How Does SUMO Participate in Spindle Organization?
Jens Lüders
Round 1
Reviewer 1 Report
Overall this review is a nice and timely summary of the role of SUMOylation in regulating cellular function, with emphasis on mitotic spindle organization. I have no major concerns, but a few comments/corrections that are listed below. A few of my comments concern inaccuracies and/or language errors. I suggest careful proofreading of the entire manuscript in this regard.
1) I would suggest to re-phrase this sentence: “Intriguingly, only a few percentage of a given SUMO substrate is SUMOylated usually, exceptions taken apart.” to “Intriguingly, with a few exceptions, only a small fraction of the total pool of a given SUMO substrate that is present in the cell is SUMOylated.”
2) The sentence “Indeed, due to the increasing sensitivity of mass spectrometry many identified targets could represent just unspecific SUMOylation, with no regulatory function.” is misleading. It reads as if the increased sensitivity of mass spec is responsible for unspecific SUMOylation. Please re-phrase.
3) “a wide-range human proteome study”: I would not use the term “wide-range” here. Do the authors mean “whole cell”?
4) Line 129: “ligae” should be “ligase”
5) Line 146: this statement may be true for NUMA, but I am pretty sure that Plk1 localizes to many core centrosomal substrates even without microtubules. Is there a reference?
6) Table 1 and Table 2: what is the difference between the two tables? Table 2 includes “proteins from Table 1, supplemented with…” – are all proteins included or only some? It should be better defined in text/titles what is included in the tables. Alternatively, a single table could be compiled.
7) The reference in the title for Table 2 is wrong. It should be clearly stated also in the text what the reference for the data in Table 2 is. Related to the previous point: what proteins were extracted from the original, published data file, what criteria where used? A more systematic approach would be preferred.
8) 179: “It is generally admitted…” – I don’t understand “admitted” in this context.
9) 242: “Another track…” – I don’t understand “track” in this context
10) 255: “most” should be “more”
11) 277: “The latter was identified in a proteomic screen of SUMOylated factors that are co-modified by SUMO and ubiquitin, interacts with RNF4, whereas RNF4 inhibition leads to reduced ubiquitylation and accumulation of its SUMOylated forms.” Please rephrase, the sentence is difficult to understand.
12) 284: “As a consequence, cells inactivate the spindle assembly checkpoint and are able to adapt and proceed into mitosis after mild replication stress.” The statement is inaccurate/difficult to understand. The spindle assembly checkpoint has nothing to do with entry into mitosis.
13) 297: “mis-happened MAPs” – do the authors mean “mis-localized MAPs”?
14) 331: instead of “assemblies that may form a different phase” I would write “assemblies that phase-separate”
15) 365: “As targets of the E1 enzyme SAE1/SAE2 one finds ginkgolic acid…” - the drug is not the target here.
Author Response
We thank the reviewer for his suggestions. We have tried to consider all of them. Here is a point by a point response:
1) I would suggest to re-phrase this sentence: “Intriguingly, only a few percentage of a given SUMO substrate is SUMOylated usually, exceptions taken apart.” to “Intriguingly, with a few exceptions, only a small fraction of the total pool of a given SUMO substrate that is present in the cell is SUMOylated.”
We have changed the sentence accordingly
2) The sentence “Indeed, due to the increasing sensitivity of mass spectrometry many identified targets could represent just unspecific SUMOylation, with no regulatory function.” is misleading. It reads as if the increased sensitivity of mass spec is responsible for unspecific SUMOylation. Please re-phrase.
We have rephrased to: Many new SUMO conjugates have been identified recently due to the increasing sensitivity of mass spectrometry and low level SUMOylation of these proteins could be just unspecific, without any regulatory function.
3) “a wide-range human proteome study”: I would not use the term “wide-range” here. Do the authors mean “whole cell”?
We have changed to: whole-cell study and added "human" in the next sentence
4) Line 129: “ligae” should be “ligase”
We have corrected the spelling
5) Line 146: this statement may be true for NUMA, but I am pretty sure that Plk1 localizes to many core centrosomal substrates even without microtubules. Is there a reference?
Indeed, this was a mistake. We have deleted the part referring to the requirement for microtubules, as this was not the main point. It now reads: At the spindle pole, studied SUMOylated substrates are PLK1 [72,73] and the MAP NuMA [70].
