Kinetochore-fiber lengths are maintained locally but coordinated globally by poles in the mammalian spindle

Article activity feed

1. 

   Version published to 10.7554/elife.85208 on eLife

   Jul 3, 2023
2. 

   eLife

   Jun 22, 2023

   Author Response

   Reviewer #1 (Public Review):

   In this study, the authors study the effect of dynactin disruption on kinetochore fiber (k-fiber) length in spindles of dividing cultured mammalian cells. Dynactin disruption is known to interfere with dynein function and hence spindle pole formation. The main findings are that poles are not required for correct average k-fiber length and that severed k-fibers can regrow to their correct length both in the presence and absence of poles by modulating their dynamic properties at both k-fiber ends. In the presence of poles, regrowth is faster and the variation between k-fiber lengths is smaller. This is a very interesting study with high-quality quantitative imaging data that provides important new insight into potential mechanisms of spindle scaling, extending in an original manner previous …

   Author Response

   Reviewer #1 (Public Review):

   In this study, the authors study the effect of dynactin disruption on kinetochore fiber (k-fiber) length in spindles of dividing cultured mammalian cells. Dynactin disruption is known to interfere with dynein function and hence spindle pole formation. The main findings are that poles are not required for correct average k-fiber length and that severed k-fibers can regrow to their correct length both in the presence and absence of poles by modulating their dynamic properties at both k-fiber ends. In the presence of poles, regrowth is faster and the variation between k-fiber lengths is smaller. This is a very interesting study with high-quality quantitative imaging data that provides important new insight into potential mechanisms of spindle scaling, extending in an original manner previous work on this topic in cultured cells and in Xenopus egg extract. The Discussion is interesting to read as several possible mechanisms for k-fiber length control are discussed. The technical quality of the study is very high, the experiments are very original, and most conclusions are well supported by the data. Especially, the experiments observing the regrowth of k-fibers after severing and the study of the dynamic properties of these k-fibers provide very novel insight. Addressing the following concerns could potentially improve the manuscript:

   We thank the reviewer for their fair, rigorous, and conceptually engaging remarks.

   (1) The phenotype generated here by disrupting dynactin via overexpressing p50 appears to be different from that caused by knocking down NuMA or dynein - as previously reported by the Dumont lab (Hueschen et al., 2019). In this study here, unfocused spindles are observed whereas earlier turbulent spindles were observed. This raises the question of whether dynein activity that contributes to pole focusing is really completely inhibited here. These discrepancies in phenotypes seem to deserve an explanation. Is k-fiber length in cultured mammalian cells only maintained in the case of this specific type of inhibition?

   We thank the reviewer for the important point about the different phenotypes observed in different dynein inhibition conditions and we refer them to our response to Essential Revision #1. In summary, we believe that different dynein inhibition phenotypes are similar. Unfocused spindles appear turbulent on longer timescales and appear to reach a steady-state on shorter timescales. The amount of pole-unfocusing also seems to correspond to the severity of dynein inhibition (Figure 1—figure supplement 1). We have chosen to study inhibited spindles that were steady-state and unfocused. We have added this discussion in line 129 as well as better characterized our system of dynein inhibition by adding two new figures (Figure 1—figure supplement 1, Figure 1—figure supplement 3).

   Furthermore, we address the question of whether dynein might still be responsible for length regulation despite poles being unfocused in line 433 of the Discussion: “recent work has revealed that mammalian spindles can achieve similar architecture whether or not dynein (or its recruiter NuMA) is knocked out (Neahring et al., 2021). This suggests that the severe defects in spindle coordination (Figure 1, Figure 5) and maintenance (Figure 2) observed in p50-unfocused spindles are more likely due to the loss of spindle poles than due to the loss of dynein activity per se.”

   We have additionally overexpressed p50 in human RPE1 cells and observed qualitatively similarly unfocused yet generally bi-oriented spindles as in rat kangaroo PtK2 cells, showing that the formation of unfocused spindles in PtK2 is not an artifact unique to that cell line (see newly added Figure 1—figure supplement 3). However, these unfocused RPE1 spindles did not have clear, resolvable k-fibers as in PtK2, so length was not quantified. The only method we are aware of that robustly unfocuses poles in PtK2 spindles is p50 overexpression.

