone B, unlike TRIM37-/- alone (Figure 4—figure supplement 3B). Similar to centrosome loss induced by PLK4 inhibition, inducible expression of TRIM37 C18R partially rescued the growth arrest phenotype caused by SASS6 loss (Figure 4G, Figure 4—figure supplement 3C,D). To induce centrosome amplification, we overexpressed PLK4 (Figure 4—figure supplement 3E). Similar to previous studies (Evans et al., 2021), we found that disruption of TRIM37 was unable to suppress growth arrest in these conditions (Figure 4H). Together, these data suggest that TRIM37 is required for growth arrest after moderate or full PLK4 inhibition and this activity is partially independent of TRIM37’s E3 ligase activity.
Growth arrest upon treatment with centrinone B does not correlate with an increase in mitotic length
Abnormal mitotic length has been put forth as an attractive model to explain growth arrest after centrosome loss (Lambrus et al., 2016). We measured mitotic length after inhibiting PLK4 at different centrinone B concentrations using live-cell imaging of WT or TRIM37-/- RPE-1 cells. Cells were treated with the indicated concentration of centrinone B for 3 days before imaging them for 24 hr, and the length of mitosis from nuclear envelope breakdown (NEBD) to telophase was quantified (Figure 5A). WT RPE-1 cells treated with centrinone B did not exhibit a significant increase in mitotic length, compared to untreated cells, until concentrations reached 150 nM. Strikingly, the mitotic length of TRIM37-/- cells treated with centrinone B was similar to that of WT cells until concentrations of 200 and 500 nM centrinone B; at which point the absence of TRIM37 partially suppressed the increase in mitotic length in WT cells (Meitinger et al., 2020). We next examined whether TRIM37 E3 activity was required to rescue the shortened mitotic length observed in TRIM37-/- cells. We induced the expression of WT TRIM37 and the C18R mutant in TRIM37-/- cells, treated them with 500 nM centrinone B and measured mitotic length. In the absence of protein induction, we observed a slight increase in mitotic length after centrinone B treatment and a larger increase after induction of WT TRIM37 (Figure 5B). In contrast, the mitotic length in the presence of centrinone B after expression of TRIM37 C18R did not change compared to similarly treated uninduced cells. Thus, the increase in mitotic length observed after full PLK4 inhibition (500 nM centrinone B) appears to be dependent on TRIM37 E3 activity.
In the absence of centrosomes, amorphous collections of primarily PCM components act as pseudo-MTOCs in cells lacking TRIM37 (Meitinger et al., 2016). We induced the expression of WT TRIM37 or TRIM37 C18R in TRIM37-/- cells, treated them with 500 nM centrinone B for 3 days before staining for CEP192 (Figure 5C) and analyzed mitotic cells for the number of MTOCs in each cell based on CEP192 distribution (Figure 5D). In DMSO-treated cells, all lines mostly formed two distinct MTOCs. In the presence of centrinone B, but in the absence of induced protein, most cells formed two fragmented MTOCs, characteristic of TRIM37-/- cells after PLK4 inhibition (Meitinger et al., 2016). Cells expressing WT TRIM37 and treated with centrinone B harbored a single fragmented MTOC or none at all. Lastly, cells expressing TRIM37 C18R and treated with centrinone B displayed a partial phenotype where some cells formed two dispersed MTOCs while others displayed a single dispersed MTOC. Thus, TRIM37 E3 ligase activity is not strictly required to suppress pseudo-MTOCs or to promote cell growth after centrosome loss.
TRIM37 affects the abundance and localization of both centriolar and PCM components in an E3-dependent manner
To assess whether TRIM37 regulates the abundance of centriolar or PCM components, we initially probed for PCM components in RPE-1 and RPE-1 TRIM37-/- lines but did not detect any significant changes in steady-state protein levels (Figure 6A and Figure 6—figure supplement 1A). To determine if overexpressed TRIM37 affected steady-state PCM levels, we again probed for PCM proteins in TRIM37-/- cells stably expressing FLAG-BirA, FB-TRIM37, or the TRIM37 E3 mutants. We observed a significant decrease in CEP192 that was E3-dependent but did not observe significant changes in PCNT or CEP215 (Figure 6B and Figure 6—figure supplement 1B). We next expressed inducible WT or C18R TRIM37 in WT RPE-1 cells (Yeow et al., 2020; Meitinger et al., 2020). After expression of WT TRIM37 for 4 or 8 hr, we detected a 50–75% decrease in total CEP192 protein (Figure 6C, left panels). In all cases the decrease in CEP192 was E3-dependent. These results are consistent with previous observations that also indicated that TRIM37 is a negative regulator of CEP192 abundance (Yeow et al., 2020; Meitinger et al., 2020). We extended these findings by using immunofluorescence to specifically detect changes at mitotic centrosomes (Figure 6—figure supplement 1C). Quantification revealed that acute expression of WT TRIM37, but not C18R decreased the intensity of CEP192 and PCNT at the centrosome (Figure 6D). Interestingly the centriole component CEP120 was similarly diminished at the centrosome indicating that this effect is not specific to PCM proteins (Figure 6D). We find that overexpression of TRIM37 affects the overall abundance of CEP192 and the mitotic accumulation of CEP192, PCNT, and CEP120 in an E3-dependent manner.
In the absence of TRIM37, some centriolar proteins form CTNROB-dependent ectopic intracellular aggregates, termed Cenpas, in interphase cells (Balestra et al., 2021; Meitinger et al., 2020). We initially observed that CEP120 was mislocalized in TRIM37-/- cells and co-localized with CNTROB (Figure 6E). Ectopic CNTROB structures remained after CEP120 depletion using siRNA suggesting that CEP120 is assembled downstream of CNTROB. (Figure 6—figure supplement 2A). We found that CEP120 and CNTROB were detected in these structures and CETN2 foci accumulated near them (Figure 6E). Notably, we did not detect PLK4 in the CNTROB/CEP120 structure. Previously, PLK4 has been observed in these structures but could only be detected using a single antibody and the signal remained after siRNA treatment (Meitinger et al., 2016; Balestra et al., 2021; Meitinger et al., 2021). To determine if PLK4 could be recruited into these structures, we expressed PLK4-3xFLAG from an inducible promoter in TRIM37-/- cells. PLK4-3xFLAG was not detected at non-centrosomal aggregates after 3 or 6 hr induction using either anti-FLAG or anti-PLK4 antibodies, despite its accumulation at the centrosome (Figure 6—figure supplement 2B). Additionally, these assemblies were not affected by the loss of PLK4 activity as they were observed in TRIM37-/- cells treated with 500 nM centrinone B for 3 days (Figure 6—figure supplement 2C, D). After stable expression of TRIM37 mutants in TRIM37-/- cells these structures disappeared in an E3-dependent manner (Figure 6F, Figure 6—figure supplement 2E). We find that CEP120 is a downstream component of the CNTROB structures formed in TRIM37-/- cells and that the suppression of their formation requires TRIM37 E3 ligase activity.
TRIM37 promotes the phosphorylation of PLK4
TRIM37 is suggested to associate with and ubiquitinate PLK4 (Meitinger et al., 2020). We performed a structure-function analysis of TRIM37 to determine which region(s) of TRIM37 were required for PLK4 complex formation (Figure 7A) We transiently expressed a series of FB-TRIM37 deletion mutants and Myc-PLK4 in RPE-1 cells, immunoprecipitated the FB-TRIM37 constructs, and probed for PLK4 (Figure 7A and B, Figure 7—figure supplement 1A). Our results confirmed that PLK4 and TRIM37 form a complex in RPE-1 cells (Meitinger et al., 2020). Further, the region from amino acids 505–709 of TRIM37 was sufficient to immunoprecipitate PLK4. Conversely, a TRIM37 mutant lacking this region (FB-Δ505–709) failed to pull down PLK4. When stably expressed in TRIM37-/- cells, FB-505–709 and FB-Δ505–709 were well expressed (Figure 7—figure supplement 1B, D) but only FB-Δ505–709 localized to centrosomes (Figure 7—figure supplement 1C, E). To determine if the PLK4/TRIM37 association was required for growth arrest activity, we performed clonogenic assays using TRIM37-/- cell lines stably expressing FB-TRIM37 505–709 and FB-TRIM37 Δ505–709 (Figure 7C, Figure 7—figure supplement 1B-E) or expressing inducible FB-Δ505–709 (Figure 7—figure supplement 1F, G). Surprisingly, the association between PLK4 and TRIM37 did not appear to be required for centrinone B-induced growth arrest.
