Interplay between Convective and Viscoelastic Forces Controls the Morphology of In Vitro Paclitaxel-Stabilized Microtubules

Round 1

Reviewer 1 Report

Reviewer Comment

Manuscript ID: crystals-636705

Title: Interplay Between Convective and Viscoelastic Forces Controls the Morphology of in vitro Paclitaxel-Stabilized Microtubules

Authors: Eftihia Barnes, Xin Guan, Erik M. Alberts, Travis L. Thornell, Christopher M. Warner, and Kevin R. Pilkiewicz

The manuscript by Barnes et al. studies the morphology of in vitro Paclitaxel-stabilized microtubules (PTX-MT) polymerized in vitro. They use several techniques to find out what mechanism is followed by the PTX-MT solutions to form the morphologies they exhibit through their birefringent patterns. They claim that the birefringent behavior of the PTX-MT crystals arises due to the combination of convective and viscoelastic forces. After reading the manuscript, I have listed up some critical issues that would concern readers with the quality of the work. 

MAJOR ISSUES:

The novelty of the work is vaguely stated in the Introduction To investigate mechanisms for birefringent stripes in polymerized MT solutions “in more detail” is not convincing enough. Moreover, the importance of studying, particularly the effect of PTX, is not adequately addressed. Recent attractions on the MT stabilization (Saez-Calvo, G. et al., Cell Chem. Biol. 2017, Nasrin S.R. et al., BBRC, 2019) are useful for the present study to impact their acceptance in a broad array of disciplines. Text on MT stabilization in lines 93-106 is more appropriate for the Introduction section rather than Results and Discussion. The 5 stages that evolve the pattern of the MT-network is also provided in the Introduction. I recommend providing it only in the discussion. AFM studies:

The height of the microtubules does not correspond to the literature value of ~25 nm. Several works have already reported the thickness of paclitaxel stabilized MTs (Keya, J.J., Sci. Rep., 2017; Ganser, C. Nanoscale, 2019) that correspond to the literature value. Therefore, unless authors give a reasonable explanation, this result may not be acceptable.

Is the FWHM of MTs double for all filaments? Authors state such an increase occurs because of sample-tip convolution effect and also drying. Therefore, how the effect of PTX on the morphology of the polymerized MTs is gauged in this study is not clear to me. It appears to me the MT samples underwent adverse handling that caused the extreme damage, depolymerization, and opening up of their cylindrical structure.

What do longer polymerization timeframes (page 3, line 117) refer to? Does it correspond to the 12h incubation of tubulins in the presence of paclitaxel for polymerization? The authors should clarify. On the other hand, if it means the same PTX-MT stock in Figure 1a or 1c was observed after 12 h, and the result was shown in Figure S1a and b respectively, then there should be severe concern with the efficiency of the MT stabilization with paclitaxel because appropriate concentration of paclitaxel should be able to keep MTs stable for several days.

The study of the gelation of the PTX-MT sample showed that the gelation occurred at ~6.5 min. It would be interesting to check if other observations are consistent with such findings, which the authors do not confirm. Time intervals in the other cases are much longer. The temperature (perhaps 37 °C ) at which the measurement was carried out should be mentioned in the relevant text. POM observations shown in Figure 3 are explained somewhat reasonably though the figure caption seems confusing. Scale bars in the POM images would be helpful for understanding the observation scale. I recommend authors to add one more row in the top of the figure showing the orientation of the cuvettes in their respective heating sources or anyhow providing complete information of the orientation of the cuvette or the temperature gradient in the MT solution.

The morphological differences of the MT solutions clearly result from the temperature of the environment surrounding the cuvettes. I request authors to consider here the heating of the solutions in different conditions and find their correlation with morphologies.

The images would be more comfortable to comprehend if schematics of cuvette orientation were provided.

The higher magnification POM images of the solution on the horizontal hotplate and vertical hotplate also show the difference in the morphology apparently due to the direction of the temperature gradient. Why bubbles are seen in Figure 4 but not in Figure 5 may use some discussion. How the bubbles arise and what is their fate may be stated.

The images in Figure S6, 5, and S7 taken after 15 min, 3.5 h, and 12 h show the morphology of the system is related to the thermal equilibrium when the cuvette is kept vertically over the hotplate. It is interesting to find that the size of the MT domains is varied due to the cuvette orientation, which may be exploited in its relevant applications. Unfortunately, not much attention is given to such observation. 

A comparison of POM studies of the same cuvette orientation (vertical) in different heating apparatus (hot plate or incubator) is missing.

