Optogenetic control of PRC1 reveals its role in chromosome alignment on the spindle by overlap length-dependent forces
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Abstract
During metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here, we develop an optogenetic approach for acute removal of PRC1 to partially disassemble bridging fibers and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and largely lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promote chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers.
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Reply to the reviewers
Reply to the Reviewers
We thank the reviewers for their thoughtful comments and suggestions how to improve our manuscript. Most of the remarks are now addressed in a new version of the manuscript with modifications marked in blue. We found especially interesting the idea to explore the changes in dynamics of microtubules that make up bridging fibers, which we will do in revision. In addition, we will perform Western blot analysis of PRC1 and acquire better images of cells with SiR-DNA for Fig. 2 A.
** Major issues: **
Reviewer #1:
The use of blue light necessary to relocate opto-PRC1 from the spindle to the membrane is a concern, specially given the …
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
Reply to the Reviewers
We thank the reviewers for their thoughtful comments and suggestions how to improve our manuscript. Most of the remarks are now addressed in a new version of the manuscript with modifications marked in blue. We found especially interesting the idea to explore the changes in dynamics of microtubules that make up bridging fibers, which we will do in revision. In addition, we will perform Western blot analysis of PRC1 and acquire better images of cells with SiR-DNA for Fig. 2 A.
** Major issues: **
Reviewer #1:
The use of blue light necessary to relocate opto-PRC1 from the spindle to the membrane is a concern, specially given the strongest phenotype associated with acute vs. constitutive inactivation of PRC1. While these differences may indeed reflect distinct cellular adaptation responses to each procedure, the authors must rule out that phototoxicity caused by blue light (e.g. see Douthwright S, Sluder G. Live Cell Imaging: Assessing the Phototoxicity of 488 and 546 nm Light and Methods to Alleviate it. J Cell Physiol. 2017 Sep;232(9):2461-2468. doi: 10.1002/jcp.25588. PubMed PMID:27608139) is not responsible for the observed stronger phenotypes. A control of U2OS cells expressing the centromere marker (without opto-PRC1) in metaphase after exposure to the same blue light regimen (i.e. 200 ms every 10 sec for 20 min and same laser power) should be provided.
Response: We thank the reviewer for raising this important point. We added the suggested experiments on U2OS cells without opto-PRC1 filmed with same blue light regimen, and updated Fig. S2 A-C, E to contain also the new measurements of inter-kinetochore distance (dKC), distance from equatorial plane (dEQ), corresponding time-lapse images, and angle between sister kinetochore axis and spindle long axis (αKC), respectively. We added the following text in Results: “The effects of PRC1 removal were found neither in control experiments without iLID, nor in a different set of control experiments where cells without opto-PRC1 and without iLID were exposed to the same laser illumination protocol (Fig. 2 B,E,F,H; Fig. S2, A-C, E), suggesting that the observed effects were not a consequence of laser photodamage (Douthwright and Sluder, 2017).”
Reviewer #1:
I could not find in the manuscript whether opto-PRC1 is RNAi resistant. I would assume so, as the authors are targeting the 3'-UTR of endogenous PRC1, but at least a western blot should be provided: 1) to properly ascertain depletion efficiency of the endogenous protein; and 2) the levels of opto-PRC1 after depletion.
__Response: We added a note in Methods that opto-PRC1 is RNAi-resistant. We have assessed the depletion efficiency of the endogenous PRC1 and the levels of opto-PRC1 after depletion by using immunofluorescence of PRC1 on the spindle (Fig. S1 A and B). Additionally, we will perform Western blot analysis to show depletion efficiency of endogenous PRC1 and the levels of transfected opto-PRC1 after depletion of endogenous PRC1. However, as we observed that the efficiency of opto-PRC1 plasmid transfection is low, Western blot analysis may provide biased levels of PRC1 in the complete population, not specific to the analyzed opto cells. __
Reviewer #1:
One aspect related with data interpretation and the proposed model: if PRC1 selectively bundles anti-parallel microtubules, how could it mechanically couple sister k-fibers that are made of parallel MTs? This should be explained in detail, ideally supported by data.
__Response: __This is an important issue, which we now explain in detail in Discussion: “As midzone-crossing microtubules associate with k-fibers on either side of the metaphase plate (O'Toole et al., 2020), PRC1 and probably also other microtubule-associated proteins crosslink antiparallel overlaps between k-fiber microtubules extending from one pole and bridging microtubules extending from the opposite spindle half, as well as antiparallel overlaps within the bridging fiber.”