6) Table 1 and Table 2: what is the difference between the two tables? Table 2 includes “proteins from Table 1, supplemented with…” – are all proteins included or only some? It should be better defined in text/titles what is included in the tables. Alternatively, a single table could be compiled.
We have changed the titles of the Tables. The first one shows all spindle proteins whose SUMOyatlion has been functionally studied. Its title reads now:
List of the SUMOylated spindle proteins for which the function of SUMOylation has been studied in the listed publications and model systems.
The second is just a selection proteins that are SUMOylated, and that we find interesting; we discuss some of them in the text. The title reads: Examples of interesting SUMOylated spindle proteins
7) The reference in the title for Table 2 is wrong. It should be clearly stated also in the text what the reference for the data in Table 2 is. Related to the previous point: what proteins were extracted from the original, published data file, what criteria where used? A more systematic approach would be preferred.
We corrected the reference; As discussed above, Table 1, shows spindle proteins that are SUMOylated, and for which there exist functional studies. Table 2 is arbitary, it presents our choice of examples, that we think are either interesting to discuss and to study. We hope that this has been clarified on the Table titles now.
8) 179: “It is generally admitted…” – I don’t understand “admitted” in this context.
Admitted was changed to 'thought"
9) 242: “Another track…” – I don’t understand “track” in this context
We changed to : could provide another explanation
10) 255: “most” should be “more”
spelling corrected
11) 277: “The latter was identified in a proteomic screen of SUMOylated factors that are co-modified by SUMO and ubiquitin, interacts with RNF4, whereas RNF4 inhibition leads to reduced ubiquitylation and accumulation of its SUMOylated forms.” Please rephrase, the sentence is difficult to understand.
We rephrased to: In addition, MIS18BP1 interacts with RNF4, and RNF4 inhibition leads to reduced ubiquitylation of the protein and accumulation of its SUMOylated forms. Lastly, MIS18BP1 becomes degraded after depletion of SENP6 and recovers after proteasomal inhibition by MG132 [80].
12) 284: “As a consequence, cells inactivate the spindle assembly checkpoint and are able to adapt and proceed into mitosis after mild replication stress.” The statement is inaccurate/difficult to understand. The spindle assembly checkpoint has nothing to do with entry into mitosis.
We rephrased to: (...) are able to adapt and proceed into anaphase
13) 297: “mis-happened MAPs” – do the authors mean “mis-localized MAPs”?
We changed mis-happened to: mislocalized
14) 331: instead of “assemblies that may form a different phase” I would write “assemblies that phase-separate”
We corrected the phrase as suggested
15) 365: “As targets of the E1 enzyme SAE1/SAE2 one finds ginkgolic acid…” - the drug is not the target here.
We corrected to: As inhibitors of the E1 enzyme SAE1/SAE2, one finds ...
Reviewer 2 Report
I think this review was very well-written and well-covered from historical to latest works of SUMO functions in spindle organization. It was very fun for me to read this review. I have very few minor comments, which should be addressed before publication.
Minor comments:
1) Line 30: MAPs should be “Microtubule Associated Proteins” and MAPs includes motors.
2) Line 38 and 41: Again MAPs includes motors.
3) Table 1: CENPI localization should be the kinetochore.
4) Table 1: Plk1 localization should be the kinetochore/centromere.
5) Table 1: Stu2 localization should be the kinetochore and microtubules.
6) Table 1: Recent study shows NUSAP1 also has an important role in mitosis (Mills CA et al., JBC, 2017).
7) Table 2: What is your definition of the kinetochore and centromere in localization in mitosis in Table 2. Why the localization of CENPH, CENPI, CENPO, CENPQ, CENPU, and CENPV is centromere, whereas Bub1, Bub1B, Bub3, Knl1 are the kinetochore? These proteins do not bind directly to centromere DNA, so should be the kinetochore.
8) Table 2: CCNB1 was recently shown at kinetochore in mitosis (Alfonso-Perez, T. et al, JCB, 2019)
9) Line 166: Delete “however’
10) Line171: This should be “ the best characterized” should be “well-characterized” or “one of the best characterized”.
11) Line210: Typo, CENPI/H/k
12) Line 287-289: Need citations.
13) Line 293: What is the defective proteins? It is too speculate.