PLK4 protein abundance is tightly controlled by multiple post-translational modifications including phosphorylation and ubiquitination (Rogers et al., 2009; Cunha-Ferreira et al., 2009; Guderian et al., 2010; Yamamoto and Kitagawa, 2019). The co-expression of Myc-PLK4 and T7-TRIM37 in HEK293T cells resulted in modification of PLK4 (Figure 7D). The modification was partially dependent on TRIM37 E3 activity, was not observed when TRIM37 505–709 was expressed, and increased in the presence of TRIM37 Δ505–709. MLN4924 is a general inhibitor of cullin-RING E3 ligases and treating cells with this compound should inhibit ubiquitination of PLK4 by SCFβ-TrCP (Soucy et al., 2009). Treatment of cells with MLN4924 resulted in stabilization of PLK4 but the modified forms remained (Figure 7D). To directly test if the observed modification was ubiquitinated PLK4, we co-expressed Myc-PLK4, T7-TRIM37, and HA-Ub in 293T cells. After immunoprecipitating PLK4, we probed for HA-Ub to detect ubiquitinated species (Figure 7E). Although we detected an E3-dependent increase in total ubiquitinated proteins in the input of cells expressing WT and Δ505–709 T7-TRIM37, we only detected low levels of HA-Ub conjugates in the anti-Myc immunoprecipitates, suggesting that PLK4 modification upon expression of TRIM37 may not be due to its ubiquitination. As an alternative possibility, we tested if the modified PLK4 bands were due to phosphorylation by treating cell lysates with λ-phosphatase (Figure 7F). The slower migrating forms of PLK4 were lost after phosphatase treatment indicating that these modifications are primarily due to phosphorylation. To identify the kinase(s) responsible for the modification, we treated cells with inhibitors targeting PLK4, AURKA, PLK1, and CDK1 for 3–6 hr and probed for PLK4. The only treatment that substantially reduced PLK4 phosphorylation was PLK4 inhibition (Figure 7G). Since we observed that TRIM37 promotes PLK4 phosphorylation (Figure 7G), we monitored GFP-PLK4 mobility by FRAP in WT, TRIM37-/- cells and after TRIM37 siRNA but did not observe any differences compared to control cells indicating that the phospho-forms of PLK4 stabilized by TRIM37 likely lie outside the phosphodegron region (Figure 7—figure supplement 1H, I). Together these data suggest that TRIM37 promotes the accumulation of phosphorylated PLK4 in an E3-dependent manner but this phenomenon does not require robust interaction with PLK4 itself.
Most animal cells harboring abnormal centrosome numbers are subject to p53-dependent growth arrest and the mechanisms of these pathways are beginning to be understood. Here, by leveraging the various phenotypes caused by treatment of cells with different concentrations of the selective PLK4 inhibitor centrinone B, we uncover multiple pathways leading to growth arrest in response to abnormal centrosome numbers. Not only does centrosome number play a role, but we hypothesize that properties or the activity of PLK4 itself can also trigger growth arrest. Curiously, we found that TRIM37 is required for growth arrest in some, but not all centrinone B concentrations tested.
Several observations support our hypothesis that differential PLK4 inhibition used for our screens resulted in distinct cellular states. First, we observed clear differences in centrosome number. Second, MDM2 was cleaved using 200 nM, but not 500 nM centrinone B consistent with excess centrosomes in the former condition. Last, the genes derived from each screen were distinct. Specifically, we identified ANKRD26/PIDDosome only in the presence of excess centrosomes and also identified centriole proteins that, when disrupted, could decrease centrosome load, although we did not formally test this. We note that 200 nM centrinone B was not optimal to induce excess centrosomes in A375 cells (compare Figure 4C with Figure 4—figure supplement 1), yet we still identified genes that overlapped with those from the comparable RPE-1 screen suggesting that these cells were subjected to similar conditions.
Centrosome amplification after partial inhibition of PLK4 has been previously observed using CFI-400495 (Mason et al., 2014), YLT-11 (Lei et al., 2018), or analog-sensitive alleles of PLK4 (Moyer et al., 2015). Current models suggest that partially inhibited PLK4 reduces its auto-phosophorylation required for its degradation. As a consequence, PLK4 accumulates and promotes centriole overduplication (Holland and Cleveland, 2014). While TRIM37 is involved in mediating growth arrest to partial PLK4 inhibition, it is not required for arrest after PLK4 overexpression, which also leads to extra centrosomes (Evans et al., 2021). The fundamental difference between centrosome amplification caused by PLK4 overexpression or by partial inhibition may relate to the per molecule activity of PLK4, perhaps pointing a role for TRIM37 in regulating PLK4 activity. Our FRAP data indicated a dose-dependent decrease in phosphorylated, and therefore active, PLK4 upon increasing centrinone B that correlated well with growth arrest activity. If altered PLK4 activity, and not extra centrosomes, is responsible for growth arrest, why would we identify proteins such as ANKRD26 and the PIDDosome that have clear roles in response to supernumerary centrosomes (Evans et al., 2021; Burigotto et al., 2021; Fava et al., 2017)? Growth suppression after PLK4 inhibition at any concentration of centrinone B was only partially TRIM37-dependent, suggesting that multiple pathways might be activated in these conditions, one dependent on centrosomes and the other dependent on PLK4. Comparing our dataset with that of the PLK4 overexpression screen (Evans et al., 2021) yields an overlap of 22 genes that we propose are involved in a response to supernumerary centrosomes (Figure 2—figure supplement 2G). We suggest that the genes unique to our dataset (i.e., low-dose centrinone B treatment) might modulate the response to inhibited PLK4. CEP85 and USP9X are such genes and both encode proteins that affect STIL, a regulator of PLK4 activity. CEP85 is required for robust STIL interaction with PLK4 while USP9X stabilizes STIL (Liu et al., 2018; Kodani et al., 2019). Interestingly, 53BP1 and USP28 were similarly dispensable for growth arrest after PLK4 overexpression (Evans et al., 2021). In this case, the MDM2 p60 fragment that is known to interact with and stabilize p53 (Oliver et al., 2011) might be redundant with the function of 53BP1-USP28. It is therefore an exciting possibility that PLK4 activity, by itself, may be a determinant of p53-dependent cell cycle arrest.
TRIM37 is proposed to mediate the degradation of CEP192 that in turn affects PCM assembly in mitotic cells treated with PLK4 inhibitors (Yeow et al., 2020; Meitinger et al., 2020), however, there is no consensus on the exact centrosome or centriole TRIM37 targets. We observed a decrease in overall CEP192 protein levels, but not those of PCNT, CEP215, or CEP120 after stable expression of FB-TRIM37 (Figure 6B). The effect of TRIM37 expression on centrosomal proteostasis is unclear. Two previous studies examining centrosomal protein abundance after TRIM37 expression detected decreases in overall CEP192, however one report did not detect changes in CEP215 or CEP152 (Meitinger et al., 2020), while another detected decreases in CEP215 and PCNT (Yeow et al., 2020). We did not detect reciprocal changes in protein levels in TRIM37-/- cells (Figure 6A) suggesting that the effects on PCM are dependent on overexpressed TRIM37 or that the TRIM37-/- cell line acquired a genetic or epigenetic change that suppresses effects on PCM proteins. TRIM37 overexpression also decreased the amount of CEP192, PCNT, and CEP120 detected at mitotic centrosomes (Figure 6D and Figure 6—figure supplement 1D). More work will be needed to identify direct and indirect targets of TRIM37 at the centrosome.
The E3 ligase activity of TRIM37 was required for changes in the bulk abundance of CEP192 and for the reduction in CEP192, PCNT, and CEP120 proteins at mitotic centrosomes. In contrast, we observed dosage-dependent phenotypes for the E3 ligase mutant TRIM37 C18R. Stable and high expression of this variant caused a strong growth arrest phenotype (Figure 4A) while lower expression using an inducible system resulted in partial growth arrest activity in response to centrinone B treatment (Figure 4E and F). To explain this phenomenon, we consider that either the expression of the C18R mutant binds to and sequesters TRIM37 targets to phenocopy the effect of degradation, or that higher levels of TRIM37 C18R drive the formation of an E3-independent complex important for growth arrest, as has been suggested for a TRIM37 ligase-independent role in autophagy (Wang et al., 2018).
Current models of TRIM37 growth arrest function after PLK4 inhibition have primarily focused on mitotic length (Lambrus et al., 2016; Meitinger et al., 2016; Yeow et al., 2020; Meitinger et al., 2020). After complete PLK4 inhibition, cells containing one or no centrosome arrested, but in these studies, mitotic length and growth arrest were not well correlated and not long enough to activate a ‘mitotic timer’ (Wong et al., 2015; Lambrus et al., 2015). This led to the speculation that small increases in mitotic length over multiple cell cycles might be equivalent to a single mitosis with a larger delay (Lambrus et al., 2016; Meitinger et al., 2016) but this possibility has not yet been tested. Here, however, we provide multiple lines of evidence that mitotic length may not be critical for growth arrest after PLK4 inhibition. First, in WT RPE-1 cells, we observed that treatment with 50 nM and 125 nM centrinone B led to ~25% and 90% decreases in cell proliferation, respectively (Figure 4B) without causing concomitant increases in mitotic length (Figure 5A). Second, when comparing WT and TRIM37-/- cells, we observed a significant difference in growth between these cell lines at 125 nM centrinone B or greater but did not detect significant differences in mitotic length until a treatment with 200 nM centrinone B. Third, induced expression of TRIM37 C18R caused a partial reduction in cell proliferation without any apparent effect on mitotic length (Figure 4E and F). We also note that PLK4 overexpression in mice or flies results in increased mitotic indices (Marthiens et al., 2013; Basto et al., 2008) indicative of lengthened mitoses, yet TRIM37 is not required for growth arrest under similar conditions in cultured cells. Mitotic length after treatment with 200 and 500 nM centrinone B is clearly affected by TRIM37 but our data does not suggest that this directly influences growth arrest.
Although others have observed TRIM37-dependent ubiquitination of PLK4 (Meitinger et al., 2020), we detected low amounts of ubiquitinated PLK4 only after co-expression with the TRIM37Δ505–709 mutant (Figure 7E). In contrast, we found that TRIM37 primarily promoted PLK4 phosphorylation in a manner that was dependent on PLK4 activity itself (Figure 7F and G). We did not find that PLK4 mobility by FRAP was affected by the loss of TRIM37 in the absence or presence of centrinone B (Figure 7—figure supplement 1H, I) suggesting that the phosphorylation sites stabilized by TRIM37 lie outside the PLK4 phosphodegron-adjacent regions monitored by this method (Yamamoto and Kitagawa, 2019). It will be of interest to determine how TRIM37 promotes PLK4 phosphorylation, which regions of PLK4 are modified, and to assess if these phosphorylation events are contributing to TRIM37-dependent growth arrest in response to abnormal centrosome numbers or altered PLK4 activity.