Reviewer #1:
The author should find a way to unequivocally demonstrate that opto-PRC1 is fully functional and can rescue depletion of endogenous PRC1. The fact that recovery of PRC1 on spindles never fully rescue spindle architecture and chromosome properties might indicate that opto-PRC1 is not fully functional. For example, can it rescue anaphase or cytokinesis roles of PRC1?
__Response: __To demonstrate functionality of opto-PRC1, we added images of cell's progression to cytokinesis in both control and opto cells in new Fig. S1D and added the following text to Results: “Importantly, after exposure to the blue light, opto cells were able to progress to cytokinesis (Fig. S1 D)”. Furthermore, as PRC1's major binding partners, Kif4A and MKLP1 (Fig. S4A, and new Fig. S4J, respectively), which depend on its localization in the spindle midzone in anaphase, are found to co-localize with opto-PRC1 in anaphase, opto-PRC1 is fully functional and rescues depletion of endogenous PRC1. We added the following text to Results: “In anaphase, MKLP1 also co-localized with opto-PRC1 in the spindle midzone (Fig. S4 J) (Gruneberg et al., 2006; Kurasawa et al., 2004)”.
** Minor issues: **
Reviewer #1:
Abstract: the authors introduce the problem by stating that chromosome position at the spindle equator is mainly regulated by forces by kMTs. We do not know this, actually there is evidence in the literature that kif4a on chromosome arms is required to maintain chromosomes aligned by exerting forces on ipMTs (e.g. Wandke et al., JCB, 2012). Along the same line, there is evidence from the Dumont lab that sister k-fibers are not mechanically coupled. These alternative views should be discussed and taken into account when formulating the problem under investigation in the present study.
__Response: __We changed the sentence in Abstract to include polar ejection forces: “During metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces”. When formulating the problem in Introduction, we discuss polar ejection forces and cite Wandke et al., 2012, and several other papers. We also discuss the findings about PRC1-mediated coupling of sister k-fibers from the Dumont lab in relation to our local effect of PRC1 removal on a fraction of sister kinetochore pairs: “This local effect is in line with weak mechanical coupling between neighboring k-fibers, yet strong coupling between sister k-fibers (Elting et al., 2017; Suresh et al., 2020).” In addition, we mention the Dumont lab results when we suggest that the persistent misorientation of kinetochores after PRC1 return to the spindle is due to perturbed overlap geometry during the absence of PRC1: “This is in agreement with a recent finding that PRC1 restricts pivoting of k-fibers near kinetochores by promoting tight coupling between sister k-fibers (Suresh et al., 2020).”
Reviewer #1:
The authors refer to kinetochore alignment or lagging kinetochores throughout the text. Although this is unquestionable, it might be more appropriate to refer to chromosome alignment or lagging chromosomes instead, as this is the object to me moved.
__Response: __We agree with the reviewer and changed this at several places throughout the text.
__Reviewer #1: __
page 2: "...PRC1 regulates forces acting on kinetochores". The authors should mention that this would be indirect, as PRC1 is not at kinetochores itself.
__Response: __This is true and therefore we added the word indirectly in this sentence.
Reviewer #1:
page 6: "PRC1 removal did not activate the spindle assembly checkpoint". Although this might be considered semantics, given that the SAC is constitutively active and needs to be satisfied, the authors might adopt a more accurate description such as "PRC1 removal did not prevent spindle assembly checkpoint satisfaction".
__Response: __We changed the sentence into the suggested one.
Reviewer #1:
page 13: the authors mention about the localization of Kif18a on bridging fibers. Was this known? From the images it is unclear if we are looking at bridging fibers or k-fibers. Co-localization with PRC1 would help clarifying this issue. If indeed associated with bridging fibers, this would raise an alternative interpretation of how Kif18a contributes to maintain chromosome alignment.
