TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin stability
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Abstract
TRIM37 is an E3 ubiquitin ligase mutated in Mulibrey nanism, a disease characterized by impaired growth and increased tumorigenesis, whose cellular etiology is poorly understood. TRIM37 depletion from tissue culture cells results in supernumerary foci bearing the centriolar protein Centrin. Here, we characterized these centriolar protein assemblies (Cenpas) to uncover the mechanism of action of TRIM37. We established that an atypical de novo assembly pathway is notably involved in forming Cenpas, which can nevertheless trigger further centriole assembly and act as MTOCs. We found also that Cenpas are present and act similarly in Mulibrey patient cells. Through correlative light electron microscopy, we uncovered that Cenpas correspond to centriole related structures and elongated electron-dense structures with stripes. Importantly, we established that TRIM37 regulates the stability and solubility of the centriolar protein Centrobin. Our findings suggest that elongated Centrobin assemblies are a major constituent of the striped electron dense structures. Furthermore, we established that Cenpas formation upon TRIM37 depletion requires PLK4 activity, as well as two parallel pathways relying respectively on Centrobin and PLK1. Overall, our work uncovers how TRIM37 prevents the formation of Cenpas that would otherwise threaten genome integrity, including possibly in Mulibrey patients.
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Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity):
The manuscript describes the formation of supernumerary centriole protein assemblies ("cenpas") upon silencing of the E3 ubiquitin ligase TRIM37. These "cenpas" resemble centrioles, centriole precursors, or electron-dense striped structures, termed "tigers". Similar observations are made in cells from patients lacking functional alleles of TRIM37. The "cenpas" usually lack the full complement of centriolar proteins, but contain increased amounts of the pro-centriole marker centrobin. It is further shown that the formation of "cenpas" depends on centrobin, or on a parallel pathway involving Plk1 and …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
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Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity):
The manuscript describes the formation of supernumerary centriole protein assemblies ("cenpas") upon silencing of the E3 ubiquitin ligase TRIM37. These "cenpas" resemble centrioles, centriole precursors, or electron-dense striped structures, termed "tigers". Similar observations are made in cells from patients lacking functional alleles of TRIM37. The "cenpas" usually lack the full complement of centriolar proteins, but contain increased amounts of the pro-centriole marker centrobin. It is further shown that the formation of "cenpas" depends on centrobin, or on a parallel pathway involving Plk1 and SAS-6. Overall, the experiments in this study are of high technical quality and most of them are carefully controlled. The discovery of centrobin-containing striped protein assemblies ("tigers") is very interesting and provokes the question of their molecular composition and their mechanistic role in centriole assembly. Since striated fibres containing the protein rootletin have a similar periodicity of stripes (75nm) as the "tigers" in this study (Vlijm et al., PNAS 2018, 115:E2246-53), I was wondering whether the authors couldn't simply test for colocalization of their "tiger"-stripes with rootletin. A potential identity of "tigers" with striated fibres would help understanding the mechanisms of "cenpas" and centriole assembly upon depletion of TRIM37: striated fibres or "tigers" might be controlling the balance of centriole cohesion vs. disengagement and thereby centriole duplication, or they might play a role in the recruitment of additional proteins involved in pro-centriole assembly.
We are grateful to the reviewer for this interesting suggestion. Accordingly, we will test the distribution of Rootletin and potentially CEP68 by immunofluorescence analysis of cells depleted of TRIM37.
In the same context, did the authors correct for the experimentally induced sample expansion in Figure 5B, when comparing inter-stripe distances between U-ExM and EM samples?
Yes, we did. We will clarify the text of the revised manuscript to make this more explicit.
Other major points: The amount of TRIM37-depletion upon siRNA-treatment should be indicated prominently. I see in the "Materials and Methods" and in Fig. S4 that quantitative RT-PCR has been performed. Could Western blotting be performed to have direct information on the protein levels? Fig. 2C demonstrates that this is possible in cells from human patients, so why are there no data on the majority of other experiments in this manuscript?