14) Line 297: Authors cited author's previous beautiful work for this statement. The paper shows that it is critical for faithful chromosome segregation to degrade Kar9 with proper timing by STUbLs’s pathway at kinetochores, however the study did not directly show whether the localization of Kar9 at kinetochore itself is wrong or not. The data show only that STUbLs’s degradation of Kar9 at kinetochores requires accurate chromosome segregation. Line 293-298 should avoid too strong speculations.
Author Response
We thank the referee for his helpful comments, that we have tried to address in their totality. Below is a point-to-point answer.
1) Line 30: MAPs should be “Microtubule Associated Proteins” and MAPs includes motors.
2) Line 38 and 41: Again MAPs includes motors.
3) Table 1: CENPI localization should be the kinetochore.
4) Table 1: Plk1 localization should be the kinetochore/centromere.
5) Table 1: Stu2 localization should be the kinetochore and microtubules.
We corrected the definition of MAPs to “Microtubule Associated Proteins” and the phrases concerning MAPs. We have revised the localizations of the suggested proteins indicated by the referee and also of the other proteins that are not clearly attached to centromeres, but are referred in the literature as centromeric, to: "centromere/kinetochore". We also changed the localization of Stu2 to "kinetochore and microtubules"
6) Table 1: Recent study shows NUSAP1 also has an important role in mitosis (Mills CA et al., JBC, 2017).
This is true. However, Table 1 shows the proteins that are SUMOylated and for which the function of SUMOyalted has been studied. This is now stated more clear on the title, that reads: List of the SUMOylated spindle proteins for which the function of SUMOylation has been studied in the listed publications and model systems. Since NuSAP has not been found to be SUMOylated, we have not put it in Table 1.
7) Table 2: What is your definition of the kinetochore and centromere in localization in mitosis in Table 2. Why the localization of CENPH, CENPI, CENPO, CENPQ, CENPU, and CENPV is centromere, whereas Bub1, Bub1B, Bub3, Knl1 are the kinetochore? These proteins do not bind directly to centromere DNA, so should be the kinetochore.
We agree with the comment, and as indicated above, we have revised the localizations of the proteins that are not clearly attached to centromeres but are often referred in the literature as centromeric to "centromere/kinetochore".
In addition, table 2 is just a selection of proteins that we find interesting to discuss or to study, This is now more clear indicated in the table title: Examples of interesting SUMOylated spindle proteins
8) Table 2: CCNB1 was recently shown at kinetochore in mitosis (Alfonso-Perez, T. et al, JCB, 2019)
We changed the localization to: ubiquitous, kinetochore, centrosome
9) Line 166: Delete “however’
We corrected
10) Line171: This should be “ the best characterized” should be “well-characterized” or “one of the best characterized”.
11) Line210: Typo, CENPI/H/k
We corrected the typo and the phrase to : one of the best characterized
12) Line 287-289: Need citations.
The citation is citation 90, which we transferred to the beginning of the paragraph
13) Line 293: What is the defective proteins? It is too speculate.
We changed defective to misfolded
14) Line 297: Authors cited author's previous beautiful work for this statement. The paper shows that it is critical for faithful chromosome segregation to degrade Kar9 with proper timing by STUbLs’s pathway at kinetochores, however the study did not directly show whether the localization of Kar9 at kinetochore itself is wrong or not. The data show only that STUbLs’s degradation of Kar9 at kinetochores requires accurate chromosome segregation. Line 293-298 should avoid too strong speculations.
We changed the phrasing to: One could speculate that STUbLs eliminate a protein that is at the wrong (the kinetochore) microtubule plus-ends. Whether STUbLs generally regulate mislocalized MAPs or misfolded kinetochore factors is however an open question. We hope that the speculation is clear now. Of note, Kar9 has never been observed at kinetochores in wild-type cells.
Reviewer 3 Report
This review is timely and covers an exciting and newly emerging area of cell biology. The review is well written, the figures are clear, and the overall message is interesting. One area that could be improved is around referencing:
There are statements in manuscript that cover various aspects of SUMO and cell division machinery that could benefit from references to reviews or primary literature. Examples of this can be found with sentences ending on line 28, 31, 41, 62, 74, etc. I would suggest a careful look over the whole manuscript to ensure proper referencing.
A reference to Meluh and Koshland 1995 should be added to line 46 with respect to the links between SMT3 and the kinetochore.
Author Response
We thank the reviewer for his suggestions. We have reviewed the manuscript and added references wherever it has been suggested (lines 28, 31, 41, 62, 74) or when they were missing. We added the reference for Meluh and Koshland 1995.