__Response: __We thank the reviewer for raising this important point. Kif18A localization in the bridge is a new observation, and to make it clearer we introduced merged images where both Kif18A and PRC1 are shown during optogenetic experiment (Fig. S4E) and four examples of enlarged regions around kinetochores with Kif18A-GFP to show its localization in the bridging fiber in mock treated cells and its lack of localization in the bridging fiber after PRC1 siRNA (Fig. S4F). We also added a discussion of a new potential role of Kif18A (and Kif4A and MKLP1) in chromosome alignment: “Interestingly, we found that Kif4A, MKLP1, and Kif18A localize in the bridging fibers in metaphase and this localization was lost after optogenetic or siRNA-mediated PRC1 removal. During anaphase, the PRC1-dependent Kif4A and MKLP1 in the bridging fibers are involved in sliding of antiparallel microtubules to elongate the spindle (Vukušić et al., 2019). Kif4A and MKLP1 may have a similar role in metaphase, and thus Kif4A removal from the bridging fibers induced by PRC1 removal may affect chromosome alignment by affecting microtubule sliding in the bridging fiber. This possibility is in agreement with previous work showing that Kif4A depletion reduces microtubule flux (Wandke et al., 2012). Similarly, Kif18A in the bridging fiber may have microtubule-sliding and crosslinking activities similar to those of the yeast kinesin-8 (Su et al., 2013), which may promote chromosome alignment. The roles of these and other motors within bridging fibers in chromosome alignment will be an intriguing topic for future studies.”
** Major concerns: **
Reviewer #2:
Regarding lagging chromosomes: Page 6 and Fig. 2F: "Kinetochore remains displaced even after opto-PRC1 return": Why is this? The reasoning in the discussion is not clear/convincing. Is it possible that these irreversible changes reflect light-induced deactivation of protein? Or, could these irreversible changes arise from a perturbation in the structure of microtubules at the end of the 'light' period? Discussion or additional supportive evidence to address this will be helpful.
__Response: __We thank the reviewer for raising this question. As we have not observed these effects in control cells with opto-PRC1 and without iLID that relocates opto-PRC1 to the membrane, we do not find light-induced deactivation of opto-PRC1 likely. We find the latter possibility more realistic, thus we added the following text to Discussion: “Kinetochore positions and orientations did not revert to the initial values within 10 min of PRC1 return. We speculate that upon PRC1 removal the geometry of the overlap structures is perturbed due to a change in the force balance in the spindle. When PRC1 returns to the perturbed overlaps, it likely confines the chromosomes in new positions and orientations. This is in agreement with a recent finding that PRC1 restricts pivoting of k-fibers near kinetochores by promoting tight coupling between sister k-fibers (Suresh et al., 2020).”
Reviewer #2:
The correlation between misaligned kinetochores and lagging chromosomes is not clear. Are lagging chromosomes more frequently attached to kinetochores that show high deq values (Fig. S2G) in metaphase?
__Response: __This is an interesting point. To clarify our results, we rewrote the text: “Opto cells that showed lagging kinetochores in anaphase had a slightly smaller inter-kinetochore distance before anaphase than opto cells without lagging kinetochores, but we did not find correlation between lagging kinetochores in anaphase and kinetochore misalignment in metaphase (Fig. S2 H).” Fig. S2 H is the old Fig. S2 G. Please note that we were not able to backtrack individual lagging kinetochores to metaphase to see if they had a higher value of d_eq. Instead, we measured mean d_eq of all kinetochores in opto cells that had a lagging chromosome and in those that did not.
Reviewer #2:
Regarding the contribution of other motors: Looking at the contributions of various other microtubule-associated proteins in accounting for effects of PRC1 removal is a good addition to the paper (Fig. 4). However, the consequences of the depletion of Kif18A and MKLP1 from the bridging fiber are not elaborated upon. Is the presence of these motors at the bridging fiber functionally important? It would be good to incorporate their known activity in the final model for how PRC1-crosslinked fibers align chromosomes. In particular, a recent biorxiv submission from this group has a thorough examination of the consequences of motor removal in anaphase, and perhaps some of their findings and other literature can be used to draw some insights into if and how the presence of these motors on PRC1-crosslinked fibers contribute to chromosome alignment.