We previously reported Western blot analysis to estimate the extent of TRIM37 depletion upon siRNA treatment (Balestra et aI., 2013). However, following the suggestion of the reviewer, we will repeat this analysis for select experiments of this study.
Moreover, what is the transfection efficiency in the siRNA experiments? Is there variability between cells that might explain variability in the "cenpas" phenotypes?
The reviewer brings up an interesting point. However, in the absence of an antibody to detect endogenous TRIM37 by immunofluorescence analysis, we cannot provide an accurate figure in this case. We will mention this limitation explicitly in the text of the revised manuscript.
Minor point: In line 353 (page 12), it is stated that centrobin in si-TRIM37 cells migrates slower (Fig. 4D), suggesting that TRIM37 regulates the post-translational state of centrobin. It looks to me as if the corresponding gel in Fig. 4D was "smiling" (see curvature of centrobin in the neighboring lane). I think that the authors should tone down their statement, or replace Fig. 4D with a more convincing image.
We thank the reviewer for having noticed this. We will provide another gel that is not “smiling” -the difference in migration has been observed in a reproducible manner.
Reviewer #1 (Significance):
The findings of this manuscript are highly significant for our understanding of centriole biogenesis. They should be of interest to a large community of cell biologists working on mitosis and on the centrosome, and they are of further importance for biomedical research related to developmental growth abnormalities (Mulibrey nanism). The manuscript shows for the first time a mechanistic link between TRIM37-dependent control of centrobin protein levels, and their impact on the formation of centriole precursors during the cell cycle. The manuscript is well presented, and the relevant scientific literature is cited correctly. However, I would prefer that a potential relationship between "cenpas", "tigers", and the welldescribed rootletin-containing striated fibres be discussed, if not controlled by additional experiments.
We thank the reviewer for her/his appreciation of our work and support for publication.
Field of expertise of this reviewer: centrosome, microtubules, mitosis, cell culture, light and electron microscopy, biochemistry.
Reviewer #2 (Evidence, reproducibility and clarity):
In this work, the authors investigated roles of TRIM37 in regulation of centriole numbers. It was previously observed that depletion of TRIM37 results in supernumerary centrioles and centriole-like structures (Balestra et al., Dev. Cell, 2013; Meitinger et. al., 2016). Here, the authors characterized these centriolar protein assemblies (Cenpas). Cenpas were formed, following an atypical de novo pathway and eventually trigger centriole assembly. They observed that Centrobin is frequently present in Cenpas from the early stage and other centriolar components are sequentially recruited. Furthermore, they established that Cenpas formation upon TRIM37 depletion requires PLK4 activity. TRIM37 depletion also activates PLK1-dependent centriole multiplication. 1.They propose that the tiger structure acts as platform for PLK4-dependent Cenpas assembly. Cenpas may evolve into centriole-like structures after a stepwise incorporation of other centriolar proteins. Fig. 6E suggests that a series of events seem to occur within G2 phase. Therefore, this reviewer suggests to perform a detailed time-course experiments at G2 phase. According to the model, the Centrobin-positive tiger structures may appear first, and then a Centrobin- and centrin-2-double positive structure starts to appear.
We fully agree with the reviewer that this is an important experiment, which we will perform by analyzing TRIM37 depleted cells at successive time points after release from a double thymidine block, using antibodies against Centrobin and Centrin.
2.They claim that Mulibrey patient cells exhibited evidence of chromosome mis-segregation, as would be expected from multipolar spindle assembly, and conclude that Cenpas are present and active also in Mulibrey patient cells. Chromosome mis-segregation may be observed in the normal cells, too. Therefore, they have to perform statistical analysis on Fig. 2D.
In response to this suggestion and to the related comment of reviewer 3 (see below), we will conduct additional immunofluorescence analysis and quantification of patient and normal cells, assessing the distribution of Centrin, Centrobin, microtubules and γ-tubulin, as well as scoring the extent of chromosome mis-segregation.
