Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule plus-ends

Growing microtubule ends organize end-tracking proteins into comets of mixed composition. Here using a reconstituted fission yeast system consisting of end-binding protein Mal3, kinesin Tea2 and cargo Tip1, we found that these proteins can be driven into liquid-phase droplets both in solution and at microtubule ends under crowding conditions. In the absence of crowding agents, cryo-electron tomography revealed that motor-dependent comets consist of disordered networks where multivalent interactions may facilitate non-stoichiometric accumulation of cargo Tip1. We found that two disordered protein regions in Mal3 are required for the formation of droplets and motor-dependent accumulation of Tip1, while autonomous Mal3 comet formation requires only one of them. Using theoretical modelling, we explore possible mechanisms by which motor activity and multivalent interactions may lead to the observed enrichment of Tip1 at microtubule ends. We conclude that microtubule ends may act as platforms where multivalent interactions condense microtubule-associated proteins into large multi-protein complexes.

I hope you are well and I apologize for the very long delay in the peer review of your submission.
Your manuscript, "Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule plus ends", has now been seen by 3 referees, who are experts in microtubules (referee 1); biomolecular condensation (referee 2); and CryoET (referee 3). As you will see from their comments (attached below) they find this work of potential interest, but have raised substantial concerns, which in our view would need to be addressed with considerable revisions before we can consider publication in Nature Cell Biology.
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In particular, it would be essential to: A) Test hypotheses generated from the modelling predictions (Reviewer #2) B) Assess potential differences in biomolecular condensation in vitro as opposed to those on microtubules (Reviewer #2) C) Characterize biomolecular condensation behaviour on microtubule behaviour with potential to be motor-activity-driven (Reviewer #2) D) Provide non-denoised CryoET images (Reviewer #3) E) All other referee concerns pertaining to strengthening existing data, providing controls, methodological details, clarifications and textual changes, should also be addressed. F) Finally please pay close attention to our guidelines on statistical and methodological reporting (listed below) as failure to do so may delay the reconsideration of the revised manuscript. In particular please provide: -a Supplementary Figure including unprocessed images of all gels/blots in the form of a multi-page pdf file. Please ensure that blots/gels are labeled and the sections presented in the figures are clearly indicated.
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Best wishes, Reviewer #1: Remarks to the Author: In their manuscript, Maan et al. determine the role of liquid-liquid phase separation in the formation of a structure known as the microtubule +TIP comet. This comet is assembled by the "master regulator" EB1 (and its homologs in different species) and assembles a number of different +TIP proteins at the growing microtubule ends. How the different proteins interact to form the +TIP comets has so far not been fully understood. The authors use components of the fission yeast +TIP tracking system to characterise their assembly of assemblies and test the hypothesis of multivalent interactions leading of liquid-liquid phase separation in this process. The work is a powerful combination of in vitro reconstitution experiments with purified proteins, cryo-EM approaches and theoretical modelling. The authors first demonstrate that Mal3, the EB-homolog, Tip1, CLIP170 homolog, and Tea2, the kinesin-7 homolog together form condensates either with or without microtubules. The condensates are liquid, can coat microtubules, and are transported by Tea2 motors towards the plus-ends of microtubules, where they form the characteristic comets. These observations where all made under crowding conditions in cell-free in-vitro reconstitutions from recombinant proteins purified from E. coli. To determine the role of the crowding agent that was necessary to see comets in light-microscopy based reconstitution, the authors turned to cryo-EM. After solving the cumbersome problem of nonspecific protein attachment to the EM grids, the authors show that the comets formed under noncrowded conditions are similar to the ones observed with crowding agents, but are more loosely structured. This explains the need to crowing agents in the fluorescent-based assays, however proves that the structures do also assemble in the absence of crowding agents. The authors next report the striking observation that the 3 components do not accumulate stoichiometrically at the microtubule plus end changing the concentration of the motor Tea2 did not affect the concentration of Mal3, but did disproportionally affect the Tip1 concentration. This nonstochiometric accumulation of the 3 proteins is another indication that they do not form defined protein complexes, but rather assemble by multivalent interactions. In the next step the authors determine the role of different domains of Mal3 in the observed cometforming behaviour. Strikingly, all deletions that prevented +TIP tracking of Mal3 also prevented droplet formation. The importance of different protein domains for protein-protein interactions was further dissected with guest-host assays. The sum of all assays performed leads the authors to the conclusion that the sum of different molecular interactions coordinated by Mal3 are essential for the formation of the +TIP comet. Finally, the authors use molecular modelling to theoretically describe the +TIP tracking of the 3 proteins. Overall, the manuscript is well-written, the experiments are described in detail to allow the reader a good understanding of the experimental settings and the results. Figures are also of high quality. However, reading the paper as a whole it becomes obvious that different chapters have been written in a different style, which is most obvious for the modelling chapter that is by far the longest and most detailed part of the result section. This inconsistent structure of the manuscript makes it difficult to follow at places, thus some streamlining of the text would be largely profitable for the storyline. Similarly, the introduction of the paper could be more concise. There is an extensive description of the results that takes almost an entire page, which is not necessary given that similar information is given in the results and the discussion. Thus, one of the central points that needs to be addressed is the writing: the manuscript could profit from being more concise, linear and written in a form easy accessible for a general public.