__Response: __We thank the reviewer for this important idea, which we now elaborate in Discussion: “Interestingly, we found that Kif4A, MKLP1, and Kif18A localize in the bridging fibers in metaphase and this localization was lost after optogenetic or siRNA-mediated PRC1 removal. During anaphase, the PRC1-dependent Kif4A and MKLP1 in the bridging fibers are involved in sliding of antiparallel microtubules to elongate the spindle (Vukušić et al., 2019). Kif4A and MKLP1 may have a similar role in metaphase, and thus Kif4A removal from the bridging fibers induced by PRC1 removal may affect chromosome alignment by affecting microtubule sliding in the bridging fiber. This possibility is in agreement with previous work showing that Kif4A depletion reduces microtubule flux (Wandke et al., 2012). Similarly, Kif18A in the bridging fiber may have microtubule-sliding and crosslinking activities similar to those of the yeast kinesin-8 (Su et al., 2013), which may promote chromosome alignment. The roles of these and other motors within bridging fibers in chromosome alignment will be an intriguing topic for future studies.”
Reviewer #2:
Page 13 and Fig. 4A & 4B: "The localization of Kif18A in the bridge was perturbed by both acute and long-term PRC1 removal." However, this is not apparent from Figures 4A and 4B. It would be helpful to clarify how this interpretation was made from the data in the figure.
__Response: __We thank the reviewer for pointing this out. To clarify this issue, we added merged-channel images of the cell from Fig. 4 A to Fig. S4 E to show colocalization of Kif18A and PRC1. Moreover, we added enlargements from spindles without and with PRC1 depletion in Fig. S4 F to show presence and absence of Kif18A in the bridging fibers, respectively. To clarify how the images were evaluated, we added the following text in Methods: “Localization test of GFP-Kif4A or Kif4A-GFP, MKLP1-GFP, Kif18A-GFP, EGFP-CLASP1, and CENP-E-GFP in the bridging fibers of either opto cells or cells treated with mock siRNA or PRC1 siRNA was performed by visually inspecting the GFP signal through the z-stack, in the region where PRC1-labeled fibers were found, i.e., in the region that spans between sister kinetochores and continues ~2 µm laterally from sister kinetochores.”
Additional suggested experiment and analysis:
Reviewer #2:
One factor that could potentially contribute to the changes in chromosome alignment and increase in lagging chromosomes upon PRC1 removal, is changes in dynamics of microtubules that make up bridging fibers. This may also provide insights on the role of associated proteins. One possible experiment is to look at tubulin turnover in the bundles (example by FRAP). Another alternative possibility is to examine EB3 comets in the presence and absence of PRC1 (note: these are just some potential suggestions; the authors may have other ways of addressing the question). Examining the dynamics would help in addressing if bridging fibers is dynamically remodeled through metaphase and early anaphase and whether the loss of PRC1 causes a change in the dynamics of these microtubules.
__Response: __We thank the reviewer for this exciting suggestion. We will perform experiments to examine EB3 comets (their numbers and velocities) in the bridging fibers in the presence and absence of PRC1.
Reviewer #2:
Do kinetochores oscillate / fluctuate about the metaphase plate over time? Does the absence of PRC1 affect these fluctuations? Since the authors already have the data (Fig. 2), they can track the trajectories of sister kinetochore displacement from the equatorial plane as a function of time from prometaphase on, both in the presence and absence of PRC1. This analysis will be informative in understanding how kinetochores and bridging fibers act together to maintain force balance in the spindle and how misalignments are corrected.
__Response: __This is an interesting point. We added the following results: “Kinetochore displacement was not a result of higher oscillation amplitude because kinetochores fluctuated to a similar extent in the presence and absence of opto-PRC1, but in its absence the displaced kinetochores fluctuated within a region that was offset from the equatorial plane (Fig. S2 D).”
** Minor points: **
Reviewer #2:
Is the number of microtubules that make up the bridging fiber the same for outermost and inner kinetochores?
__Response: __This is an interesting question. Even though we could not measure this here, we suggest that the outermost bridges may have more microtubules: “The misaligned kinetochores were found in the inner part of the spindle, where PRC1 signal disappeared faster than on the outer part, which indicates that the inner bridging fibers were more severely affected by PRC1 removal and/or that they are made up of fewer microtubules than the outer bridges.”
Reviewer #2:
The quantity dax that is plotted in fig. S2F has not been defined in the text.
__Response: __We now define dAX in the caption of Fig. S2 F: “Graphs show aKC versus corresponding dEQ and the distance from the midpoint between sister kinetochores to the long spindle axis, dAX (left), ...”
Reviewer #2:
Discussion of these findings in the context of recent work from the lab of Sophie Dumont will be interesting (Suresh et al. eLife 2020;9:e53807).