   (2) p50 addition and also p150-cc1 addition was often used in Xenopus egg extract in order to inhibit dynein function. Considerably larger concentrations of p50 than p150-cc1 needed to be used. Can the authors estimate the level of overexpression of p50 in the cells they study? It seems that could be possible given that a mCherry fusion protein can be overexpressed. Was it necessary to select cells with a particular level of mCherry-p50 overexpression to observe the reported phenotypes?

   We thank the reviewers for the suggestion to quantify p50 expression and have added Figure 1—figure supplement 1. Due to gradual red laser power loss over months, data from a single day were plotted for proper comparison, but trends were always consistent within any given day. As discussed above, we observed that higher levels of mean p50 intensity corresponded to unfocused spindles. We have clarified that we chose to study these highly overexpressing unfocused spindles in the text and methods, and we speculate that level of p50 overexpression correlates with amount of dynein inhibition and subsequent pole-unfocusing. This is also consistent with the higher concentrations of p50 needed to inhibit dynein in Xenopus.

   (3) Some comparison to previous experiments using p50 and p150-cc1 addition to Xenopus egg extract spindles could put this study better into the context of the available literature. It seems from previous publications that the p50 addition produced short, unfocused, barrel-shaped spindles, indicating that spindle length is maintained without poles, whereas the p150-cc1 addition produced elongating spindles (e.g. Gaetz & Kapoor, 2004).

   We appreciate the reviewer’s discussion of dynein inhibition in the Xenopus context.

   While Xenopus has been used to study spindle size regulation, it has not been as useful to study k-fiber length regulation, which we focus on. Xenopus spindles have a different architecture, with k-fibers that are not discrete and continuous like in mammalian spindles. Indeed, while p50 and p150-CC1 overexpression alter spindle length in Xenopus, they do not have the same effect in mammalian spindles. Additionally, p150-CC1 does not robustly unfocus poles in mammalian spindles as it does in Xenopus; instead, it leads to an inconsistent variety of spindle disorganization phenotypes with frequently focused poles in PtK2 (data not shown). We speculate this variety of spindle phenotypes arise from a different mechanism of dynein inhibition that does not fully target pole-focusing.

   However, we agree that referencing prior Xenopus work establishes important context and precedent. In line 95 of the Introduction, we state “…inhibiting dynein unfocuses poles but spindles still form albeit with altered lengths in Drosophila (Goshima et al., 2005) and Xenopus (Gaetz and Kapoor, 2004; Heald et al., 1996; Merdes et al., 1996), and without a clear effect on mammalian spindle length (Guild et al., 2017; Howell et al., 2001),” addressing the different effects of dynein inhibition in Xenopus compared to mammalian spindles. We have also added direct mentions of p50 in Xenopus in line 129 (see Essential Revision #1 response).

   Finally, we have added a figure showing overexpression of p50 in a human RPE1 cells to show reproducibility of pole unfocusing across other mammalian cell types (see newly added Figure 1—figure supplement 3).

   (4) In this context, it seems that some more explanation is required for the observations presented in Fig. 1D and 1E. It appears that spindle length and k-fiber length have been measured quite differently. Not much information is provided for how spindle length was defined and measured (please expand this part of the Methods). Could the two different methods of measurement be the reason for the mean k-fiber length remaining unaltered in dynactin-disrupted spindles, whereas the spindle length increases in these cells? If not, do non-k-fiber microtubules contribute to unfocused spindles being longer or are chromosomes not aligned in the metaphase plate causing the increase in spindle length by misalignment of k-fiber sister pairs?

   We thank the reviewers for pointing out the lack of clarity in Figures 1D and 1E. We have expanded and clarified the Methods section describing how spindle axes were measured and how k-fiber lengths were measured, as well as included examples and cartoons to illustrate them (see newly added Figure1—figure supplement 4).