Reviewer #2: Remarks to the Author: In the article entitled "Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule ends," Maan, Reese, Volkov and colleagues investigate the interactions between Mal3, Tea2, and Tip1 that give rise to condensates that form and localize at the plus end of microtubules. Using a combination of in vitro reconstitution, cryoET, and computational modeling, the authors identified two intrinsically disordered regions in Mal3; both regions were required for Tip1 localization at the plus end, but only one region was necessary for condensate (comet) formation on microtubules. The authors conclude that the combination of motor activity and multivalent interactions between Mal3, Tea2, and Tip1 drive their condensation at the plus ends of microtubules.
This article provides stunning structural insight into the Mal3 and Mal3, Tip1, Tea2 network of proteins localized to the plus-end of microtubules. Their work to understand the phase separation of Mal3 in the presence of crowding agents provides important insight into its phase behavior. In addition to these observations, the liquid like behavior of plus-end condensates is extremely important for the fields of phase separation and cytoskeletal biology. Finally, the theoretical model provides insight into a potential mechanism that can explain the experimental data presented in this manuscript. Overall, this is an excellent manuscript that provides a unique picture into mechanisms by which plus-end condensates can be regulated. However, as will be discussed below, it would beneficial for the authors to test their model using an experimental system in which they perturb both model and experiments to confirm that their model accurately describes the biological system. Otherwise, it could be argued that the presented model is simply a data-fitting algorithm rather than a bona fide model that can be used to predict plus-end condensate dynamics with other binding partners. If this concern and other minor comments can be addressed, this manuscript is a strong candidate for publication in Nature Cell Biology.
Major Comment: 1) As noted above, the authors have set up their model as a mathematical description rather than a tool to predict testable experimental manipulations. It would be of great benefit to test some of their model predictions, such as motor slowdown. The authors should perform experiments with Tea2 that has either high or low velocity along the microtubule to test whether the prediction that motor speed will alter Mal3 condensation at the plus-end.
Minor Comments: 1) In Fig 1H and video S2, it appears that condensates wet the microtubules only in the plus-end direction. This seems to suggest that condensate wetting is at least partially motor-driven rather than passive wetting as seen by condensates made by non-active matter. The authors should note this more explicitly in lines 153-156. 2) In lines 167-170, the authors posit that Plateau-Rayleigh instability is responsible for the observations in Figure 1I. This would require that the condensate on the microtubule is a single condensate that only forms discrete droplets after falling off the end of the microtubule. Another potential explanation for these observations is that discrete droplets exist on the microtubule, fall off the end of the moving microtubule, and remain discrete droplets on the substrate. Is there any additional evidence that can be used to support Plateau-Rayleigh instability? 3) In lines 226-230, the authors describe the macroscale difference between in vitro condensates and condensates on the microtubule. But what about the structure on the microscale? Based on the CryoET images, it appears that the microtubule-bound networks are like the larger scale in vitro networks within the condensates. Could this be possible? If this is the case, what are the biological implications or condensates with the same internal structure, yet scaled at different sizes? Would the authors expect the experiments with the larger condensates to be predictive of the smaller condensates on the microtubules if the internal environment of condensates is similar? 4) Starting in line 303, the authors should use the common "scaffold / client" terminology (See PMID 28225081 for further details).
Reviewer #3: Remarks to the Author: This is a very nicely designed study to gain clarity on the increased concentrations and phase separation of several microtubule plus-end proteins. The study uses a variety of methods including fluorescence, CryoET and simulations. Overall the methodology seems well designed and properly carried out, and the conclusions justified by the data. This study is likely to be of interest primarily to microtubule specialists, but as a non-specialist, I still found it interesting to read. I believe the publication to be suitable for publication, with two critical modifications. I do not believe either of these changes would require re-review of the manuscript.
First, I cannot find figure captions for the supplementary figures anywhere. While I could figure out what was going on well enough for the review, clearly this needs to be addressed.
Second, the CryoCARE method used for denoising the tomograms is quite new, and not yet widely accepted in the community. Deep learning based denoising methods can modify raw data in unpredictable and potentially undesirable ways. Certainly the procedure is acceptable for purposes of making annotation easier, if the annotations are then presented on top of the original data. However, unlike standard operations like low-pass filters which are well understood, and cannot remove discrete objects, presenting denoised data as the primary result without also presenting the original data without denoising is not acceptable. I do believe it is very unlikely that the presentation of reconstructions without denoising would alter any of the conclusions in this particular study, but sideby-side presentation of all presented data with/without denoising is absolutely necessary.