We described three distinct growth arrest phases after PLK4 inhibition characterized by cellular centrosome number abnormalities (Figure 4E). Our FRAP assays indicated that the mobility of PLK4 decreased after centrinone B treatment in a dose-dependent manner that mirrored the growth arrest activity of WT cells. We therefore propose that a direct aspect of PLK4 activity, either PLK4 itself or a substrate of PLK4, underlies the growth arrest after PLK4 inhibition (Figure 8). A level of PLK4 inhibition that does not affect centriole number initiates p53 arrest independent of TRIM37, but cell arrest after further PLK4 inhibition becomes dependent on TRIM37. TRIM37 itself promotes phosphorylation of PLK4 and can affect the abundance and/or localization of CEP192, PCNT, and CEP120, but the effect of these functions is not clear. SASS6 was required for growth arrest at all centrinone B concentrations, (Figure 4—figure supplement 3) suggesting that centrioles themselves may play a role to integrate the growth arrest signal.
In closing, we used CRISPR/Cas9 screening to characterize the global, dose-dependent response to PLK4 inhibition. While previous studies focused on complete PLK4 inactivation and mitotic length after centrosome loss as a potential mechanism driving p53-dependent cell cycle arrest, we found that the loss of PLK4 activity better correlates with the subsequent growth arrest. Proteomic analysis of PLK4 substrates and the cellular aggregates that form in the absence of TRIM37 will be required to provide mechanistic details of this pathway and may yield the identification of PLK4 substrates underpinning this response.
Materials and methods
Cell culture and drug treatmentsRequest a detailed protocol
All cell lines were cultured in a 5% CO2 humidified atmosphere at 37°C. HEK293T (female, human embryonic kidney epithelial), hTERT RPE-1 (female, human epithelial cells immortalized with hTERT), and A375 cells (female, human malignant melanoma epithelial) are from ATCC. hTERT RPE-1 and A375 stably expressing Cas9 were from D Durocher (Hart et al., 2015; Zimmermann et al., 2018). All references to RPE-1 and A375 cells herein refer to hTERT RPE-1 or A375 stably expressing Cas9. RPE-1, HEK293T, and A375 were grown in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco) and 2 mM Glutamax (Gibco). PLK4 inhibitor centrinone B (Tocris) was used as described. Nutlin-3a (Cayman Chemical) was used at 600 nM. The CDK1, PLK1, and Aurora A kinase inhibitors RO-3306 (Selleck Chemicals), BI-2536 (ChemieTek), and MLN8237 (Selleck Chemicals) were used at 10 μM, 100 nM, and 200 nM, respectively. MG132 (Selleck Chemicals) was used at 10 μM. G418 (WISENT Bioproducts) was used at 600 μg/mL for cell selection and 200 μg/mL for routine culture. All cell lines used have been authenticated by STR profiling and tested negative for mycoplasma contamination.
Plasmid constructionRequest a detailed protocol
TRIM37 C18R was created with pcDNA5 FB TRIM37 as a template using site-directed mutagenesis (QuikChange, Agilent). Truncation mutants were created by PCR using pcDNA FB TRIM37 as a template and ligated into pcDNA5 FLAG-BirA digested with NotI and AscI. Internal deletions were created using pcDNA5 FB TRIM37 as a template using the Q5 Site-Directed Mutagenesis kit (NEB). pSIN constructs were created by amplifying the insert from the corresponding pcDNA5 plasmid and using Gibson cloning (NEB) to ligate into pSIN previously digested with BamHI and NotI. To create T7-tagged TRIM37, first FLAG-BirA was removed from pcDNA5 Flag-BirA by PCR. The appropriate TRIM37 mutant was amplified with primers encoding the T7 tag and inserted in the pcDNA5 template using Gibson cloning. PLK4 was amplified from cDNA and ligated into pcDNA5-FRT/TO-Myc using AscI and XhoI. sgRNA guide sequences were cloned into pLentiguidePuro as described (Sanjana et al., 2014).
Virus productionRequest a detailed protocol
To produce lentivirus, 4 × 106 HEK293T were seeded in a T-75 flask and subsequently transfected with 4 μg of the appropriate transfer vector, 3 μg psPAX2, and 2 μg pCMV-VSV-G using 18 µL each Lipofectamine 3000/P3000 reagent (Invitrogen). After 24 hr, growth medium was replaced with fresh medium containing 30% FBS and viral supernatant was collected after a further 48 hr. Virus was stored at –80°C.
CRISPR/Cas9 screeningRequest a detailed protocol
CRISPR screens were performed as described (Hart et al., 2015; Zimmermann et al., 2018). Briefly, Cas9-expressing cells were transduced with the TKOv1 viral library (~90 k sgRNA) (Hart et al., 2015) at low MOI (~0.3) in the presence of 4 μg/mL polybrene. RPE-1 cells were selected as described (Olivieri and Durocher, 2021). A375 cells were selected using 2 μg/mL puromycin. 10× 106 cells were harvested 4 days post-transduction and represents day 0. Cells were grown for 6 days before being split for drug treatment in technical triplicate and further grown for 21 days. A library coverage of >100 cells/sgRNA was maintained at each step. gDNA from cell pellets was isolated using a QIAamp Blood Maxi Kit (Qiagen) and genome-integrated sgRNA sequences were amplified using the KAPA HiFi HotStart ReadyMix (Kapa Biosystems). Sequencing libraries were made by addition of i5 and i7 multiplexing barcodes in a second round of PCR and the product gel purified using QIAquick Gel Purification kit (Qiagen). Libraries were sequenced using Illumina HiSeq2500 or NextSeq500. Sequence data was analyzed using MAGeCK (Li et al., 2014) to determine sgRNA distribution among the samples. Drug-treated samples at 21 days post-drug addition were compared to DMSO-treated cells at 12 days post-drug addition to equalize the number of cell doublings. Genes with FDR <0.05 were used for further analysis. The significant gene list for the RPE-1 200 nM screen is the union from two independent biological replicates.
Network analysis and gene enrichmentRequest a detailed protocol
High-scoring genes from MAGeCK analysis were visualized using Cytoscape (Shannon et al., 2003). General node arrangement was performed using the yFiles Organic Layout and manually modified to facilitate visualization. Each screen condition (200 nM centrinone B, 500 nM centrinone B, and Nutlin-3a) was considered as a source node, corresponding hits as target nodes, and FDR as edge attributes. Genes from the indicated datasets were analyzed using the ClueGo app within Cytoscape (Bindea et al., 2009). Enrichments for Biological Function (circles) or Cellular Component (hexagons) based on all experimental evidence was determined. Only pathways with p-value < 0.05 are shown. Nodes arranged using the yFiles Organic Layout.
CRISPR/Cas9 gene disruptionRequest a detailed protocol
For lentivirus-mediated gene disruption of TRIM37, sgRNA sequences were cloned into plentiGuide-Puro as described (Sanjana et al., 2014). RPE-1 Cas9 cells were infected with lentiviral particles and selected as described above for CRISPR/Cas9 screening. A Clonal TRIM37-/- line was generated using in vitro transcribed (IVT) sgRNA. IVT templates were created by PCR using cr_tracrRNA, IVT forward, IVT reverse, and sgRNA-specific oligonucleotides (TRIM37 sgRNA 1). PCR products were used directly as templates for IVT using HiScribe T7 transcription kit (NEB). Resulting RNA was purified using RNAClean XP beads (Beckman Coulter) and used to transfect RPE-1 cells using RNAiMAX (ThermoFisher) according to the manufacturer’s instructions. Clonal lines were generated by limiting dilution and assessed for gene disruption by Western blot and TIDE (Brinkman et al., 2014) or Synthego ICE (Synthego Performance Analysis, ICE Analysis. 2019. v2.0. Synthego; accessed 9/19/2018) analyses. TRIM37-/- pools in RPE-1 and A375 cells were generated similarly using an sgRNA targeting exon 5. After transfection, cells were grown in medium containing 500 nM centrinone B for 2 weeks to select for TRIM37 disruption before growth in normal medium.
Stable cell line generationRequest a detailed protocol
To generate cell lines, 200,000 cells were seeded with serial aliquots of viral supernatant and 4 μg/mL polybrene (MilliporeSigma) in one well of a six-well plate. Medium was changed after 24 hr and appropriate drug selection was added after an additional 24 hr where required. For stable expression of FLAG-BirA rescue constructs, immunofluorescence was performed to ensure all cells expressed the appropriate transgene. Doxycycline-inducible lines were selected with 600 μg/mL G418 until control cells died. We used pools that showed approximately 30% survival after initial selection.
siRNA conditionsRequest a detailed protocol
For siRNA knockdown experiments, 200 k cells were seeded per well of a six-well plate. Cells were reverse transfected using the indicated siRNA trigger (Horizon Discovery, Dharmacon; Supplementary file 3). For each well, 5 μL of 20 μM siRNA was combined with 3 μL Lipofectamine RNAiMAX (ThermoFisher) in 125 µL OPTIMEM medium (Gibco). Media was replaced after 24 hr and cells processed after 72 hr.