   To clarify, we did not intend to directly measure spindle length, but we did approximate the size of each spindle’s “footprint” in Figure 1D as well as measure individual k-fiber length in Figure 1E. It is now clarified in the Methods line 898 as “Spindle minor and major axes lengths were determined by cropping, rotating, then thresholding spindle images with the Otsu filter using SciKit. Ellipses were fitted to thresholded spindles to approximate the length of their major and minor axes using SciKit’s region properties measurement (Figure1—figure supplement 4A). In control spindles, the major axis corresponded to spindle length along the pole-to-pole axis, and the minor axis corresponded to spindle width along the metaphase plate axis. However, unfocused spindles were disorganized along both axes to the extent where the minor axis did not always correspond to the metaphase plate axis. Thus, Figure 1D reports ”spindle minor axis length” and “spindle major axis length” rather than “spindle width” and “spindle length”. Furthermore, it is worth noting that in unfocused spindles, spindle length is decoupled from k-fiber length because of k-fiber disorganization along both axes. Thus, spindle length was not measured in unfocused spindles...”

   We additionally removed the potentially confusing terminology of “wider” and “longer” in the Results section to make clear that we are approximating spindle size, not spindle length and width, and we now state in line 168,“ k-fibers were more spread out in the cell, with spindles covering a larger area compared to control along both its major and minor axes (Figure 1D).”

   We believe our clarification and expansion of the Methods section, as well as inclusion of a new supplementary figure and cartoon address the reviewer’s points, and we thank them for pointing out the lack of clarity.

   (5) It seems that in the Discussion it is implied that k-fibers can respond to severing in both focused and unfocused spindles by modulating their dynamics at both ends of the k-fibers, but in the Results section the wording is more cautious because of the difference in 'flux' in severed and unsevered unfocused spindles is not significant (Fig. 4D, blue data). It appears indeed that there is also a difference in flux between severed and unsevered unfocused spindles, but the number of data points is too small. Depending on how difficult these experiments are, it could be worth increasing the size of the data set to come to a clear conclusion, given that the data shown in Figs. 3 and 4 are quite remarkable and form the core of the study.

   We appreciate the reviewer’s close reading and pertinent suggestions.

   As detailed in our response to Essential Revision #3, we did not increase the sample size for unfocused spindles since it would not be reasonably feasible to show significant differences in flux. However, we performed more ablations and photomarking in control spindles as detailed in our response to this reviewer’s point 6 below, a different but related point.

   (6) Can the authors exclude that the stopping of 'flux' at minus ends after severing is due to some sort of permanent damage induced by ablation? In other words, do severed spindles begin to flux again once they have regrown to their original length?

   We thank the reviewer for their important points.

   We have addressed this question in the newly added Figure 4—figure supplement 1 as described in our response to Essential Revision #3 to show that flux resumes after length recovery. In summary, we observed no adverse effects of ablation on k-fiber minus-ends. Severed k-fibers have restored lengths, and minus-end dynamics several minutes after ablation.

   (7) To this reader, the conceptualization of distinguishing between 'global' and 'local' effects/behavior was a little confusing, both in the title and also later in the text. The concept of 'local' regulation of k-fiber length appears to contradict the observation that k-fiber length can be regained after severing by changes in the dynamics at both ends (so at two very different locations) which is a rather remarkable finding. Maybe distinguishing between 'individual' and 'collective' k-fiber behavior could be clearer.

   We appreciate the reviewer’s consideration of terminology. We have addressed this by clearly defining our use of ‘local’ to refer to individual k-fibers as a unit where appropriate in the text (lines 271, 449). We chose these terms since they can help describe individual versus collective properties, while simultaneously emphasizing the aspects of global architecture and spatial organization in the spindle.

   (8) Can the authors exclude that some of the differences between unfocused and focused spindles could be due to altered dynein activity at kinetochores? Or due to the dynein-dependent accumulation of certain spindle proteins along microtubules towards the minus ends of k-fibers or other spindle microtubules, instead of being due to only the presence versus absence of poles? Could this be tested by ablating both poles? If this is too challenging, a discussion of these possibilities could be justified.