__Response: __We thank the reviewer for reminding us to discuss this highly relevant recent paper by the Dumont lab. We included a discussion of the findings about PRC1-mediated coupling of sister k-fibers in relation to our local effect of PRC1 removal on a fraction of sister kinetochore pairs: “This local effect is in line with weak mechanical coupling between neighboring k-fibers, yet strong coupling between sister k-fibers (Elting et al., 2017; Suresh et al., 2020).” In addition, we mention these results when we suggest that the persistent misorientation of kinetochores after PRC1 return to the spindle is due to perturbed overlap geometry during the absence of PRC1: “This is in agreement with a recent finding that PRC1 restricts pivoting of k-fibers near kinetochores by promoting tight coupling between sister k-fibers (Suresh et al., 2020).”
Reviewer #3:
Some of the images are sub-optimal. For example Fig 2A, there doesn't seem to be much/any PRC1 on the spindle in the "Dark 0 min" condition, although some is visible after the reversal. Do the authors have a better example to show here? In Figures 1 and 2 we can see the removal clearly yet in later images, the spindle is zoomed such that the relocation cannot be observed.
__Response: __We agree that PRC1 is not properly visible in Fig. 2 A and we will do new experiments to obtain better images. Regarding the later images in Figs. 3 and 4 that are zoomed, they are displayed in this manner to show the localization of proteins in the bridging fiber and/or at the ends of kinetochore fibers. In these experiments, the removal of PRC1 was the same as in earlier images, which is visible in examples shown in Figs. S3 and S4.
Reviewer #3:
Have the authors looked at whether the cells progress normally after removal and reversal of PRC1? In the paper the authors describe how the knockdown and re-expression of opto-PRC1 does not interfere with mitotic progression, but we wondered whether cells recover after the optogenetic operation, compared to a control with similar illumination.
__Response: __We found that cells were able to progress to cytokinesis and added an example to Fig. S1 D and the following text to Results: “Importantly, after exposure to the blue light, opto cells were able to progress to cytokinesis (Fig. S1 D).”
Reviewer #3:
Is there a reason why no Dark-state images are shown in Fig 2C and I?
__Response: __We swapped those images with Dark-state images.
Reviewer #3:
For some of the plots, the y-axis is not shown scaled from 0. This is misleading because it exaggerates differences. Examples are 2E,F,G,H, 3G,J, S3E,G,H, S5C,D,E,F,G.
__Response: __We agree and we now show graphs with the y-axis starting at 0 for Fig. 2 F,H, Fig. S2 B,E, Fig. 3 G,J, Fig. S3 G, and Fig. S5 C,F,G. However, we did not change the graphs for d_kc, theta, spindle length and width, and widths of bridging and k-fibers, because these values span a rather narrow range, which is far from zero. Because the differences are statistically significant, we chose a scale at which they can be easily visualized.
Reviewer #3:
In the legend, formulae should be written in correct notation.
__Response: __Corrected: Formulae y=A*exp(-τ*x) and y=A*exp(-τ*x)+c were used for opto-PRC1 removal and return, respectively.
__Reviewer #3: __
In Fig 2 legend it says that a 0.5-pixel-radius Gaussian blur is applied. Doesn't the kernel for transformation result in an identity matrix?
__Response: __To clarify this, we replaced “0.5-pixel-radius Gaussian blur” with “0.5-pixel-sigma Gaussian blur” in figure captions and added the following to Methods: “To remove high frequency noise in displayed images a Gaussian blur filter with a 0.5-pixel sigma (radius) was applied where stated”.
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Referee #3
Evidence, reproducibility and clarity
This manuscript by Jagrić et al. shows a role for PRC1 at metaphase by using an optogenetic method to rapidly remove PRC1 from bridging fibres in the mitotic spindle. They show that this methodology (which uses light to relocate PRC1 temporarily on the plasma membrane) is superior to long-term depletion by siRNA and, because it is reversible, has advantages over chemically-induced protein translocation. They put the method to use to examine PRC1's role in bridging fibres the results are consistent with siRNA approaches but cleaner due to the acute nature of the method. Overall the paper is convincing and is likely to be of interest …
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Referee #3
Evidence, reproducibility and clarity
This manuscript by Jagrić et al. shows a role for PRC1 at metaphase by using an optogenetic method to rapidly remove PRC1 from bridging fibres in the mitotic spindle. They show that this methodology (which uses light to relocate PRC1 temporarily on the plasma membrane) is superior to long-term depletion by siRNA and, because it is reversible, has advantages over chemically-induced protein translocation. They put the method to use to examine PRC1's role in bridging fibres the results are consistent with siRNA approaches but cleaner due to the acute nature of the method. Overall the paper is convincing and is likely to be of interest to cell biologists working on mitosis.