3.In Fig. 2A, They claimed that mitotic microtubules were disrupted with the cold treatment for 30 min. In our experience, cold treatment for 30 min is not sufficient to disrupt mitotic microtubules. They may show control panel before microtubule regrowth.
We will show the control panel as requested.
Reviewer #2 (Significance):
Significance of this work resides in identification and description of Cenpas as a novel centriole assembly pathway. The authors used cutting-edge microscopy techniques to visualize Cenpas. The manuscript raised more questions than answers. Nonetheless, it is worth to publish the manuscript after revision.
We thank the reviewer for supporting publication after revision.
Reviewer #3 (Evidence, reproducibility and clarity):
Balestra and colleagues investigate the function of Trim 37 in centrosome biogenesis. Trim 37 is a ubiquitin ligase that has previously been identified by the authors as a regulator of centriole duplication. Mutations in Trim37 cause a rare syndrome named Mulibrey that is responsible for a severe form of dwarphism Here they show that depletion of Trim37 in human cells results in the assembly of structures that they name Cenpas. They follow the possibility that Trim37 localises to the centrosome, which might inhibit the assembly of these structures. Further they show that Trim37 depleted cells (or in patient fibroblasts ) assemble multipolar mitosis. Further analysis shows that what the authors defined as abnormal centriole structures are formed in Trim37 depleted cells. These structures recruit centrobin, a daughter centriole component and this process requires the activity of PLK4 and PLK1. Major comments: This study characterizes Trim37 and its possible role in centriole biogenesis. Most conclusions are convincing, although some of the claims taken by the authors might require more data to be corroborated.
1)The major point to be taken into consideration in my opinion relates with the Cenpas structure. According to the beautiful cryo-EM data shown on Fig 3, I wonder why the authors describe these structures as centriole like- or centriole related. I think these appear very different from centrioles and this might be even quite interesting if these structures nucleate microtubules and can participate in mitotic spindle assembly.
We have a different opinion on this point. Most of the “centriole-like” or “centriole-related” structures do resemble the organelle, in that they contain microtubule bundles and are of a related size (in addition to bearing centriolar markers). However, recognizing that the distinction between these two categories of structures is somewhat arbitrary, we will combine them into the most prudent term “centriole-related”, and further explain in the revised manuscript that they comprise a range of structures.
The authors correlate these non-canonical centriole structures as possible microtubule nucleators that might be responsible for multipolar configurations like in Fig 2D. This correlation has to be established. In Figure 2D, the authors analyze configurations of mitotic cells in terms of centrosome number and characterized frequency of extra foci. To me the foci they show are quite different in nature. Poles 1 and 3 have both centrin and g-tubulin (presumably centrioles), pole 2 has only a tiny amount of centrin and no g-tubulin, while pole 4 appears to contain both but less of each protein. So the question is are they all nucleating microtubules and participating in spindle assembly? This is particularly important in light of what the authors then mention, which is the occurrence of chromosome mis-segreation in patient cells (this is not shown). Also they describe these extra poles, and then say that Cenpas are active in patient cells. But, active in which manner? By nucleating microtubules? First, in either siRNA cells or in patient cells the authors should analyze microtubules and show that all the extra poles (made of non-canonical centriole) nucleate microtubules and participate in spindle assembly.
In response to this suggestion and to the related comment of reviewer 2 (see above), we will conduct additional immunofluorescence analysis and quantification of patient and normal cells, assessing the distribution of Centrin, Centrobin, microtubules and γ-tubulin, as well as scoring the extent of chromosome mis-segregation.
If they want to propose that this might be the cause of genome integrity loss in patients (as stated in the abstract and suggested a few times throughout the paper) they have to show that cells divide abnormally and generate aneuploidy progeny.
See response just above.