Immunofluorescence staining and microscopyRequest a detailed protocol
Cells were grown as indicated on No. 1.5 coverslips, washed once with PBS, and fixed with –20°C methanol for at least 10 min. All subsequent steps performed at room temperature (RT). Coverslips were rinsed with PBS and blocked with antibody solution (PBS, 0.5% (w/v) BSA and 0.05% Tween-20) for 15–30 min. Samples were incubated with primary antibodies (Supplementary file 3) for 1 hr, washed 3 × 5 min and incubated with secondary antibodies (Supplementary file 3) and DAPI (0.1 μg/mL) for 45 min. Coverslips were washed 3 × 5 min and mounted on slides using Prolong Gold (Invitrogen). Deconvolution wide-field microscopy was performed using the DeltaVision Elite system equipped with an NA 1.42 60× PlanApo objective (Olympus) and an sCMOS 2048 × 2048 camera (Leica Microsystems). Each field was acquired with a z-step of 0.2 μm through the entire cell and deconvolved using softWoRx (v6.0, Leica Microsystems). Maximum intensity projections are shown (0.1080 μm/pixel). Display levels are the same for all images in a panel unless otherwise indicated.
For live imaging, 15,000 cells were seeded per well in an eight-well Lab-Tek II chamber slide. The next day fresh medium containing drug was added and cells incubated for 3 days. Fresh medium containing indicated drug and 200 nM SiR-DNA (Spirochrome) was added for 2 hr before imaging. Microscopy was performed using the DeltaVision Elite system equipped with an NA 0.75 U Plan S-Apo objective (Olympus) and an sCMOS 2048 × 2048 camera. Each field was acquired with 6 × 2 μm z-step every 5 min for 24 hr. The time between NEBD and full chromosome separation judged by nuclear morphology was quantified.
Super-resolution microscopy was performed on a three-dimensional structured-illumination microscope (3D-SIM) (OMX Blaze v4, Leica Microsystems) as described (Mojarad et al., 2017).
Image analysisRequest a detailed protocol
All automated quantification pipelines were created using CellProfiler 3.0 (McQuin et al., 2018) (http://www.cellprofiler.org/).
Figure 1A and B: Nuclei were detected using the DAPI channel and objects subsequently used as a mask to measure intensity in p53 or p21 channels. An arbitrary cut-off based on the distribution of p21 or p53 intensities in untreated cells was used to score positive cells.
Figure 3J, Figure 3—figure supplement 1, Figure 4—figure supplement 2: A centrosomal (γ-tubulin) or centriole marker (CEP120) were used to define centrosome regions. The TRIM37 images were masked by the centrosome objects and total intensity was measured.
Figure 3—figure supplement 2 and Figure 7—figure supplement 1D: Nuclei were detected using the DAPI channel. The nuclear objects were expanded and a ring surrounding each nucleus was used as a mask to measure the total intensity in the BirA channel. The mean and standard deviation of the measured intensities of control cells was determined and a cut-off of the mean + 2.5× the standard deviation was used to score positive cells.
Figure 6D and Figure 6—figure supplement 1D: Each image was manually cropped to include a single mitotic cell. Each channel was background subtracted using the lower quartile intensity of the entire image and each channel was segmented into objects using a robust background thresholding and the integrated intensity of each object was measured.
Western blotRequest a detailed protocol
Cells were grown as indicated, washed once with PBS and resuspended directly in 2× SDS-PAGE sample buffer containing Benzonase (0.25 U/μL, MilliporeSigma) and heated at 95°C for 5 min. Proteins (typically 10–20 μg) were separated by SDS-PAGE and transferred to PVDF using a wet transfer apparatus (Bio-Rad). Total protein was detected by staining with PonceauS (MilliporeSigma) and scanning. All steps performed at RT unless indicated. Blocking and primary antibody incubations were performed using TBS-T (TBS + 0.05% Tween-20) with 0.5% skim-milk powder (Bioshop). Membranes were blocked for 30 min and incubated with primary antibody (Supplementary file 3) overnight at 4°C. After washing 3 × 5 min, membranes were incubated with secondary antibodies for 45. HRP-conjugated secondary antibodies (Bio-Rad) were incubated in TBS-T/milk for 45 min and washed 3 × 5 min with TBS-T before detecting using a Chemidoc imager (Bio-Rad). NearIR-conjugated secondary antibodies (LI-COR Biosciences) were incubated in TBS-T/milk + 0.015% SDS for 45, washed 3 × 5 min with TBS-T and 1 × 5 min with TBS before drying the membrane for 2 hr at RT. Dried blots were imaged using an Odyssey CLx imager (LI-COR Biosciences).
Quantification of Western blots were performed on images obtained using NearIR secondary antibodies. Images were quantified using Image Studio software (LI-COR Biosciences) and normalized to Ponceau S or Coomassie Blue staining of the same lane.
Clonogenic survival assaysRequest a detailed protocol
Two-hundred and fifty RPE-1 or 200 A375 cells were seeded in either a 10 cm dish or six-well plate. The next day medium was removed and medium containing the indicated drug was added. For experiments using doxycycline-inducible cell lines, the media was refreshed every 3–4 days to ensure continued expression of the induced proteins. After 12–14 days, plates were rinsed once with PBS and fixed and stained with 0.5% crystal violet (MilliporeSigma) in 20% methanol for at least 20 min. Plates were washed extensively with water, dried, and scanned. Images were segmented using the Trainable Weka Segmentation tool (Arganda-Carreras et al., 2017) in ImageJ. A new model was built for each replicate if required. The resulting segmentation image was thresholded and used as a mask to overlay the original image that was inverted and background subtracted using a 50-pixel rolling circle, or the average of a region not containing colonies. The colony intensity per well or dish was then measured within the masked region.
Immunoprecipitation and protein treatmentsRequest a detailed protocol
To detect complex formation between PLK4 and TRIM37, 2 × 106 RPE-1 cells were seeded per 10 cm dish and transfected with 3.75 μg Myc-PLK4 and 3.75 μg pcDNA5 FLAG-BirA construct using 15 μL Lipofectamine 3000/P3000 (ThermoFisher). Cells were harvested 24 hr post-transfection, washed once with PBS, and resuspended in lysis buffer (50 mM HEPES pH 8; 100 mM KCl; 2 mM EDTA; 10% glycerol; 0.1% NP-40; 1 mM DTT; protease inhibitors [Roche] phosphatase inhibitor cocktail 3 [MilliporeSigma]) for 30 min on ice. Lysates were frozen in dry ice for 5 min, then thawed and centrifuged for 20 min at 16,000× g at 4°C. An aliquot representing the input was removed before cleared supernatants were incubated with equilibrated anti-FLAG M2 Affinity Gel (MilliporeSigma) for 1–2 hr at 4°C. Beads were washed three times with lysis buffer before resuspension in 2× SDS-PAGE sample buffer. Samples were heated at 95°C for 5 min.
To probe PLK4 modification in Figure 7D,F,G, 350 k HEK293T cells were seeded per well of a six-well plate and subsequently transfected with 0.67 μg Myc-PLK4 and 1 μg T7-TRIM37 construct using 3.34 μL Lipofectamine 3000/P3000. Medium was changed after 6 hr and cells incubated for 48 hr in total before sampling. For Figure 7G, the indicated drug was added 3 and 6 hr before collection. Cells were collected directly in 2× SDS-PAGE sample buffer for Figure 7D and G. For Figure 7F, cells were collected and washed once with PBS. Cells were resuspended in a modified TNTE buffer (10 mM Tris-HCl, pH = 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1% TX-100; protease inhibitors; ±phosphatase inhibitor cocktail 3) and incubated for 30 min on ice before addition of MnCl2 and λ-phosphatase (Bio-Rad) to the appropriate samples for 30 min at 30°C. The soluble fractions were obtained by centrifugation at 16,000× g for 30 min. To probe for PLK4 ubiquitination, 1.5 × 106 HEK293T cells were seeded in a 10 cm dish and transfected with 2 μg Myc-PLK4, 2 μg HA-ubiquitin, and 2 μg eGFP or T7-TRIM37 with 12 μL Lipofectamine 3000/P3000. The medium was changed after 16 hr and cells harvested after 48 hr. Cells were washed once with PBS and resuspended in modified TNTE buffer and the soluble fraction was obtained as described above. Lysates were incubated with 3 μg anti-Myc antibodies (Supplementary file 3) and incubated for 1 hr at 4°C. Equilibrated Protein G Sepharose 4 Fast Flow beads (Cytiva) were added and samples further incubated for 1 hr at 4°C. Immunoprecipitates were washed 3× with modified TNTE buffer and eluted by addition of 2× SDS-PAGE sample buffer and heating at 95°C for 5 min.
FRAP analysisRequest a detailed protocol
For experiments using disruption lines, 62.5 k cells were seeded per well of an eight-well LabTekII chamber. Cells were transfected with 400 ng pcDNA5 GFP-PLK4 or pcDNA5 GFP-PLK4 kin + L1 using 0.8 and 0.6 µL P3000/Lipofectamine 3000. Media was removed after 6 hr and replaced with media containing DMSO or centrinone B. Cells were incubated approximately 16 hr before imaging using a Nikon A1R-HD25 scanning laser confocal microscope with a LUN4 laser unit and GaAsP PMT. A single Z-slice was imaged in Galvano mode, 1.2 μs dwell time using a 488 nm excitation wavelength, a 521/42 bandpass emission filter, and a 60× NA 1.2 water immersion objective. GFP-PLK4 condensates were imaged at 0, 4, and 8 s before bleaching for 500 ms using 60% 488 laser power and 16 fps scan speed. Images were acquired every 4 s for a total of 90 s after bleaching. Imaging parameters were adjusted as needed between replicates (typically 1.2% laser power with gain setting of 10). Analysis was performed using NIS-elements ‘time measurement’ module. For each image an ROI was drawn around the targeted area, a similar unbleached area, and a background region. Where appropriate, the ROI was moved to track the structure of interest. The signal from the targeted area (ROI1) was background subtracted (ROI3) and then normalized using the unbleached area (ROI2) to correct for photobleaching during imaging. Ten to 15 GFP-PLK4 condensates from different cells were analyzed per condition per replicate.