We have only minor comments that can be easily addressed during the current crisis. Note that we covered this paper in our lab journal club when it went up on bioRxiv and our comments in that pre-pandemic time were the same as now.
1.Some of the images are sub-optimal. For example Fig 2A, there doesn't seem to be much/any PRC1 on the spindle in the "Dark 0 min" condition, although some is visible after the reversal. Do the authors have a better example to show here? In Figures 1 and 2 we can see the removal clearly yet in later images, the spindle is zoomed such that the relocation cannot be observed.
2.Have the authors looked at whether the cells progress normally after removal and reversal of PRC1? In the paper the authors describe how the knockdown and re-expression of opto-PRC1 does not interfere with mitotic progression, but we wondered whether cells recover after the optogenetic operation, compared to a control with similar illumination.
3 Is there a reason why no Dark-state images are shown in Fig 2C and I?
4.For some of the plots, the y-axis is not shown scaled from 0. This is misleading because it exaggerates differences. Examples are 2E,F,G,H, 3G,J, S3E,G,H, S5C,D,E,F,G
5.In the legend, formulae should be written in correct notation.
6.In Fig 2 legend it says that a 0.5-pixel-radius Gaussian blur is applied. Doesn't the kernel for transformation result in an identity matrix?
Significance
We thought the paper is likely to be of significant interest to cell biologists working on mitosis.
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Referee #2
Evidence, reproducibility and clarity
In this manuscript, Jagric and colleagues adapt an optogenetic method for acute and reversible removal of spindle associated proteins to the cell membrane. They apply this technique to deplete the microtubule crosslinking protein PRC1 from the metaphase spindle with high temporal accuracy. They establish that the spindle localization of PRC1 can be perturbed in a fast and reversible manner, on a timescale of minutes, using this method.
Next, they use this system to show that acute depletion of PRC1, which has previously been shown to localize to the bridging fibers that link kinetochore pairs. They find that PRC1 depletion modestly …
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Referee #2
Evidence, reproducibility and clarity
In this manuscript, Jagric and colleagues adapt an optogenetic method for acute and reversible removal of spindle associated proteins to the cell membrane. They apply this technique to deplete the microtubule crosslinking protein PRC1 from the metaphase spindle with high temporal accuracy. They establish that the spindle localization of PRC1 can be perturbed in a fast and reversible manner, on a timescale of minutes, using this method.
Next, they use this system to show that acute depletion of PRC1, which has previously been shown to localize to the bridging fibers that link kinetochore pairs. They find that PRC1 depletion modestly disrupts chromosome alignment on the metaphase plate and results in an increased frequency of lagging kinetochores during anaphase. The advantage of the optogenetic system is that they can look at the reversibility and compare the effects of acute depletion to long-time course methods such as RNAi. This comparison is well done and well presented in the paper. The authors further probe the mechanism underlying the defects associated with PRC1 depletion and find a decrease in the number of microtubules that make up a bridging fiber. The localization of other proteins to the kinetochores are not affected by the removal of PRC1, but the localization of Kif18a and MKLP1 to bridging fibers is disrupted. Together, the authors propose a model where the movement of bi-oriented chromosomes is restricted to the region containing PRC1-crosslinked bridging fibers, and this buffering is important in maintaining chromosome alignment.
Overall, the paper is well written, and the schematics, figures and descriptions of experiments are easy to follow. The microscopy experiments and data analysis are carefully performed and thorough, and the representation of data in figures and tables is very clear. The comparison between RNAi and opto-depletion has been well executed and a great addition. The advance in this paper is the establishment of an optogenetics system to selectively and reversibly perturb PRC1. While the method is not novel (Guntas et al., 2015), its development and application to a spindle protein will be of interest to researchers in the field, and I expect this work to be a major resource in that regard. I am less enthusiastic about the biological findings as the effects of PRC1-removal from the bridging fiber are modest. In addition, some effects, such as kinetochore misalignment and decrease in the number of microtubules in the bridging fiber, are not reversible, which raises some concerns about whether these effects are directly mediated by specific protein depletion. I have outlined my specific comments below:
Major concerns:
1 . Regarding lagging chromosomes:
•Page 6 and Fig. 2F: "Kinetochore remains displaced even after opto-PRC1 return": Why is this? The reasoning in the discussion is not clear/convincing. Is it possible that these irreversible changes reflect light-induced deactivation of protein? Or, could these irreversible changes arise from a perturbation in the structure of microtubules at the end of the 'light' period? Discussion or additional supportive evidence to address this will be helpful.