- Another important point that is only partially addresses is the function of Trim37 in stabilizing centrobin. Does Trim37 ubiquitinates centrobin? While the western blot on Figure 4 shows an increase at 8hrs in Trim37 RNAi, this is also the case for tubulin (Fig 4E). But the overall levels appear only slightly increased when compared to its levels at time point zero (Fig. 4F). I can see that in siRNA Ctrl Trim 37 levels go down, but it is still present so how do they explain the lack of Cenpas in this case? Is there a threshold that supports centriole duplication without any major defect but accumulation of a certain level of centrobin then generates Cenpas? Can the authors generate Cenpas just by over-expressing centrobin directly?
It appears from the comment of the reviewer that we were not sufficiently clear here. The experiment reported in Figure 4E and 4F is done in the presence of cycloheximide to analyze the half-life of Centrobin in control conditions and upon TRIM37 depletion. We will clarify the text in the revised manuscript to facilitate understanding.
In Figure 2, they analyze configurations of mitotic cells in terms of centrosome number and characterized frequency of extra foci. To me the foci they show are quite different in nature. Poles 1 and 3 have both centrin and g-tubulin (presumably centrioles), pole 2 has only a tiny mount of centrin and no g-tubulin, while pole 4 appears to contain both but less of each protein. So the question is are they all nucleating microtubules and participating in spindle assembly? This is particularly important in light of what the authors then mention, which is the occurrence of chromosome mis-segreation in patient cells without showing it. Also they describe these extra poles, and then say that Cenpas are active in patient cells. But, active in which manner? By nucleating microtubules? This has to be shown. Also analysis of mitosis should be included to back up a defect in chromosome segregation and also to identify which type of defect.
The above section is a copy/paste mistake (as indicated also in a correspondence between Review Commons and the reviewer).
So in conclusion, the link between Cenpas and multipolarity has to be better investigated in my opinion. This should not be time consuming and also not extremely costly. Authors should label spindle MTs in patient fibroblasts to show that indeed Cenpas are nucleating microtubules. Ideally Cenpas would be distinguished by centrobin labeling. In siRNA depleted cells maybe time lapse microscopy can be used to image mitosis and show a correlation between Cenpas and multipolarity?
As mentioned above, we will conduct additional immunofluorescence analysis and quantification of patient and normal cells, assessing the distribution of Centrin, Centrobin, microtubules and γ-tubulin, as well as scoring the extent of chromosome mis-segregation.
The data is presented without statistical analysis on the figures only on Fig legends, This is really difficult for the reader. The number of experiments and cells analyzed maybe should be also included in each Figure.
We had kept this information to the legends merely to have lean figures, but will consider moving it to the figure panels in the revised manuscript.
Minor comments: Some picture lack scale bars
Apologies. This will be fixed.
the localization of GFP-Trim37. On Figure 1 the authors describe a different localization when fused to a NES localization. It is true that a dotty signal is seen on the panel of NES (Figure 1D), but a nuclear signal is not seen on Trim-GFP in any of the images provided. Shouldn't this be the case?
There is some GFP-TRIM37 nuclear signal in the left panel of Figure 1D, although it is very weak. We will explore the possibility of providing an inset with adjusted brightness/contrast to emphasize this point.
Fig 1C is missing a siCtrl.
The control quantification will be included (no extra centrioles are present in this case).
Why Trim37GFP does not rescue completely the assembly of the extra foci?
In general, there can be many reasons why rescue in such an experimental setting is not complete, including slightly different protein levels, distribution, or interaction with partner proteins. Such possibilities will be discussed explicitly in the revised manuscript.
In Fig 6E, are the authors sure that in the condition of siTRim3 plus si Centrobin and Plk1 inhibition, cells are not stuck in S-phase? This might explain the lack of being in a permissive G2 phase to generate Cenpas?
Although Plk1 inhibition is not expected to block cells in S phase, we cannot rule out this possibility from the data currently available. Therefore, we plan to conduct FACS analysis in a repeat of this experiment to assess cell cycle status.
The data is presented without statistical analysis on the figures. This can be found on figure legends, but it is better to include on the figures to facilitate the reader's job. The number of experiments and cells analyzed maybe should be also included in each Figure?