All data generated or analyzed during this study are included in the manuscript and supporting files. Source data and unaltered Western blots have been provided for all Figures. CellProfiler analysis pipelines available from Zenodo (https://zenodo.org/record/6532747).
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Jens LüdersReviewing Editor; Institute for Research in Biomedicine, Spain
Anna AkhmanovaSenior Editor; Utrecht University, Netherlands
In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.
[Editors' note: this paper was reviewed by Review Commons.]https://doi.org/10.7554/eLife.73944.sa1
Reviewer 1, Major points
1. Previous data suggested that an important role of TRIM37 was to limit accumulation of CEP192 levels, yet here CEP192 levels appeared unchanged in TRIM37 knockout cells that stably express wild-type or RING domain mutant TRIM37. However, in agreement with previous work, transient expression of TRIM37 reduced CEP192 levels along with those of other PCM and centriole components in an E3-dependent manner. These data are rather confusing in light of the literature, and the current report does not really deal with these discrepancies but to me they suggest that high levels of TRIM37 can target multiple centrosome components for degradation, but this may be an experimental artefact.
We thank the reviewer for pointing this out. To investigate this further, we performed additional replicates for the experiments examining the total cellular levels of PCM components in WT RPE-1, TRIM37-/-, and TRIM37-/- stably overexpressing FLAG-BirA(FB) TRIM37. We now find that stable overexpression of FB-TRIM37 also results in lower steadystate levels of CEP192 (but not PCNT or CEP215). These results are found in Figure 6A and Figure 6B and Figure 6 —figure supplement 1A and B and are now consistent with the literature showing that induced overexpression of TRIM37 affects CEP192 levels. However, we still do not observe reciprocal changes (i.e. an increase in abundance of CEP192) after TRIM37 disruption. We also performed Western blot and immunofluorescence experiments using WT RPE-1 and TRIM37-/- cells induced to express TRIM37-3xFLAG or stably expressing FB-TRIM37 to compare overall protein levels and centrosomal levels more directly (Figure 4 —figure supplement 2A-C). We find that induced TRIM37-3xFLAG is expressed at 4- to 5-fold higher levels than the endogenous protein while stably expressed FB-TRIM37 is expressed at 150- to 200-fold higher levels. We discuss that overexpressed TRIM37 may account for some of the observed phenotypes and/or our TRIM37-/- clonal line may have acquired a mutation that suppresses the loss of TRIM37 (Page 14, paragraph 1) of the revised manuscript (also see Reviewer 1, major point 4).
In addition, we determined that the amount of CEP192 at mitotic centrosomes decreases after 4 or 8 hours of acute induction of TRIM37 and found that the abundance of another PCM component, PCNT, and a non-PCM centriole component, CEP120 are also diminished at mitotic centrosome after 4 or 8h acute TRIM37 expression. These data indicate that the effect of induced overexpression of TRIM37 is not restricted to CEP192 (Figure 6D). We now further discuss that the exact substrates, or proteins affected by TRIM37 expression or depletion remain to be fully delineated. Further work will be required to delineate direct versus indirect effects of TRIM37 and the mechanisms that control these functions (Page 14, paragraph 1).
2. The choice of cells for particular experiments is not always stated or explained. For instance, in Figure 3A: Trim37 KO pool used while in Figure 3B TRIM37 single KO. These are then combined with both transient and stable expression of TRIM37 mutants.
We apologize for not always stating / explaining this well enough. In principle, however, we find this to be a strength of our work. Our key results can be reproduced using TRIM37-/- lines generated by different methods and using different sgRNAs ensuring that we are not observing artefacts due to clonal selection or off-target effects. We first confirmed our screen results by infecting cells with virus to deliver two independent guides against TRIM37. Having confirmed the screen results, we generated a clonal TRIM37-/- line. To further ensure the reproducibility of our observations we created additional TRIM37-/- pools in two different cell lines. All experiments in our manuscript after the initial confirmation (Figure 3A) are now performed using our clonal TRIM37-/- line except for those in Figure 4B and C and Figure 4 —figure supplement 1D and E, where we use pools for the specific reason of disrupting TRIM37 in an independent manner. This is now more clearly noted in the revised version of the manuscript (Page 8, paragraph 3).
3. Two different concentrations (200 nM and 500 nM) of centrinone were used to compare responses of too many or no centrosomes in RPE1 and A375. While these concentrations result in centrosome amplification (200 nM) and loss (500 nM) in RPE1 cells, the phenotypes seem much less clear-cut in A375 cells. At 200nM 70% of cells have 0 or 1 centrioles (~35% each category) and only about 15% have centrosome amplification, whereas centrosome amplification occurs in 30% of RPE1 with 0-1 centrioles seen in fewer than 10% (Figure 4—figure supplement 1H). Hence the different outcomes of centrinone treatment makes conclusions about cell-type specific responses difficult. This difference may be due to differences in drug uptake/efflux, PLK4 activity or in expression of other components of these pathways. In fact, 167nM centrinone B in A375 cells would have been a much closer match to the 200nM treatment of RPE-1. These points should be discussed as they impact the conclusions.
The reviewer rightly points out that the response to centrinone appears to differ between cell types, as shown previously (Meitinger et al., 2020 and Yeow et al., 2020), and that this difference may impact our conclusions. We concur that 167 nM centrinone B in A375 cells would have been a better match to 200 nM centrinone B in RPE-1 cells based on our centrosome count experiment (Figure 4C and Figure 4 —figure supplement 1E). However, the screen results still reveal similar differential responses to the two centrinone B doses used. This caveat is now explicitly discussed in the revised manuscript (Page 13, paragraph 2). The 200 nM centrinone B screens in both cell lines identified genes known to respond to supernumerary centrosomes (ex. ANKRD26, CASP2, PIDD1, CRADD) and centriole components that when disrupted might decrease centrosome load (ex. STIL, CENPJ). Interestingly, the A375 screen identifies more genes in the latter group than the RPE-1 screen (ex. CEP120, CEP135, CEP152, SASS6; see Figure 2 —figure supplement 2C and F).
4. I find the different outcomes of stable versus acute expression of TRIM37 ligase mutant confusing. Here, stable expression of TRIM37 ligase mutant increases mitotic length compared to that of TRIM37 wild-type, which contradicts a recent report by (Meitinger et al. 2021). What could be the potential reason for these differences?
Our initial experiments were performed using a cell line stably expressing FB-TRIM37 C18R. We performed new experiments to compare mitotic length in TRIM37-/- cells induced to express TRIM37 C18R-3xFLAG. Using this system, we find that, like Meitinger et al., 2021, induced TRIM37 C18R-3xFLAG fails to increase mitotic length in the presence of centrinone (Figure 5B). This contrasts with stably overexpressed FB TRIM37 C18R that increases mitotic length that was presented in former Figure 5C. Although the data using the stable cell lines we included in the original manuscript with the rescue mutant is still valid (former Figure 5D), we decided to remove it from the revised manuscript since the inducible system provided more robust results and was used in subsequent experiments. We offer two possible explanations for this observation. First, similar to what we observed with wild type TRIM37, the expression level of FB-TRIM37 C18R is higher than induced TRIM37 C18R-3xFLAG. We discuss that high amounts of the E3-ligase mutant might drive the interaction of a critical complex stabilized by TRIM37 or that the catalytic inactive mutant might interact with a substrate that is normally degraded by wild type TRIM37, thus phenocopying the mitotic arrest phenotype (Page 14, paragraph 2). However, we still find that the induced expression of TRIM37 C18R-3xFLAG partially rescues the growth arrest phenotype in clonogenic assays. We present these new data in Figure 4E and F. To date, the model for growth arrest after PLK4 inhibition has focused on mitotic length. We provide multiple pieces of evidence, including those just described, that challenge this model. We discuss our findings that are inconsistent with a mitotic length model on Page 14, paragraph 3.
What could be the mechanism for TRIM37 action in regulating spindle assembly/mitotic duration and cell proliferation upon centrosome loss? How do those acentrosomal MTOCs form that decrease mitotic duration and promote proliferation?
These are exciting questions that explore details of TRIM37 mechanism that are not addressed in the current manuscript. Our data challenges the view that mitotic length contributes to cell growth arrest mediated by TRIM37. Rather, we highlight the interplay between PLK4, TRIM37 and growth arrest. This does not mean that TRIM37 doesn’t contribute to ectopic mitotic structures or mitotic length, only that mitotic length is perhaps not the sole determinant of growth arrest. This is now better discussed on Page 14, paragraph 4 of the manuscript and included in our working model (Figure 8).
Do the authors find a difference in the % of cells expressing TRIM37 mutants upon stable or acute expression? This part needs a better summary, and again a table would help. I also wonder about protein expression levels; wild-type FB-TRIM37 seems to be expressed at much lower levels than the mutants in Figure 5B.