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Reviewers' Comments:
We would like to thank all three reviewers for their constructive comments, which helped us improve our manuscript. In addition to rewriting parts of the text (see for details below), we added new data to Figure 2 and Supplementary Fig. S3 (previously Fig. S2) (both a new condition and increased statistics for previously shown data). We also added a new Supplementary Fig. S2 in response to reviewer 3. Below we respond in detail to each of the reviewers' comments.
Reviewer #1: Remarks to the Author: In their manuscript, Maan et al. determine the role of liquid-liquid phase separation in the formation of a structure known as the microtubule +TIP comet. This comet is assembled by the "master regulator" EB1 (and its homologs in different species) and assembles a number of different +TIP proteins at the growing microtubule ends. How the different proteins interact to form the +TIP comets has so far not been fully understood. The authors use components of the fission yeast +TIP tracking system to characterise their assembly of assemblies and test the hypothesis of multivalent interactions leading of liquid-liquid phase separation in this process. The work is a powerful combination of in vitro reconstitution experiments with purified proteins, cryo-EM approaches and theoretical modelling.
The authors first demonstrate that Mal3, the EB-homolog, Tip1, CLIP170 homolog, and Tea2, the kinesin-7 homolog together form condensates either with or without microtubules. The condensates are liquid, can coat microtubules, and are transported by Tea2 motors towards the plus-ends of microtubules, where they form the characteristic comets. These observations where all made under crowding conditions in cell-free in-vitro reconstitutions from recombinant proteins purified from E. coli. To determine the role of the crowding agent that was necessary to see comets in light-microscopy based reconstitution, the authors turned to cryo-EM. After solving the cumbersome problem of non-specific protein attachment to the EM grids, the authors show that the comets formed under non-crowded conditions are similar to the ones observed with crowding agents, but are more loosely structured. This explains the need to crowing agents in the fluorescent-based assays, however proves that the structures do also assemble in the absence of crowding agents.
The authors next report the striking observation that the 3 components do not accumulate stoichiometrically at the microtubule plus end changing the concentration of the motor Tea2 did not affect the concentration of Mal3, but did disproportionally affect the Tip1 concentration. This nonstochiometric accumulation of the 3 proteins is another indication that they do not form defined protein complexes, but rather assemble by multivalent interactions.
In the next step the authors determine the role of different domains of Mal3 in the observed cometforming behaviour. Strikingly, all deletions that prevented +TIP tracking of Mal3 also prevented droplet formation. The importance of different protein domains for protein-protein interactions was further dissected with guest-host assays. The sum of all assays performed leads the authors to the conclusion that the sum of different molecular interactions coordinated by Mal3 are essential for the formation of the +TIP comet.
Finally, the authors use molecular modelling to theoretically describe the +TIP tracking of the 3 proteins. Overall, the manuscript is well-written, the experiments are described in detail to allow the reader a good understanding of the experimental settings and the results. Figures are also of high quality.
However, reading the paper as a whole it becomes obvious that different chapters have been written in a different style, which is most obvious for the modelling chapter that is by far the longest and most detailed part of the result section. This inconsistent structure of the manuscript makes it difficult to follow at places, thus some streamlining of the text would be largely profitable for the storyline.
Similarly, the introduction of the paper could be more concise. There is an extensive description of the results that takes almost an entire page, which is not necessary given that similar information is given in the results and the discussion. Thus, one of the central points that needs to be addressed is the writing: the manuscript could profit from being more concise, linear and written in a form easy accessible for a general public.
We thank the reviewer for the positive comments. We appreciate that the writing can be improved and have thus significantly shortened both the introduction and the modeling section. See also the response to the comment about modeling by reviewer 2 below.
Reviewer #2: Remarks to the Author: In the article entitled "Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule ends," Maan, Reese, Volkov and colleagues investigate the interactions between Mal3, Tea2, and Tip1 that give rise to condensates that form and localize at the plus end of microtubules. Using a combination of in vitro reconstitution, cryoET, and computational modeling, the authors identified two intrinsically disordered regions in Mal3; both regions were required for Tip1 localization at the plus end, but only one region was necessary for condensate (comet) formation on microtubules. The authors conclude that the combination of motor activity and multivalent interactions between Mal3, Tea2, and Tip1 drive their condensation at the plus ends of microtubules.
This article provides stunning structural insight into the Mal3 and Mal3, Tip1, Tea2 network of proteins localized to the plus-end of microtubules. Their work to understand the phase separation of Mal3 in the presence of crowding agents provides important insight into its phase behavior. In addition to these observations, the liquid like behavior of plus-end condensates is extremely important for the fields of phase separation and cytoskeletal biology. Finally, the theoretical model provides insight into a potential mechanism that can explain the experimental data presented in this manuscript. Overall, this is an excellent manuscript that provides a unique picture into mechanisms by which plus-end condensates can be regulated. However, as will be discussed below, it would beneficial for the authors to test their model using an experimental system in which they perturb both model and experiments to confirm that their model accurately describes the biological system. Otherwise, it could be argued that the presented model is simply a data-fitting algorithm rather than a bona fide model that can be used to predict plus-end condensate dynamics with other binding partners. If this concern and other minor comments can be addressed, this manuscript is a strong candidate for publication in Nature Cell Biology.
Major Comment: 1) As noted above, the authors have set up their model as a mathematical description rather than a tool to predict testable experimental manipulations. It would be of great benefit to test some of their model predictions, such as motor slowdown. The authors should perform experiments with Tea2 that has either high or low velocity along the microtubule to test whether the prediction that motor speed will alter Mal3 condensation at the plus-end.