As mentioned above also, we had kept this information to the legends merely to have lean figures, but will consider moving it to the figure panels in the revised manuscript.
Reviewer #3 (Significance):
Interesting findings and quite novel since a role for Trim 37 in centriole biogenesis has never been reported. Also quite interesting the possible link between multipolarity (needs better characterization) and Mulibrey syndrome.
We thank the reviewer for recognizing the interest and novelty of our work
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #3
Evidence, reproducibility and clarity
Balestra and colleagues investigate the function of Trim 37 in centrosome biogenesis. Trim 37 is a ubiquitin ligase that has previously been identified by the authors as a regulator of centriole duplication. Mutations in Trim37 cause a rare syndrome named Mulibrey that is responsible for a severe form of dwarphism Here they show that depletion of Trim37 in human cells results in the assembly of structures that they name Cenpas. They follow the possibility that Trim37 localises to the centrosome, which might inhibit the assembly of these structures. Further they show that Trim37 depleted cells (or in patient fibroblasts ) assemble …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
Balestra and colleagues investigate the function of Trim 37 in centrosome biogenesis. Trim 37 is a ubiquitin ligase that has previously been identified by the authors as a regulator of centriole duplication. Mutations in Trim37 cause a rare syndrome named Mulibrey that is responsible for a severe form of dwarphism Here they show that depletion of Trim37 in human cells results in the assembly of structures that they name Cenpas. They follow the possibility that Trim37 localises to the centrosome, which might inhibit the assembly of these structures. Further they show that Trim37 depleted cells (or in patient fibroblasts ) assemble multipolar mitosis. Further analysis shows that what the authors defined as abnormal centriole structures are formed in Trim37 depleted cells. These structures recruit centrobin, a daughter centriole component and this process requires the activity of PLK4 and PLK1.
Major comments:
This study characterizes Trim37 and its possible role in centriole biogenesis. Most conclusions are convincing, although some of the claims taken by the authors might require more data to be corroborated.
- The major point to be taken into consideration in my opinion relates with the Cenpas structure. According to the beautiful cryo-EM data shown on Fig 3, I wonder why the authors describe these structures as centriole like- or centriole related. I think these appear very different from centrioles and this might be even quite interesting if these structures nucleate microtubules and can participate in mitotic spindle assembly. The authors correlate these non-canonical centriole structures as possible microtubule nucleators that might be responsible for multipolar configurations like in Fig 2D. This correlation has to be established. In Figure 2D, the authors analyze configurations of mitotic cells in terms of centrosome number and characterized frequency of extra foci. To me the foci they show are quite different in nature. Poles 1 and 3 have both centrin and g-tubulin (presumably centrioles), pole 2 has only a tiny amount of centrin and no g-tubulin, while pole 4 appears to contain both but less of each protein. So the question is are they all nucleating microtubules and participating in spindle assembly? This is particularly important in light of what the authors then mention, which is the occurrence of chromosome mis-segreation in patient cells (this is not shown). Also they describe these extra poles, and then say that Cenpas are active in patient cells. But, active in which manner? By nucleating microtubules? First, in either siRNA cells or in patient cells the authors should analyze microtubules and show that all the extra poles (made of non-canonical centriole) nucleate microtubules and participate in spindle assembly. If they want to propose that this might be the cause of genome integrity loss in patients (as stated in the abstract and suggested a few times throughout the paper) they have to show that cells divide abnormally and generate aneuploidy progeny.
- Another important point that is only partially addresses is the function of Trim37 in stabilizing centrobin. Does Trim37 ubiquitinates centrobin? While the western blot on Figure 4 shows an increase at 8hrs in Trim37 RNAi, this is also the case for tubulin (Fig 4E). But the overall levels appear only slightly increased when compared to its levels at time point zero (Fig. 4F). I can see that in siRNA Ctrl Trim 37 levels go down, but it is still present so how do they explain the lack of Cenpas in this case? Is there a threshold that supports centriole duplication without any major defect but accumulation of a certain level of centrobin then generates Cenpas? Can the authors generate Cenpas just by over-expressing centrobin directly?