•The correlation between misaligned kinetochores and lagging chromosomes is not clear. Are lagging chromosomes more frequently attached to kinetochores that show high deq values (Fig. S2G) in metaphase?
2 . Regarding the contribution of other motors
•Looking at the contributions of various other microtubule-associated proteins in accounting for effects of PRC1 removal is a good addition to the paper (Fig. 4). However, the consequences of the depletion of Kif18A and MKLP1 from the bridging fiber are not elaborated upon. Is the presence of these motors at the bridging fiber functionally important? It would be good to incorporate their known activity in the final model for how PRC1-crosslinked fibers align chromosomes. In particular, a recent biorxiv submission from this group has a thorough examination of the consequences of motor removal in anaphase, and perhaps some of their findings and other literature can be used to draw some insights into if and how the presence of these motors on PRC1-crosslinked fibers contribute to chromosome alignment.
•Page 13 and Fig. 4A & 4B: "The localization of Kif18A in the bridge was perturbed by both acute and long-term PRC1 removal." However, this is not apparent from Figures 4A and 4B. It would be helpful to clarify how this interpretation was made from the data in the figure.
Additional suggested experiment and analysis:
1 . One factor that could potentially contribute to the changes in chromosome alignment and increase in lagging chromosomes upon PRC1 removal, is changes in dynamics of microtubules that make up bridging fibers. This may also provide insights on the role of associated proteins. One possible experiment is to look at tubulin turnover in the bundles (example by FRAP). Another alternative possibility is to examine EB3 comets in the presence and absence of PRC1 (note: these are just some potential suggestions; the authors may have other ways of addressing the question). Examining the dynamics would help in addressing if bridging fibers is dynamically remodeled through metaphase and early anaphase and whether the loss of PRC1 causes a change in the dynamics of these microtubules.
2 . Do kinetochores oscillate / fluctuate about the metaphase plate over time? Does the absence of PRC1 affect these fluctuations? Since the authors already have the data (Fig. 2), they can track the trajectories of sister kinetochore displacement from the equatorial plane as a function of time from prometaphase on, both in the presence and absence of PRC1. This analysis will be informative in understanding how kinetochores and bridging fibers act together to maintain force balance in the spindle and how misalignments are corrected.
Minor points:
1 . Is the number of microtubules that make up the bridging fiber the same for outermost and inner kinetochores?
2 . The quantity dax that is plotted in fig. S2F has not been defined in the text.
3 . Discussion of these findings in the context of recent work from the lab of Sophie Dumont will be interesting (Suresh et al. eLife 2020;9:e53807).
Significance
The advance in this paper is the establishment of an optogenetics system to selectively and reversibly perturb PRC1. While the method is not novel (Guntas et al., 2015), its development and application to a spindle protein will be of interest to researchers in the field, and I expect this work to be a major resource in that regard. I am less enthusiastic about the biological findings as the effects of PRC1-removal from the bridging fiber are modest.
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Referee #1
Evidence, reproducibility and clarity
The manuscript by Jagric et al. investigates the role of PRC1 in the maintenance of chromosome alignment at the spindle equator using acute inactivation by optogenetic control. This is an elegant system based on the iLID system that recruits a protein of interest to the cell membrane in a reversible way. Accordingly, acute removal of PRC1 resulted in reduction of bridging fibers and decreased inter-kinetochore distances, while widening the metaphase plate and increasing the frequency of lagging chromosomes in anaphase. The authors investigate whether acute PRC1 removal from bridging fibers compromise other proteins and conclude that …
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 by Jagric et al. investigates the role of PRC1 in the maintenance of chromosome alignment at the spindle equator using acute inactivation by optogenetic control. This is an elegant system based on the iLID system that recruits a protein of interest to the cell membrane in a reversible way. Accordingly, acute removal of PRC1 resulted in reduction of bridging fibers and decreased inter-kinetochore distances, while widening the metaphase plate and increasing the frequency of lagging chromosomes in anaphase. The authors investigate whether acute PRC1 removal from bridging fibers compromise other proteins and conclude that PRC1 acts essentially by coupling bridging and kinetochore fibers. They propose that PRC1 uses this role to buffer kinetochore movements to promote chromosome alignment. Overall, this is a very high quality study that adds to our knowledge about the roles of PRC1 and bridging fibers in spindle mechanics and will be of interest to a specialized readership of mitosis researchers. Nevertheless, there are still few remaining issues, mostly concerning additional controls and interpretations that should be addressed prior to publication.