The differences in overall abundance are not due to heterogenous expression within the population. The TRIM37 mutants are expressed in all cells after stable expression (Figure 3 —figure supplement 2B and C, and Figure 7 —figure supplement 1B and D). Although we did not formally test this in the induced cell lines, these lines are selected using G418. TRIM37 is proposed to negatively regulate its own expression in an E3-dependent manner (Meitinger et al. 2021, Figure 3f). Our results are consistent with this as the TRIM37 C18R and TRIM37 DRING mutants have a higher overall abundance compared to TRIM37 or TRIM37 D505709 using either stable or induced cell lines. Some of the functional differences we initially observed can be explained by stable overexpression of FB-TRIM37 C18R versus induced expression of TRIM37 C18R3xFLAG. We have redone experiments using an inducible system and our results are now more consistent with those in the literature. We provide a table in Figure 7 —figure supplement 2A to summarize the TRIM37 mutants we used and their phenotypes.
5. Other means of centrosome depletion (Cenpj, SAS6 etc) would have been useful to include in the manuscript in support of E3 ligase dependent and independent roles of TRIM37. It is not essential to perform these experiment but if data are available, including these would improve the paper.
To eliminate centrioles using another approach, we created an RPE-1 TRIM37-/- SASS6-/- cell line. Disruption of SASS6 suppresses growth arrest at all centrinone concentrations tested, including 125 nM centrinone B where arrest is largely TRIM37-independent (Figure 4 —figure supplement 3A and B). We find that similar to centriole loss induced by 500 nM centrinone B, TRIM37 C18R-3xFLAG partially rescues growth arrest in these conditions (Figure 4F). These data together suggest that TRIM37 catalytic activity is not strictly required for growth arrest function.
6. The authors show that TRIM37 regulates PLK4 phosphorylation and that this modification could only be observed in HEK293T and not in RPE1. Why would there be a difference between HEK293 and RPE1?
In our original manuscript we did not directly compare 293T and RPE-1 using the same experimental conditions. We were referring to the appearance of PLK4 in our immunoprecipitation experiments using RPE-1 cells (Figure 7B; former Figure 4B) compared to PLK4 observed in (Figure 7D-G; former Figure 7A-D). There are key differences in how these experiments were performed including a longer transfection time and the presence of MG132 using 293T cells that might affect PLK4 modification. We were incorrect to try and make the comparison and apologize for this indiscretion.
However, we have now performed experiments using 293T, HeLa and RPE-1 cells. We find marked PLK4 modification after co-expressing TRIM37 and PLK4 in 293T and HeLa cells. However, such modifications are much less evident in RPE-1 cells.
We speculate that the presence of the modified forms will depend on rates of modification (phosphorylation, ubiquitination or other) and de-modification (dephosphorylation, deubiquitination, etc.) and on any proteins that might also form a complex with PLK4 and TRIM37. Interestingly, 293T and HeLa cells both contain extra copies of chromosome 17 that harbors the TRIM37 locus. Since we observe modification in at least two of the cell lines tested, we conclude that this is not cell type specific. We present these data in Author response image 1 to the editor and reviewers but did not include them in our revised manuscript.
7. Statistical analysis for graphs should be included. Figure 5 is ok but graphs in Figures 3, 4, 6, 7 would benefit.
This point is well taken. Statistical analyses have now been performed for the data in these figures as indicated and significant (p < 0.05) values clearly noted to support our conclusions.
8. The authors characterise TRIM37 localisation. They detect it at centrosomes (as shown by Yeow et al. 2021) and more specifically at the PCM, but apparently the signal is not present in all cells. They should also provide a quantification of the % of cells with centrosomal TRIM37 signal and compare this to cells expressing Flag-tagged Trim37. The specificity of the antibody signal using TRIM37-/- should be confirmed.
We improved our immunofluorescence protocol to include a pre-extraction step and TRIM37 signal at the centrosome is now evident in a majority of cells. We confirmed the specificity of the antibody using this new protocol by staining TRIM37-/- cells and cells depleted for TRIM37 using siRNA (Figure 3 —figure supplement 1E-G). We found that the TRIM37 signal was significantly decreased at the centrosome in interphase cells and mitotic cells, but the overall decrease was lower in mitotic cells. We therefore restricted our analysis to interphase cells. We also provide a more detailed analysis of TRIM37 localization based on cell cycle and mother/daughter centriole (Figure 3D-G). Finally, we quantify the centrosomal intensity of wild type FB-TRIM37, the E3-ligase mutants C18R and DRING (Figure 3I-J), and the PLK4 interaction mutants 505-709 and D505-709 (Figure 7 —figure supplement 1C and E).
Reviewer 1, Minor points
1. Page 3: "A recent screen for mediators of supernumerary centrosome-induced arrest identified PIDDosome/p53 and placed the distal appendage protein ANKRD26 within this pathway ". It appears that the reference for Burigotto et al. is missing.
This reference has been inserted on Page 3, paragraph 2 of the revised manuscript.
2. Page 6: The authors state that: TP53BP1, USP28 and CDKN1A are also suppressors in the Nutlin-3a screen and suggest that they act in a general p53 pathway. However Meitinger et al. (2016) showed that depletion of TP53BP1 or USP28 did not affect the upregulation of p53 and p21 upon Mdm2 inhibition.
We first note that we used Nutlin-3a to stabilize p53 while Meitinger et al. use a different inhibitor, although they both act by blocking the interaction between p53 and Mdm2. The data from Meitinger et al. explore the abundance of p53 and p21 and cell growth after Mdm2 inhibition. 53BP1 and USP28 are thought to affect p53 transcription (Cuella-Martin R et al., 2016). As such, p53 abundance should not be affected. Without quantification it is difficult to assess the relative abundance of the p53 transcriptional target, p21. The abundance of p21 appears to be lower in USP28-/- cells, and possibly in TP53BP1-/- cells. We note that our data is consistent with a previous report indicating that TP53BP1 and USP28 are required for cell arrest after Nutlin-3a treatment (Cuella-Martin R et al. 2016). We, and Cuella-Martin et al. use clonogenic assays to assess cell growth in the presence of Nutlin-3a. It is possible that this is a more sensitive assay than serial regrowth performed by Meitinger et al. Clonogenic assays allow for continuous growth while slow growth might not be well captured by continuously re-seeding cells. We mention that our results are consistent with those of Cuella-Martin et al. (Page 6, paragraph 1), but do not discuss this point further due to space constraints.
3. Page 9: "First, we performed live cell imaging to measure mitotic length in cells grown in centrinone". For consistency the authors should say centrinone B here as well.
We now use ‘centrinone B’ throughout the manuscript.
4. Page 9: "Cells lacking TRIM37 suppressed the growth arrest from 150 to 500 nM centrinone B in RPE-1 and 167 to 500 nM in A375 cells". The growth data for the A375 cells seem to be missing from the figures.
The text for the results has changed significantly to improve the clarity of our manuscript. The growth data for A375 cells is now presented in Figure 4 —figure supplement 1C.
5. Page 10: "Our results confirmed that PLK4 and TRIM37 form a complex in RPE-1 cells (Figure 3G)" It appears the authors referred to the wrong figure, it should be Figure 4B.
Our apologies. The text of the results has changed significantly to improve the clarity of our manuscript. This statement now refers to the correct figure panel (Figure 7B)
6. Figure 1C: The nuclear p53 signal is not apparent with 500 nM centrinone B in the exemplary cells. Did the authors use thresholding to quantify p53/p21 positive cells?
To quantify the data, we used automated image analysis and set a cut off based on p53 intensity in DMSO-treated cells to indicate p53-positive cells. The p53 staining in centrinone-treated cells is variable and we initially chose the same cells to show p53 staining and centrosome number. We improved the figure by showing these data in separate panels. We repeated the experiment and captured a lower magnification image to show a more representative field of cells stained for p53 (Figure 1C) and separate high magnification to show examples of centrosome number (Figure 1D). The quantification pipeline is now better explained in the methods section.
7. Figure 4D and Figure 4—figure supplement 1G: The graph is misleading and should not be presented as a continuous line.
We apologize for this. We have now changed the way this data is presented to make it easier to understand and to facilitate indicating statistical differences (Figure 4B and Figure 4 —figure supplement 1C). Instead of a scatter plot of all the data, we now present the data from individual replicates as open circles with the mean and standard deviation at each centrinone B concentration with statistical differences indicated. We hope this addresses any confusion regarding these data.
8. Figure 5A and C: A direct and statistical comparison mitotic timing upon expression different Trim37 mutants to wildtype and trim37-/- cells is missing
In Figure 5A we compare RPE-1 WT to TRIM37-/- at each centrinone B concentration and within each line we compare each centrinone B concentration to DMSO. Perhaps we do not understand the reviewer’s concern here, but we do not think any comparisons are missing from the data panel. The data originally in Figure 5C has been removed and replaced by the data in Figure 5B. Appropriate statistical tests were made among the samples. We hope this addresses this concern.
9. Figure 6B: A loading control/Ponceau staining is missing as well as the quantification of protein levels
This experiment was repeated for proper quantification with an appropriate loading control for our representative results (Figure 6B and Figure 6 —figure supplement 1B).
10. Figure 6D: It is unclear if the centrosomal signal intensity was quantified in interphase or mitotic cells
The centrosomal signal was quantified in mitotic cells only. This results and figure legend have been updated to more clearly indicate this.
11. Figure 7C: A loading control/Ponceau staining is missing
The experiment was repeated and a Coomassie Blue stained membrane is provided to indicate the input amounts for each sample (Figure 7F).
12. Figure 2—figure supplement 2F and G: It would help if the authors could highlight the cell line, e.g. RPE-1 (F) or A375 (G) in the venn diagrams.