So in conclusion, the link between Cenpas and multipolarity has to be better investigated in my opinion. This should not be time consuming and also not extremely costly. Authors should label spindle microtubules in patient fibroblasts to show that indeed Cenpas are nucleating microtubules. Ideally Cenpas would be distinguished by centrobin labeling. In siRNA depleted cells maybe time lapse microscopy can be used to image mitosis and show a correlation between Cenpas and multipolarity?
Minor comments:
Some picture lack scale bars
the localization of GFP-Trim37. On Figure 1 the authors describe a different localization when fused to a NES localization. It is true that a dotty signal is seen on the panel of NES (Figure 1D), but a nuclear signal is not seen on Trim-GFP in any of the images provided. Shouldn't this be the case?
Fig 1C is missing a siCtrl. Why Trim37GFP does not rescue completely the assembly of the extra foci?
In Fig 6E, are the authors sure that in the condition of siTRim3 plus si Centrobin and Plk1 inhibition, cells are not stuck in S-phase? This might explain the lack of being in a permissive G2 phase to generate Cenpas?
The data is presented without statistical analysis on the figures. This can be found on figure legends, but it is better to include on the figures to facilitate the reader's job. The number of experiments and cells analyzed maybe should be also included in each Figure?
Significance
Interesting findings and quite novel since a role for Trim 37 in centriole biogenesis has never been reported. Also quite interesting the possible link between multipolarity (needs better characterization) and Mulibrey syndrome.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
In this work, the authors investigated roles of TRIM37 in regulation of centriole numbers. It was previously observed that depletion of TRIM37 results in supernumerary centrioles and centriole-like structures (Balestra et al., Dev. Cell, 2013; Meitinger et. al., 2016). Here, the authors characterized these centriolar protein assemblies (Cenpas). Cenpas were formed, following an atypical de novo pathway and eventually trigger centriole assembly. They observed that Centrobin is frequently present in Cenpas from the early stage and other centriolar components are sequentially recruited. Furthermore, they established that Cenpas …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
In this work, the authors investigated roles of TRIM37 in regulation of centriole numbers. It was previously observed that depletion of TRIM37 results in supernumerary centrioles and centriole-like structures (Balestra et al., Dev. Cell, 2013; Meitinger et. al., 2016). Here, the authors characterized these centriolar protein assemblies (Cenpas). Cenpas were formed, following an atypical de novo pathway and eventually trigger centriole assembly. They observed that Centrobin is frequently present in Cenpas from the early stage and other centriolar components are sequentially recruited. Furthermore, they established that Cenpas formation upon TRIM37 depletion requires PLK4 activity. TRIM37 depletion also activates PLK1-dependent centriole multiplication.
- They propose that the tiger structure acts as platform for PLK4-dependent Cenpas assembly. Cenpas may evolve into centriole-like structures after a stepwise incorporation of other centriolar proteins. Fig. 6E suggests that a series of events seem to occur within G2 phase. Therefore, this reviewer suggests to perform a detailed time-course experiments at G2 phase. According to the model, the Centrobin-positive tiger structures may appear first, and then a Centrobin- and centrin-2-double positive structure starts to appear.
- They claim that Mulibrey patient cells exhibited evidence of chromosome mis-segregation, as would be expected from multipolar spindle assembly, and conclude that Cenpas are present and active also in Mulibrey patient cells. Chromosome mis-segregation may be observed in the normal cells, too. Therefore, they have to perform statistical analysis on Fig. 2D.
- In Fig. 2A, They claimed that mitotic microtubules were disrupted with the cold treatment for 30 min. In our experience, cold treatment for 30 min is not sufficient to disrupt mitotic microtubules. They may show control panel before microtubule regrowth.