Major issues:
1- The use of blue light necessary to relocate opto-PRC1 from the spindle to the membrane is a concern, specially given the strongest phenotype associated with acute vs. constitutive inactivation of PRC1. While these differences may indeed reflect distinct cellular adaptation responses to each procedure, the authors must rule out that phototoxicity caused by blue light (e.g. see Douthwright S, Sluder G. Live Cell Imaging: Assessing the Phototoxicity of 488 and 546 nm Light and Methods to Alleviate it. J Cell Physiol. 2017 Sep;232(9):2461-2468. doi: 10.1002/jcp.25588. PubMed PMID:27608139) is not responsible for the observed stronger phenotypes. A control of U2OS cells expressing the centromere marker (without opto-PRC1) in metaphase after exposure to the same blue light regimen (i.e. 200 ms every 10 sec for 20 min and same laser power) should be provided.
2- I could not find in the manuscript whether opto-PRC1 is RNAi resistant. I would assume so, as the authors are targeting the 3'-UTR of endogenous PRC1, but at least a western blot should be provided: 1) to properly ascertain depletion efficiency of the endogenous protein; and 2) the levels of opto-PRC1 after depletion.
3- One aspect related with data interpretation and the proposed model: if PRC1 selectively bundles anti-parallel microtubules, how could it mechanically couple sister k-fibers that are made of parallel MTs? This should be explained in detail, ideally supported by data.
4- The author should find a way to unequivocally demonstrate that opto-PRC1 is fully functional and can rescue depletion of endogenous PRC1. The fact that recovery of PRC1 on spindles never fully rescue spindle architecture and chromosome properties might indicate that opto-PRC1 is not fully functional. For example, can it rescue anaphase or cytokinesis roles of PRC1?
Minor issues:
1- Abstract: the authors introduce the problem by stating that chromosome position at the spindle equator is mainly regulated by forces by kMTs. We do not know this, actually there is evidence in the literature that kif4a on chromosome arms is required to maintain chromosomes aligned by exerting forces on ipMTs (e.g. Wandke et al., JCB, 2012). Along the same line, there is evidence from the Dumont lab that sister k-fibers are not mechanically coupled. These alternative views should be discussed and taken into account when formulating the problem under investigation in the present study.
2- The authors refer to kinetochore alignment or lagging kinetochores throughout the text. Although this is unquestionable, it might be more appropriate to refer to chromosome alignment or lagging chromosomes instead, as this is the object to me moved.
3- page 2: "...PRC1 regulates forces acting on kinetochores". The authors should mention that this would be indirect, as PRC1 is not at kinetochores itself.
4- page 6: "PRC1 removal did not activate the spindle assembly checkpoint". Although this might be considered semantics, given that the SAC is constitutively active and needs to be satisfied, the authors might adopt a more accurate description such as "PRC1 removal did not prevent spindle assembly checkpoint satisfaction".
5- page 13: the authors mention about the localization of Kif18a on bridging fibers. Was this known? From the images it is unclear if we are looking at bridging fibers or k-fibers. Co-localization with PRC1 would help clarifying this issue. If indeed associated with bridging fibers, this would raise an alternative interpretation of how Kif18a contributes to maintain chromosome alignment.
Significance
If additional controls are provided, this manuscript represents a significant technical advance in the study of PRC1 function. The results however are just incremental relative to previous state-of-the-art and will be of interest to more specialized researchers working on mitosis and spindle architecture. The concept of buffer for kinetochore movement is interesting, but how exactly PRC1 contributes to this is not addressed in the present work. Maybe some modeling would help test some ideas.
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