We appreciate the comment that testable model predictions and a direct quantitative comparison between model and experiments would in principle be desirable. We however run into the problem that our experimental system is highly complex: plus-end accumulation of the three MPET network components (Mal3, Tea2 and Tip1) is a result of both motor-driven transport towards the plus end and autonomous interaction of Mal3 with the comet region near the growing MT ends. Furthermore, varying the concentration of each of the components in the assay is likely to change the balance of complex formation both in solution and on the microtubule lattice making straightforward predictions about the resulting effects on both lattice coverage and end-accumulation non-trivial.
Since we do not have enough information to model the complete system and make direct quantitative comparisons, we feel we can realistically only use modelling to gain intuition on how different assumptions about motor (Tea2) and cargo (Tip1) behavior may affect end-accumulation, complementing a series of previously published models of (single component) motor traffic jams (see e.g. Leduc et al, 2012). We specifically focus on the accumulation of the cargo Tip1 which experimentally appears to increase with motor concentration in a non-stoichiometric way (Fig. 3E). For Mal3 it is known that adding motors is not necessary to obtain end-accumulation and we furthermore show that in the presence of motors the amount of Mal3 at the tip does not increase when more motors are added (Fig.  3D). Mal3 is therefore not explicitly included in the model. Instead, its effect is indirectly included when we assume cargo molecules to form multi-component clusters.
Using our modeling efforts, we first of all argue that slow-down of motors near microtubule ends (due to effects that are specific for the comet region such as a different nucleotide state and/or molecular crowding) is a sufficient condition to accumulate cargo at microtubule ends, even if motors run off freely when they reach the very tip of the microtubule. This is a relevant feature of our experiments and complements previous models where it was shown that end-accumulation (or "spikes") may also (or in addition) result from a reduced motor detachment rate at the microtubule end (and we don't exclude that both effects play a role in our system). Changing the motor velocity on the whole microtubule as suggested by the reviewer has no effect on this phenomenon, since it is the change in motor velocity when reaching the comet region near the growing end that is responsible for accumulation. Removing the end-specific change in velocity can be achieved by growing microtubules with a non-hydrolysable analogue such as GTPgammaS (Fig. S6A). Indeed, no clear end-accumulation is observed under these conditions. Note however, that the motor density is very high on these filaments due to increased lattice affinity which may lead to the absence of "spikes" that would result from a potebtial end-specific motor detachment rate (Leduc al, Fig. 4; see also our additional experiments below).
Nevertheless, to explore in more detail the effect of a boundary between two microtubule regions on motor/cargo behavior, we performed additional experiments where we created microtubule lattices composed of three different regions with three different nucleotide states: we used stabilized GMPCPP seeds to first nucleate dynamic microtubules in the presence of GTP (as elsewhere in our paper). We then replaced the solution with a solution containing tubulin and GTPgammaS to grow long stable microtubule regions at both the plus-and minus-ends of the microtubules. We then added our three MPET components as before and followed the behavior of Tip1 when reaching the boundaries between these various segments leading to the following observations (see Figure below): -The landing rate of motor/cargo complexes was highest on the gammaS regions (consistent with our observations in Fig. 6SA