   We appreciate the reviewer’s consideration of kinetochore activity as well as other methods of removing poles. However, p50 overexpression is currently the only method to robustly unfocus spindles in PtK2 cells – ablating poles or removing pole-associated structures such as centrosomes does not abolish pole-focusing in this system (Khodjakov et al., 2000). Furthermore, we now discuss the possibility that altered dynein activity (such as activity at kinetochores) may give rise to the phenotypes we describe in our work in line 433: “…recent work has revealed that mammalian spindles can achieve similar architecture whether or not dynein (or its recruiter NuMA) is knocked out (Neahring et al., 2021). This suggests that the severe defects in spindle coordination (Figure 1, Figure 5) and maintenance (Figure 2) observed in p50-unfocused spindles are more likely due to the loss of spindle poles than due to the loss of dynein activity per se. Though we cannot exclude it, this also suggests that the findings we make in unfocused spindles are not due changes in activity of the dynein population at kinetochores.”

   Reviewer #2 (Public Review):

   The mitotic spindle of eukaryotic cells is a microtubule-based assembly responsible for chromosome segregation during cell division. For a given cell type, the steady-state size and shape of this structure are remarkably consistent. How this morphologic consistency is achieved, particularly when one considers the complex interplay between dynamic microtubules, spatial and temporal regulation of microtubule nucleation, and the activities of several microtubule-based motor proteins, remains a fundamental unanswered question in cell biology. In this work by Richter et al., the authors use biochemical and biophysical perturbations to explore the feedback between mitotic spindle shape and the dynamics of one of its main structural elements, kinetochore fibers (k-fibers) - bundles of microtubules that extend from kinetochores to spindle poles. Overexpression of the p50 dynactin subunit in mammalian tissue culture cells (Ptk2) was used to inhibit the microtubule motor cytoplasmic dynein resulting in misshapen spindles with unfocused poles. Measurements of k-fiber lengths in control and unfocused conditions showed that although mean k-fiber length was not statistically different, the variation of length was significantly higher in unfocused spindles, suggesting that k-fiber length is set locally, occurring in the absence of focused poles. With a clever combination of live-cell imaging with photoablation and/or photobleaching of fluorescently-labeled k-fibers, the authors went on to explore the mechanistic bases of this length regulation. K-fiber regrowth following ablation occurred in both conditions, albeit more slowly in unfocused spindles. Paired ablation and localized photobleaching on the same k-fiber revealed that microtubule dynamics, specifically those at the plus-end, can be tuned at the level of individual k-fiber. Lastly, the authors show that chromosome segregation is severely impaired when cells with unfocused spindles are forced to enter mitosis. The work's biggest strength is the application of an innovative experimental approach to address thoughtful and well-articulated hypotheses and predictions. Conclusions stemming from the experiments are generally well-supported, though the experiments addressing the "tuning" of k-fiber dynamics could be bolstered by additional data points and perhaps better presented. The manuscript would also benefit from the inclusion of some investigation of spatial differences in the observed effects as well as the molecular and biophysical basis of the observed feedback between k-fiber length and focused poles.

   We appreciate the reviewer providing pertinent, rigorous, and intellectually astute suggestions.

   Comments/Concerns/Questions:

   1. In the discussion, the authors acknowledge that the changes in spindle morphology resulting from p50 overexpression are likely also causing changes in the well-characterized RanGTP/SAF gradients that radiate from chromosome surfaces. Why did the authors did not include an analysis of k-fiber length as a function of positioning within the spindle? The inclusion of this data would not require more experimentation and could be added as a plot showing K-fiber length versus distance from the geometric center of the spindle (defined by the intersection of the major and minor axes perhaps?).

   We thank the reviewer for this pertinent suggestion and refer them to our response to Essential Revision #2. Briefly, we have added the recommended analyses to Figure 1—figure supplement 6 by correlating k-fiber length to position along the spindle’s longitudinal and latitudinal axes.

   1. The authors also acknowledge the established relationship between MT length and MT end dynamics, yet in their ablation studies, the average initial k-fiber length at ablation in control spindles was higher than that for k-fibers in unfocused spindles. It seems that this difference makes the interpretation of the data, particularly the conclusion that fiber growth rates differ due to the absence of focused poles, a bit tenuous. To address this, the authors should consider including plots of grow-back rates versus k-fiber length (again, this should not require additional experiments, just more analysis).