In Figure 2 —figure supplement 2F we now highlight the genes found in RPE-1 and A375 screens only in the overlap of the Venn diagram using font colour. We now colour code the hits from each cell line in panels (F) and (G). We thank the reviewer for this suggestion.
13. Figure 4—figure supplement 1E: it appears that the BirA antibody gives only an unspecific signal. It would be useful to show if the different TRIM37 variants are able to localise to the centrosomes. Furthermore it appears that centrosomes are missing in the C18R and 505-709 variants. It would be useful if the authors quantify centrosome numbers upon expression of different Trim37 variants as shown in Figure 4—figure supplement 1. To make the identification of the cell easier it would help to include a DNA signal or indicate the outline of the cell.
The anti-BirA antibody does give a diffuse signal, although we feel it is specific considering that the BirA signal is only observed in cells expressing FLAG-BirA alone or BirA fusion proteins.
Having said this, we have changed the way we present this data. For the stable rescue lines generated using the RPE-1 TRIM37-/- clone, we present detailed characterization including percentage of cells expressing the fusion protein using the BirA antibody (Figure 3 —figure supplement 2B and C; Figure 7 —figure supplement 1B and D) and the total centrosomal intensity using FLAG or TRIM37 antibodies (Figure 3I and J; Figure 7 —figure supplement 1C and E). We provide Western blots for the stable expression of the fusion proteins in RPE1 and A375 TRIM37-/- pools in Figure 4 —figure supplement 1A and B.
For images showing centrosomes and ectopic centrosomal structures/Cenpas, we used a closed arrowhead to indicate the centrosome and caret mark (open arrowhead) to indicate the Cenpas structure (Figure 6F; Figure 6 —figure supplement 2A-C). Additionally, these phenotypes are quantified to show penetrance (Figure 6 —figure supplement 2E). We hope this improves our presentation in these figure panels.
14.The generation of stable and dox-inducible cell lines is missing in the material and methods
We apologize for this omission. This information has now been added.
Reviewer 2, Major points
1. The centrosomal localization of endogenous TRIM37 should be validated by comparing control and knockout/knockdown cells.
We have performed these experiments as outlined in response to Reviewer 1, Major point 8.
We improved our immunofluorescence protocol to include a pre-extraction step and TRIM37 signal at the centrosome is now evident in a majority of cells. We confirmed the specificity of the antibody using this new protocol by staining TRIM37-/- cells and cells depleted for TRIM37 using siRNA (Figure 3 —figure supplement 1E-G). We found that the TRIM37 signal was significantly decreased at the centrosome in interphase cells and mitotic cells, but the overall decrease was lower in mitotic cells. The general cytoplasmic signal was also qualitatively decreased although we did not measure the signal in this compartment. We therefore restricted our analysis to interphase cells.
2. Some of the quantifications are derived from only two experiments and in many cases no statistical testing was done. The authors should test the observed effects and add extra replicates to make the data more robust, where required.
This issue was also raised by Reviewer #1, major point 7. We added replicate experiments where required and statistical analyses performed are indicated with significant (p < 0.05) values included to support our conclusions..
3. Figure 5 supplements: panels showing effects on marker proteins in cells by IF lack quantification of the claimed effects. Without providing some type of quantifications for key findings, it is unclear how strong or penetrant the effects are.
Figure 5 —figure supplement 1C has been removed. We provide quantification for former Figure 5 —figure supplement 1D, now Figure 6 —figure supplement 2C and D. Data replacing former Figure 5 —figure supplement 2A and B has been quantified and is now displayed in Figure 5C and D. See also comment to Reviewer 1, Major point 7.
Reviewer 2, Minor points
I would suggest a final, summarizing schematic that illustrates the main findings in a cartoon/flow chart manner.
We have extensively reworked the discussion of our main findings, provided a summary table in Figure 7 —figure supplement 2A, and provided a summary model in Figure 8 to increase the clarity of our manuscript.
1. Please revise incorrect abstract sentence: "We identify TRIM37 as a key mediator of growth arrest when PLK4 activity is partially or fully inhibited but is not required for growth arrest triggered by supernumerary centrosomes."
In our screens, we find that TRIM37 is required for growth arrest after treating cells with 200 and 500 nM centrinone B. Treatment of cells with 200 nM centrinone B causes centriole overduplication and our initial hypothesis was that centriole overduplication alone is inducing growth arrest. To test this in a parallel manner, we also overexpressed PLK4 to induce centriole overduplication. Surprisingly, but consistent with recently published results (Evans et al., 2020), TRIM37 was not required for growth arrest after PLK4 overexpression (Figure 4G). Thus, TRIM37 is required for growth arrest after 200 nM centrinone treatment, but not PLK4 overexpression, yet both conditions result in centriole overduplication. This concept is further highlighted, discussed and clarified in the discussion (Page 13, paragraph 3). We have also removed the confusing sentence in the abstract since it was rewritten anew during the revision process.
Please also see similar comment to Reviewer 3, Major point 1.
2. In various figures and supplements showing centrosome and condensates/Cenpas, these are very difficult to distinguish due to their small size. I suggest to magnify regions of interest and/or add arrowheads in different colors marking the specific structures.
This comment is similar to Reviewer 1, Minor point 13. We remade the image panels, cropping as tightly to the desired structures as possible and enlarged the images as much as we could to fit the figure panel. Additionally, we used arrowheads to indicate centrosomes and caret marks (open arrowheads) to indicate condensates/Cenpas (Figure 6F; Figure 6 —figure supplement 2A-C). Additionally, these phenotypes are quantified to show penetrance (Figure 6 —figure supplement 2E). We hope this improves the clarity of these figure panels.
3. Figure 2A: What is the purpose of the schematics on the right of panel A? The labels in the graph are unreadable and the network diagram without any labels is also not very useful. This could be removed.
The schematics on the right indicate a ‘generic analysis’ using the NGS sequencing data. We agree it is not essential and have removed it the revised version of the manuscript.
4. Figure 2B: The network presentation is not very easy to read. What are the functional groups/pathways here? The clusters should be labeled accordingly. What is the meaning of the different sizes of the circles? Maybe key interactions (e.g. TRIM37) could be indicated in a different color shade to highlight these?
In our figure we tried to highlight (1) the connectivity among screening conditions and (2) complexes that were identified by the screens. Each node (other than the six hub nodes that denote a screen condition) represents a hit from the screens. Thus, the nodes are connected by edges only to the screening conditions, not to each other. In this scenario, highlighting TRIM37 ‘interactions’ would only highlight the screening conditions for which TRIM37 was a hit (200 nM RPE-1, 500 nM RPE-1, 200 nM A375, 500 nM A375). We could try to overlay functional enrichment data on the graph, but this data is presented separately in Figure 2 —figure supplement A-D. To distinguish the ‘hub’ nodes, representing the screen conditions, from the others we have made them larger and are outlined with yellow. The large circles represent hits found in previous PLK4 inhibition screens; we now indicate this both in the legend in the figure panel (Figure 2B) and in the written figure legend (Page 26).
Reviewer 3, Major points
1. The presentation throughout the manuscript sometimes made it difficult to follow exactly what the authors meant when they referred to the various doses of Centrinone used in their experimentsoften using the terms "low" or "high" without specifying exactly what they mean. In Figure 1A, for example, they present a growth inhibition curve using a log10 scale of Centrinone concentration, and they conclude that growth was inhibited "at concentrations above 150nM, with full inhibition observed at concentrations greater than 200nM". I presume this is just sloppy language, as it appears that growth is significantly inhibited at 150nM and full growth inhibition is achieved at 200nM. However, in Figure 4D, the authors show another growth inhibition curve (this time presented on a linear scale) where significant growth inhibition is seen well below 100nM and full inhibition appears to be achieved at ~125nM. The discrepancy between these experiments is not noted, nor any reason for it explained.
We agree with the reviewers and apologize for using ‘low’ and ‘high’ as they are ambiguous. We now refer to specific centrinone B concentrations or to inhibition ‘phases’ based on TRIM37-dependent growth arrest and centrosome number. The direct comparison between former Figure 1A and former Figure 4D (now Figure 4B) is not straightforward. The experiments presented were performed approximately 6 years apart and in slightly different ways. As reviewer 3 indicates, Figure 1A is presented in a log scale; this makes it difficult for the reader to determine the exact concentrations of centrinone B used. For this panel, we used, 0 (DMSO), 10, 30, 75, 165, 200 and 500 nM centrinone B. For former Figure 4D, we used 0, 50, 125, 150, 167, 200 and 500 nM. The only point that might be anomalous is 75 nM in Figure 1A. We do see approximately 25% inhibition using 50 nM centrinone B in Figure 4D, but no inhibition using 75 nM in Figure 1A. We can offer two explanations for this discrepancy. First, we noticed small deviations in the potency of centrinone B batches. Second, for Figure 1A, cells were assayed using a passaging assay where they are continuously plated, counted and re-seeded. Cells in Figure 4D were assayed using a clonogenic assay where cells are plated at low density and allowed to grow over the course of approximately two weeks. It is possible that a combination of these factors led to the discrepancy pointed out by the reviewer. To streamline this aspect of the manuscript, we have made the following change: we repeated the growth experiment for Figure 1A and now present the data by plotting the results from the individual replicates with the mean and standard deviation. We show only the 200 and 500 nM centrinone B concentrations since we derive our basic conclusions from these conditions (i.e. both concentrations result in p53dependent growth arrest where centrioles are overduplicated after 200 nM centrinone B, while centrioles are lost after treatment with 500 nM) and these are the conditions we subsequently used in our screens. We hope that this explanation and changes satisfy this reviewer.