), although the overall density on gammaS was lower in these new experiments (potentially because of a different distribution of the different components between different possible complexes in solution and on the different microtubule lattices).
-While not forming steady comets with roughly constant intensity as observed in our normal assays (Fig. 1B), Tip1 clusters were able to (transiently) accumulate at growing ends even though there was no boundary between lattice types near the microtubule ends in these experiments. We assume this to be the result of an end-specific motor off-rate (see Leduc, 2012). -Tip1 was also sometimes observed to accumulate at the minus ends of the GMPCPP seeds, but only when a GDP segment at the minus end was missing (bottom row in figure below). This

shows that the boundary between a gammaS region (which mimics the GDP-Pi comet region on a normal growing MT) to a GMPCPP region (which mimics the very GTP tip of growing MT) may
also contribute to accumulation at growing microtubule ends. The same was not observed when a GDP segment was present between the gammaS segment and the seed (top row in figure below).

Figure: two-color kymographs showing transport of labeled Tip1 (turquoise) on segmented microtubules. Only the GDP segments of the microtubules are labeled (red). GMPCPP seeds (dark areas) may be flanked by GDP segments on their plus and/or minus ends.
These observations illustrate the different effects that boundaries between different microtubule lattice regions as well as lattice ends may have on motor/cargo behavior, further emphasizing the complexity of the experimental system.
In addition, we were interested in finding a simple mechanism that could explain the non-linear motordependence of Tip1 accumulation at the microtubule end. Using our model, we show that simply allowing cargo to form clusters does not lead to enhanced accumulation (compare Fig. 6B to Fig. 6C). Instead, a clustering-dependent decrease in the motor off-rate (or a clustering-dependent increase in the residence time) does lead to non-linear effects (Fig. 6DEF). Note that this is only a first natural step in increasing the complexity of the model which gives us insight in a possible mechanism behind our experimental observations. Additional complexity could be incorporated by also making the cargo offrate clustering-dependent which will add additional non-linear effects. However, as explained above, we restrict ourselves to moderate changes to existing models as the full complexity of the system is beyond reach of our modeling efforts.
Realizing all these limitations of our modelling efforts, one may wonder why to include them at all. We firmly believe however, that simple extensions of existing models that include relevant features of real experimental systems contributes to the field in a broader sense, as they may also help understand experimental systems other than the specific one described here.
To better explain the merit of our modelling efforts and what it can and cannot predict given the complexity of our experimental system, we have rewritten (and shortened) this section of the paper (also in response to the comment made by reviewer 1).
Minor Comments: 1) In Fig 1H and video S2, it appears that condensates wet the microtubules only in the plus-end direction. This seems to suggest that condensate wetting is at least partially motor-driven rather than passive wetting as seen by condensates made by non-active matter. The authors should note this more explicitly in lines 153-156.