Significance
Significance of this work resides in identification and description of Cenpas as a novel centriole assembly pathway. The authors used cutting-edge microscopy techniques to visualize Cenpas. The manuscript raised more questions than answers. Nonetheless, it is worth to publish the manuscript after revision.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The manuscript describes the formation of supernumerary centriole protein assemblies ("cenpas") upon silencing of the E3 ubiquitin ligase TRIM37. These "cenpas" resemble centrioles, centriole precursors, or electron-dense striped structures, termed "tigers". Similar observations are made in cells from patients lacking functional alleles of TRIM37. The "cenpas" usually lack the full complement of centriolar proteins, but contain increased amounts of the pro-centriole marker centrobin. It is further shown that the formation of "cenpas" depends on centrobin, or on a parallel pathway involving Plk1 and SAS-6.
Overall, the experiments in …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The manuscript describes the formation of supernumerary centriole protein assemblies ("cenpas") upon silencing of the E3 ubiquitin ligase TRIM37. These "cenpas" resemble centrioles, centriole precursors, or electron-dense striped structures, termed "tigers". Similar observations are made in cells from patients lacking functional alleles of TRIM37. The "cenpas" usually lack the full complement of centriolar proteins, but contain increased amounts of the pro-centriole marker centrobin. It is further shown that the formation of "cenpas" depends on centrobin, or on a parallel pathway involving Plk1 and SAS-6.
Overall, the experiments in this study are of high technical quality and most of them are carefully controlled. The discovery of centrobin-containing striped protein assemblies ("tigers") is very interesting and provokes the question of their molecular composition and their mechanistic role in centriole assembly. Since striated fibres containing the protein rootletin have a similar periodicity of stripes (75nm) as the "tigers" in this study (Vlijm et al., PNAS 2018, 115:E2246-53), I was wondering whether the authors couldn't simply test for co-localization of their "tiger"-stripes with rootletin. A potential identity of "tigers" with striated fibres would help understanding the mechanisms of "cenpas" and centriole assembly upon depletion of TRIM37: striated fibres or "tigers" might be controlling the balance of centriole cohesion vs. disengagement and thereby centriole duplication, or they might play a role in the recruitment of additional proteins involved in pro-centriole assembly. In the same context, did the authors correct for the experimentally induced sample expansion in Figure 5B, when comparing inter-stripe distances between U-ExM and EM samples?
Other major points:
The amount of TRIM37-depletion upon siRNA-treatment should be indicated prominently. I see in the "Materials and Methods" and in Fig. S4 that quantitative RT-PCR has been performed. Could Western blotting be performed to have direct information on the protein levels? Fig. 2C demonstrates that this is possible in cells from human patients, so why are there no data on the majority of other experiments in this manuscript? Moreover, what is the transfection efficiency in the siRNA experiments? Is there variability between cells that might explain variability in the "cenpas" phenotypes?
Minor point:
In line 353 (page 12), it is stated that centrobin in si-TRIM37 cells migrates slower (Fig. 4D), suggesting that TRIM37 regulates the post-translational state of centrobin. It looks to me as if the corresponding gel in Fig. 4D was "smiling" (see curvature of centrobin in the neighboring lane). I think that the authors should tone down their statement, or replace Fig. 4D with a more convincing image.
Significance
The findings of this manuscript are highly significant for our understanding of centriole biogenesis. They should be of interest to a large community of cell biologists working on mitosis and on the centrosome, and they are of further importance for biomedical research related to developmental growth abnormalities (Mulibrey nanism). The manuscript shows for the first time a mechanistic link between TRIM37-dependent control of centrobin protein levels, and their impact on the formation of centriole precursors during the cell cycle. The manuscript is well presented, and the relevant scientific literature is cited correctly. However, I would prefer that a potential relationship between "cenpas", "tigers", and the well-described rootletin-containing striated fibres be discussed, if not controlled by additional experiments.
Field of expertise of this reviewer: centrosome, microtubules, mitosis, cell culture, light and electron microscopy, biochemistry.
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