   We thank the reviewer for their critical thinking about experiments. We would like to clarify to the reviewer that initial k-fiber lengths within unfocused spindles preceding ablation were not actually longer on average compared to the average length of control k-fibers from Figure 1E (Figure 2—figure supplement 1). We apologize that this unexpected artifact was not clear in the text and have now reworded line 232 to be more straightforward: “Mean k-fiber lengths in unfocused spindles before ablation appeared to be shorter (Figure 2D); however, this was due to not capturing the full length of k-fibers in a single z-plane while imaging ablated k-fibers. Indeed, length analysis of full z-stacks from unfocused spindles before ablation yielded an indistinguishable mean k-fiber length compared to control k-fibers in Figure 1E (Figure 2—figure supplement 1). Thus, ablated k-fibers were compared to their unablated neighbors as internal controls.”

   We believe that this language clearly calls out the perceived inconsistency, and that our use of internal controls overcomes this confounding factor to make meaningful conclusions. We address the relationship of k-fiber length and growth rate in our response to Essential Revision #2. We are not including it in the manuscript based on our inability to make any meaningful conclusion to either support or exclude the possibility of length-dependent growth rates.

   1. As presented, the data shown in Figure 4 is confusing and does not seem very compelling. The relationship between the kymographs and time series is unclear as is the relationship between the dashed lines in the kymographs and the triangles and the plots in the 4B time series and 4C, respectively. Furthermore, it's not always clear what the triangles are pointing to (e.g. in the unfocused condition time series). The authors might want to consider reworking this figure and providing more measurements of flux following ablation in both the control and unfocused conditions. Lastly, the authors should clarify what negative displacement means.

   We apologize for the unclear figure annotations and thank reviewers for their suggestions. As discussed in our response to Essential Revision #3, we believe we have improved the clarity and presentation of figures and kymographs. More measurements of flux after ablation in unfocused spindles was not feasible as discussed; however, we have performed these measurements in control spindles and added Figure 4—figure supplement 1 to strengthen conclusions about turning flux off/on after ablation.

   We have additionally clarified axis titles by replacing “negative displacement” with the more intuitive descriptor “photomark position relative to minus-end” and clearly defining it in the figure legends in line 565 as follows: “Figure 3 […] (D) Minus-end dynamics, where photomark position over time describes how the mark approaches the k-fiber’s minus-end over time in control and unfocused k-fibers.”

   We thank reviewers for their suggestions to improve clarity and bolster our conclusions.

   Read the original source
3. 

   eLife

   Jun 17, 2023

   eLife assessment

   The authors compellingly demonstrate that k-fiber length and dynamics are regulated at the level of individual fibers, even in the absence of focused poles, but that unfocused spindles fail to accurately segregate chromosomes, suggesting that coordination of k-fiber length by pole focusing is important for spindle function. This study provides important new information on spindle scaling, extending in an original manner previous work on this topic.

   Read the original source
4. 

   eLife

   Jun 17, 2023

   Reviewer #1 (Public Review):

   In this study, the authors study the effect of dynactin disruption on kinetochore fiber (k-fiber) length in spindles of dividing cultured mammalian cells. Dynactin disruption is known to interfere with dynein function and hence spindle pole formation. The main findings are that poles are not required for correct average k-fiber length and that severed k-fibers can regrow to their correct length both in the presence and absence of poles by modulating their dynamic properties at both k-fiber ends. In the presence of poles, regrowth is faster and the variation between k-fiber lengths is smaller. This is a very interesting study with high-quality quantitative imaging data that provides important new insight into potential mechanisms of spindle scaling, extending in an original manner previous work on this topic …

   Reviewer #1 (Public Review):

   In this study, the authors study the effect of dynactin disruption on kinetochore fiber (k-fiber) length in spindles of dividing cultured mammalian cells. Dynactin disruption is known to interfere with dynein function and hence spindle pole formation. The main findings are that poles are not required for correct average k-fiber length and that severed k-fibers can regrow to their correct length both in the presence and absence of poles by modulating their dynamic properties at both k-fiber ends. In the presence of poles, regrowth is faster and the variation between k-fiber lengths is smaller. This is a very interesting study with high-quality quantitative imaging data that provides important new insight into potential mechanisms of spindle scaling, extending in an original manner previous work on this topic in cultured cells and in Xenopus egg extract. The Discussion is interesting to read as several possible mechanisms for k-fiber length control are discussed. The technical quality of the study is very high, the experiments are very original, and most conclusions are well supported by the data. Especially, the experiments observing the regrowth of k-fibers after severing and the study of the dynamic properties of these k-fibers provide very novel insight. Addressing the following concerns could potentially improve the manuscript:

   (1) The phenotype generated here by disrupting dynactin via overexpressing p50 appears to be different from that caused by knocking down NuMA or dynein - as previously reported by the Dumont lab (Hueschen et al., 2019). In this study here, unfocused spindles are observed whereas earlier turbulent spindles were observed. This raises the question of whether dynein activity that contributes to pole focusing is really completely inhibited here. These discrepancies in phenotypes seem to deserve an explanation. Is k-fiber length in cultured mammalian cells only maintained in the case of this specific type of inhibition?

   (2) p50 addition and also p150-cc1 addition was often used in Xenopus egg extract in order to inhibit dynein function. Considerably larger concentrations of p50 than p150-cc1 needed to be used. Can the authors estimate the level of overexpression of p50 in the cells they study? It seems that could be possible given that a mCherry fusion protein can be overexpressed. Was it necessary to select cells with a particular level of mCherry-p50 overexpression to observe the reported phenotypes?

   (3) Some comparison to previous experiments using p50 and p150-cc1 addition to Xenopus egg extract spindles could put this study better into the context of the available literature. It seems from previous publications that the p50 addition produced short, unfocused, barrel-shaped spindles, indicating that spindle length is maintained without poles, whereas the p150-cc1 addition produced elongating spindles (e.g. Gaetz & Kapoor, 2004).

   (4) In this context, it seems that some more explanation is required for the observations presented in Fig. 1D and 1E. It appears that spindle length and k-fiber length have been measured quite differently. Not much information is provided for how spindle length was defined and measured (please expand this part of the Methods). Could the two different methods of measurement be the reason for the mean k-fiber length remaining unaltered in dynactin-disrupted spindles, whereas the spindle length increases in these cells? If not, do non-k-fiber microtubules contribute to unfocused spindles being longer or are chromosomes not aligned in the metaphase plate causing the increase in spindle length by misalignment of k-fiber sister pairs?

   (5) It seems that in the Discussion it is implied that k-fibers can respond to severing in both focused and unfocused spindles by modulating their dynamics at both ends of the k-fibers, but in the Results section the wording is more cautious because of the difference in 'flux' in severed and unsevered unfocused spindles is not significant (Fig. 4D, blue data). It appears indeed that there is also a difference in flux between severed and unsevered unfocused spindles, but the number of data points is too small. Depending on how difficult these experiments are, it could be worth increasing the size of the data set to come to a clear conclusion, given that the data shown in Figs. 3 and 4 are quite remarkable and form the core of the study.

   (6) Can the authors exclude that the stopping of 'flux' at minus ends after severing is due to some sort of permanent damage induced by ablation? In other words, do severed spindles begin to flux again once they have regrown to their original length?

   (7) To this reader, the conceptualization of distinguishing between 'global' and 'local' effects/behavior was a little confusing, both in the title and also later in the text. The concept of 'local' regulation of k-fiber length appears to contradict the observation that k-fiber length can be regained after severing by changes in the dynamics at both ends (so at two very different locations) which is a rather remarkable finding. Maybe distinguishing between 'individual' and 'collective' k-fiber behavior could be clearer.

   (8) Can the authors exclude that some of the differences between unfocused and focused spindles could be due to altered dynein activity at kinetochores? Or due to the dynein-dependent accumulation of certain spindle proteins along microtubules towards the minus ends of k-fibers or other spindle microtubules, instead of being due to only the presence versus absence of poles? Could this be tested by ablating both poles? If this is too challenging, a discussion of these possibilities could be justified.