While discrepancies such as this may seem trivial, they make it hard to interpret some of the authors conclusions. For example, in their initial screen, the "low" dose of Centrinone (200nM) leads to centriole amplification and genes that block centriole duplication or PIDDosome function (which normally signals the presence of extra centrioles) are required for the growth arrest triggered by this concentration of the drug (Figure 1B). To me, this suggests that centriole amplification is required for this growth arrest at 200nM. However, when the authors test a more graded series of concentrations they conclude "excess centrioles might not be the trigger for this arrest at low Centrinone B concentrations". I assume they are using "low" here to indicate concentrations at or below 150nM (even though they use low to mean 200nM in their initial screen)? In the Discussion, they state that TRIM37 is "required for the growth arrest in response to partially or fully inhibited PLK4, but this activity was independent of the presence of excess centrioles". Again, it is not clear to which experiments they are referring when they talk about "partially" or "fully" inhibited PLK4, but, if this is correct, then why are genes required for centriole duplication and PIDDosome function identified in their initial screen as being required for the growth arrest at 200nM but not 500nM? Do they consider 200nM to be fully inhibiting PLK4?
We apologize for not being clearer about these points. We now characterize the doseresponse to centrinone B based on growth arrest, centrosome number and TRIM37 dependence (Page 8, paragraph 3, Figure 4E). Briefly, we observe cell arrest in the following phases: (1) without major centrosome abnormalities at 125 nM centrinone B (Phase I); (2) with supernumerary centrosomes at 150-200 nM centrinone B (Phase IIa); (3) with centrosome loss at 500 nM (Phase IIb). Phase I growth arrest is independent of TRIM37 while Phase II is dependent on TRIM37. Moreover, where required, we refer to specific concentrations of centrinone B, rather than use the relative terms ‘low’ and ‘high’.
We initially observed that cells arrested after treatment with either 200 or 500 nM centrinone B. Additionally, we observed centriole over-duplication in cells treated wtih 200 nM but centriole loss with 500 nM. Our initial hypothesis was that either centriole overduplication or loss resulted in growth arrest. To determine if centriole overduplication caused by 200 nM centrinone B triggers growth arrest, we induced centriole overduplication using a parallel method by overexpressing PLK4 and, surprisingly, TRIM37 was not required for growth arrest in these conditions, similar to that observed by Evans et al., 2020. Thus, there are two conditions where centriole overduplication is observed and the growth arrest in only one condition is dependent on TRIM37. This is an important difference that we now better highlight in our revised manuscript on Page 13, paragraph 3. We now present a model (new Figure 8) and a table (Figure 7 —figure supplement 2A) that summarizes our results.
Briefly, it is thought that partially inhibited PLK4 blocks its own auto-phosphorylation and therefore blocks its degradation. The overall abundance of PLK4 therefore increases under these conditions and overduplication occurs. In our hands, we consider PLK4 to be partially inhibited in RPE-1 or A375 cells at any concentrations of centrinone B at 200 nM or lower.
We agree that it is interesting we detect genes required for centriole duplication and PIDDosome function in our 200 nM screen. We now discuss this more explicitly. We note that TRIM37 suppression of growth arrest is only partial, suggesting another pathway might be active. We suggest that at 200 nM there is a centrosome-dependent response characterized by the ANKRD26/PIDDosome pathway and a PLK4 activity pathway that TRIM37 participates in. We compare our screen results to those of Evans et al. to identify genes potentially involved in these two different responses (Page 13, paragraph 3).
Please also see similar comment to Reviewer 2, Minor point 1.
Presumably it will only require textual changes to address this point, but it is hard to assess the broader significance of the paper until these points are clarified: is the main point of this paper that the cells response to Centrinone treatment is complicated and the role of TRIM37 equally so; or, is there a narrative that leads to a clear hypothesis that can explain these surprising findings?
The current model suggests that TRIM37 E3-dependent remodeling of CEP192 and
subsequent mitotic length changes underlies its growth arrest activity after centriole loss. We have data that is consistent with previously published results and data that challenges some of these recent reports. Importantly, our data does not support a model where mitotic length is the sole determinant of growth arrest and provide a new model for growth arrest and TRIM37 function (Figure 8). While revising our manuscript we performed an additional set of experiments to test this by indirectly monitoring PLK4 activity after centrinone B treatment in the presence or absence of TRIM37. To do so, we expressed GFPPLK4kin+L1 (containing the PLK4 kinase domain plus the unstructured L1 region or full length GFP-PLK4; see Yamamoto S et al., 2019) and performed FRAP analyses (Figure 4D; Figure 7 —figure supplement 1H and I). In this assay, PLK4 mobility is linked to its phosphorylation status near the phosphodegron where decreased mobility corresponds decreased phosphorylation. We find that PLK4 mobility decreases in a dose-dependent manner after treatment with centrinone B mirroring the cell arrest phenotype leading us to suggest cell arrest is directly dependent on PLK4 activity. We hypothesize that PLK4, or a substrate of PLK4 is important for the growth arrest signal (Figure 8).
2. It seems a striking omission that the authors show that p53 and p21 are induced by 200nM and 500nM Centrinone (Figure 1D), but they don't assay these proteins at any concentration lower than this. Perhaps they are saving this data for a subsequent manuscript, but the authors certainly seem to draw conclusions from several experiments they perform at concentrations below 200nM, so they should at least explain why they don't assay p53 and p21 status in these experiments.
We apologize for not including this data in the original version of the manuscript. We find that p53 and p21 increase in a dose-dependent manner and that this response is attenuated in TRIM37-/- cells. We now include that data in Figure 4 —figure supplement 1D.
Reviewer 3, Minor points
1. In the abstract the authors claim that the way in which altered centrosome numbers cause a p53-dependent growth arrest is evolutionarily conserved. This is misleading, as it implies that the loss and gain of centrosomes trigger the same arrest (which is probably not correct), and most of the data to date suggests that flies and worms (two popular models for centrosome research) do not have such a growth-arrest pathway.
This is a good point. We modified this statement to indicate that p53-dependent arrest is confined to mammalian cells: “Altered centrosome numbers in mouse and human cells cause p53-dependent growth arrest through poorly defined mechanisms”.
Reviewer 3, comment in ‘significance’
I could not discern, however, whether one could draw any broader conclusions than this, in part due to the presentation problems described above. Moreover, in the abstract the authors propose that altering PLK4 activity alone is sufficient to signal growth arrest. This would be an important conclusion, and I presume this refers to the very low dosage Centrinone experiments that trigger growth arrest without altering centrosome numbers and which does not require TRIM37? If so, this arrest is poorly characterised here and will be the subject of a future investigation, so it seems to strange to have this as a major conclusion in the abstract.
We agree. Since we now have some evidence to support this model we have included this information in the abstract and it is now part of the model we now include in Figure 8 (see also our response to Major point 1) and discussed on Page 14, paragraph 4.
Meitinger, Franz et al. “TRIM37 controls cancer-specific vulnerability to PLK4 inhibition.” Nature vol. 585,7825 (2020): 440-446. doi:10.1038/s41586-020-2710-1
Yeow, Zhong Y et al. “Targeting TRIM37-driven centrosome dysfunction in 17q23-amplified breast cancer.” Nature vol. 585,7825 (2020): 447-452. doi:10.1038/s41586-020-2690-1
Cuella-Martin, Raquel et al. “53BP1 Integrates DNA Repair and p53-Dependent Cell Fate Decisions via Distinct Mechanisms.” Molecular cell vol. 64,1 (2016): 51-64.
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Yamamoto, Shohei, and Daiju Kitagawa. “Self-organization of Plk4 regulates symmetry breaking in centriole duplication.” Nature communications vol. 10,1 1810. 18 Apr. 2019, doi:10.1038/s41467-019-09847-xhttps://doi.org/10.7554/eLife.73944.sa2
Article and author information
- Laurence Pelletier
Canadian Institutes of Health Research (CIHR FDN 167279)
- Laurence Pelletier
Ontario Research Foundation
- Laurence Pelletier
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank members of the Pelletier Lab for scientific feedback during the preparation of this work. We are grateful to Sally Cheung and Suzanna Prosser for proofreading the manuscript. The rabbit and rat CEP120 antibodies and CEP135 antibody were generous gifts from Li-Heui Tsai, Moe Mahjoub, and Alex Bird, respectively. The TKOv1 virus library was generously provided by Jason Moffat. The pcDNA3 HA-ubiquitin plasmid was constructed by Abdallah Al-Hakim. pcDNA5 GFP-PLK4, and GFP-PLK4kin + L1 were gifts from Daiju Kitagawa. This work was funded through a Canadian Institute for Health Research Foundation Grant, an ORF RE-5 grant from the Ontario Ministry for Research, and a grant-in-aid from the Krembil Foundation. LP holds a Tier 1 Canada Research Chair in Centrosome Biogenesis and Function. The Network Biology Collaborative Centre at the LTRI is supported by the Canada Foundation for Innovation, the Ontario Government, and Genome Canada and Ontario Genomics (OGI-139).
- Anna Akhmanova, Utrecht University, Netherlands
- Jens Lüders, Institute for Research in Biomedicine, Spain
- Preprint posted: June 25, 2021 (view preprint)
- Received: September 16, 2021
- Accepted: June 4, 2022
- Version of Record published: June 27, 2022 (version 1)
© 2022, Tkach et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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