We have made the suggested change in the text
2) In lines 167-170, the authors posit that Plateau-Rayleigh instability is responsible for the observations in Figure 1I. This would require that the condensate on the microtubule is a single condensate that only forms discrete droplets after falling off the end of the microtubule. Another potential explanation for these observations is that discrete droplets exist on the microtubule, fall off the end of the moving microtubule, and remain discrete droplets on the substrate. Is there any additional evidence that can be used to support Plateau-Rayleigh instability?
In our videos (such as video S2) we observe that the motor-driven condensates increase in size over time while maintaining an apparent spherical shape. We interpret this as evidence that a single condensate is formed.
3) In lines 226-230, the authors describe the macroscale difference between in vitro condensates and condensates on the microtubule. But what about the structure on the microscale? Based on the CryoET images, it appears that the microtubule-bound networks are like the larger scale in vitro networks within the condensates. Could this be possible? If this is the case, what are the biological implications or condensates with the same internal structure, yet scaled at different sizes? Would the authors expect the experiments with the larger condensates to be predictive of the smaller condensates on the microtubules if the internal environment of condensates is similar?
We thank the reviewer for this observation and suggestions. The internal (microscale) structure of both condensates and comets in Fig. 2) becomes clearly visible only after the denoising procedure that removes any high-resolution information from the tomograms (see also new Supplementary Fig. S2). We therefore hesitate to make conclusions about the microscale structure of the microtubule-bound comets in comparison with condensates formed in presence of crowding agents. However, to illustrate the difference between these two structures within one sample, we performed additional experiments where we first polymerized microtubules with end-tracking Mal3/Tip1/Tea2 in the absence of PEG, and then added PEG-35k + Mal3/Tip1/Tea2 (no additional tubulin) to the same sample. We also generated more data in absence of PEG to increase the statistics. These new data are added in Figure 2 and Supplementary Figures S2 (new)  We thank the reviewer and made the suggested changes.
Reviewer #3: Remarks to the Author: This is a very nicely designed study to gain clarity on the increased concentrations and phase separation of several microtubule plus-end proteins. The study uses a variety of methods including fluorescence, CryoET and simulations. Overall the methodology seems well designed and properly carried out, and the conclusions justified by the data. This study is likely to be of interest primarily to microtubule specialists, but as a non-specialist, I still found it interesting to read. I believe the publication to be suitable for publication, with two critical modifications. I do not believe either of these changes would require rereview of the manuscript.
First, I cannot find figure captions for the supplementary figures anywhere. While I could figure out what was going on well enough for the review, clearly this needs to be addressed.
We apologize for this omission which occurred due to a mistake during the submission process. The supplementary figure captions are now properly included in the Supplementary Information file.
Second, the CryoCARE method used for denoising the tomograms is quite new, and not yet widely accepted in the community. Deep learning based denoising methods can modify raw data in unpredictable and potentially undesirable ways. Certainly the procedure is acceptable for purposes of making annotation easier, if the annotations are then presented on top of the original data. However, unlike standard operations like low-pass filters which are well understood, and cannot remove discrete objects, presenting denoised data as the primary result without also presenting the original data without denoising is not acceptable. I do believe it is very unlikely that the presentation of reconstructions without denoising would alter any of the conclusions in this particular study, but sideby-side presentation of all presented data with/without denoising is absolutely necessary.
We thank the reviewer for the suggestion to emphasize the validation of results obtained using cryoCARE. In the new Supplementary Figure S2 we provide slices from denoised and non-denoised tomograms side by side with the same slices taken from tomograms processed using nonlinear anisotropic diffusion algorithm (NAD). Raw, non-denoised tomograms are also uploaded to EMDB and will be released upon the publication of this paper.

Decision Letter, first revision:
Our ref: NCB-A46790A 12th July 2022 Dear Dr. Dogterom, I hope you are well and I apologize for the delay. Thank you for submitting your revised manuscript "Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule plus ends" (NCB-A46790A). It has now been seen by the original referees and their comments are below. The reviewers find that the paper has improved in revision, and therefore we'll be happy in principle to publish it in Nature Cell Biology, pending minor revisions to satisfy the referees' final requests and to comply with our editorial and formatting guidelines.
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Minor Comments: 1) If space allows a concluding / summary sentence in the abstract would be helpful.
Our current word count for the abstract is 147. Since the maximum is 150, we did not change the abstract 2) In line 61, 'unstructured' should be 'disordered', as all proteins have some structure, whether primary, secondary, etc.

We have made this change
3) In line 282, 'truncates' should be 'truncations'.

We have made this change
Reviewer #3: Remarks to the Author: The revised manuscript addresses the issues I raised in the original review, and I believe it is suitable for publication. I have no further comments to add.

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