   Read the original source
5. 

   eLife

   Jun 17, 2023

   Reviewer #2 (Public Review):

   The mitotic spindle of eukaryotic cells is a microtubule-based assembly responsible for chromosome segregation during cell division. For a given cell type, the steady-state size and shape of this structure are remarkably consistent. How this morphologic consistency is achieved, particularly when one considers the complex interplay between dynamic microtubules, spatial and temporal regulation of microtubule nucleation, and the activities of several microtubule-based motor proteins, remains a fundamental unanswered question in cell biology. In this work by Richter et al., the authors use biochemical and biophysical perturbations to explore the feedback between mitotic spindle shape and the dynamics of one of its main structural elements, kinetochore fibers (k-fibers) - bundles of microtubules that extend from …

   Reviewer #2 (Public Review):

   The mitotic spindle of eukaryotic cells is a microtubule-based assembly responsible for chromosome segregation during cell division. For a given cell type, the steady-state size and shape of this structure are remarkably consistent. How this morphologic consistency is achieved, particularly when one considers the complex interplay between dynamic microtubules, spatial and temporal regulation of microtubule nucleation, and the activities of several microtubule-based motor proteins, remains a fundamental unanswered question in cell biology. In this work by Richter et al., the authors use biochemical and biophysical perturbations to explore the feedback between mitotic spindle shape and the dynamics of one of its main structural elements, kinetochore fibers (k-fibers) - bundles of microtubules that extend from kinetochores to spindle poles. Overexpression of the p50 dynactin subunit in mammalian tissue culture cells (Ptk2) was used to inhibit the microtubule motor cytoplasmic dynein resulting in misshapen spindles with unfocused poles. Measurements of k-fiber lengths in control and unfocused conditions showed that although mean k-fiber length was not statistically different, the variation of length was significantly higher in unfocused spindles, suggesting that k-fiber length is set locally, occurring in the absence of focused poles. With a clever combination of live-cell imaging with photoablation and/or photobleaching of fluorescently-labeled k-fibers, the authors went on to explore the mechanistic bases of this length regulation. K-fiber regrowth following ablation occurred in both conditions, albeit more slowly in unfocused spindles. Paired ablation and localized photobleaching on the same k-fiber revealed that microtubule dynamics, specifically those at the plus-end, can be tuned at the level of individual k-fiber. Lastly, the authors show that chromosome segregation is severely impaired when cells with unfocused spindles are forced to enter mitosis. The work's biggest strength is the application of an innovative experimental approach to address thoughtful and well-articulated hypotheses and predictions. Conclusions stemming from the experiments are generally well-supported, though the experiments addressing the "tuning" of k-fiber dynamics could be bolstered by additional data points and perhaps better presented. The manuscript would also benefit from the inclusion of some investigation of spatial differences in the observed effects as well as the molecular and biophysical basis of the observed feedback between k-fiber length and focused poles.

   Comments/Concerns/Questions:

   1. In the discussion, the authors acknowledge that the changes in spindle morphology resulting from p50 overexpression are likely also causing changes in the well-characterized RanGTP/SAF gradients that radiate from chromosome surfaces. Why did the authors did not include an analysis of k-fiber length as a function of positioning within the spindle? The inclusion of this data would not require more experimentation and could be added as a plot showing K-fiber length versus distance from the geometric center of the spindle (defined by the intersection of the major and minor axes perhaps?).
   2. The authors also acknowledge the established relationship between MT length and MT end dynamics, yet in their ablation studies, the average initial k-fiber length at ablation in control spindles was higher than that for k-fibers in unfocused spindles. It seems that this difference makes the interpretation of the data, particularly the conclusion that fiber growth rates differ due to the absence of focused poles, a bit tenuous. To address this, the authors should consider including plots of grow-back rates versus k-fiber length (again, this should not require additional experiments, just more analysis).
   3. As presented, the data shown in Figure 4 is confusing and does not seem very compelling. The relationship between the kymographs and time series is unclear as is the relationship between the dashed lines in the kymographs and the triangles and the plots in the 4B time series and 4C, respectively. Furthermore, it's not always clear what the triangles are pointing to (e.g. in the unfocused condition time series). The authors might want to consider reworking this figure and providing more measurements of flux following ablation in both the control and unfocused conditions. Lastly, the authors should clarify what negative displacement means.

   Read the original source
6. 

   Version published to 10.1101/2022.11.26.517738v1 on bioRxiv

   Nov 26, 2022