The study did not find well oriented MT bundles observed by Kakugo, A. et al., JACS 2009 (ref 30), Shikinaka, K., et al., Soft matter, 2015, Liu J.H., et al., Actuator B-Chem., 2019 (ref 49), I am interested in knowing the factors that may have caused such difference. I am having a problem in agreeing to the statement “MT polymerization kinetics have a strong dependence on the amount of PTX present in the solutions, with more PTX content leading to faster polymerization, consistent with the earlier rheological experiments.” (Line 251-253) and therefore request authors to explain. Paclitaxel serves for stabilizing MTs but not polymerize them. The “small” particles, movement of which is monitored, need further information (particle size, material, density, and so on.) It is interesting to find how high PTX content causes increased viscosity, and thus, the particle ceases to move faster. To better illustrate the finding, authors may make use of the software ImageJ (plugin: MTrackJ) that provides the chance to track a moving particle in space with time and present it in a plot that should show displacement of the particle with time. With time how the MT polymerization with different PTX concentrations are changed is an interesting observation. I appreciate how authors describe each phenomenon taking place with time. The PTX-MT polymerization on a vertical hotplate is similar to the system in reference 30; I am curious to know how their observation was different/novel.

What happens if the cuvette is kept in the horizontal orientation is not discussed. 

The 5 stage interpretation of PTX-MT morphology in the vertical cuvette is acceptable to me. But there seems to be no explanation on the difference of the morphologies inside the horizontally oriented cuvette. I strongly recommend authors in providing a schematic illustration that explains the speculation authors make in explaining the mechanism at the end of the discussion. While the approach by changing the orientation of cuvettes, using different PTX concentrations, etc., to investigate the mechanism is interesting, authors could not fully exploit their observations in the interpretation. I recommend carefully correlating the results form each observation and summarizing the findings. The combination of convective and viscoelastic forces are stated to give rise to the birefringent patterns. Is there a competition between the forces? If so, what causes them to arise?

MINOR ISSUES:

There are a few more issues I would like the authors to care of:

Many sentences are too long to comprehend. For example, the very first sentence of the Abstract is 37 words long. This becomes difficult for the reader to understand the sense of the statement. There are even sentences with 41 or 47 words. I request authors to consider the readability of their manuscript while revising. MT polymerization is optimum at 37 °C; why the polymerization was carried out at 41°C is not clear to me. Therefore, the explanation of this will be appreciated. The two species under study should be addressed as PTX-MT and PTX free-MT uniformly throughout the text. Some odd grammatical mistakes appear throughout the text, which should be taken care of. Most of the references cited in the manuscript are old. I recommend the authors citing some latest work so as to picture the present condition of the relevant research. Image sequences are not followed in the text. Mention of Figure 1 c comes before Figure 1 b, 3f before 3b,c,d, or e. and so on. Figures need attention. The font size of the labels (1,2 ...) of MTs in the AFM images is too small. Captions should accurately state what is shown in the figures for ease of readership. Example: Figure 1. AFM imaging of (a) PTX-MTs and (c) PTX-free MTs at 30 min. of what?, Figure 2. Evolution of storage modulus (G’) and loss modulus (G”) as function of time for PTX-MTs and PTX-MTs? Careful in using abbreviations. Line 35 (GTP) but line 45 guanosine triphosphate (GTP)?, line 68 (PTX appears for the first time in the main text), line 117 (microtubule), line 129 (PT-MT), line 233 (microtubules), line 311 (Taxol), etc. I could not find captions for the supporting videos anywhere.

Overall, I find the study interesting and essential for understanding the mechanism in the PTX-MT crystals.

Author Response

Please see attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

This study was well written and had large amount of data. This study dissects the effects of taxol, a microtubule polymerizer, which is commonly used for cancer chemotherapy. This paper used biophysical approach and provided careful analysis. This work is suitable for Crystals, but it requires to address following concerns.

Major concern,

Recent study shows that clinical concentration of taxol in cancer patients is 1~10 nM range, which is at least 50-2500-fold lower than concentration used in this study. How about the effect for polymerization, morphology, birefringence if the clinical concentration of PTX is used? Authors clearly showed taxol promotes both microtubule nuclearization and growth. However, results in this paper did not separate the affect for nuclearization and growth. Authors should test the PTX-effect when the seed (skip nuclearization step) is provided. Authors also should test effect of PTX with clinical dose when the microtubule seed is provided. Although the lower dose of taxol may not work well for microtubule nuclearization, it may promote growth similar to the higher dose of taxol.

Minor concerns,

Line 40: miss typo in citation number. Figure 1. Figures should be called a, b, c, d…sequentially in the text. Line124-127: Authors should explain more details like Line137-144. Figure 3: same as above 2) Line 204: remove “very”. Line 230: “he” is not appropriate.

Author Response

Please see attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Manuscript ID: crystals-636705

Title: Interplay Between Convective and Viscoelastic Forces Controls the Morphology of in vitro Paclitaxel-Stabilized Microtubules

Authors: Eftihia Barnes, Xin Guan, Erik M. Alberts, Travis L. Thornell, Christopher M. Warner, and Kevin R. Pilkiewicz

The revised version of the manuscript by Barnes et al. comes in excellent shape. I thank the authors for taking all the comments seriously and putting efforts to work on them. Although the line or page numbers mentioned in the answers did not match the revised manuscript, however, I could find significant improvement in the quality of the manuscript. The introduction now clearly reflects the motivation and the concept of the work. The revised Figure 3 is much easier to comprehend. The results are now all well correlated and explained nicely in the discussion. I would like to especially appreciate the self-explaining scheme showing the proposed schematic of the morphological evolution of PTX-MT solution polymerization in different orientations.

The newly added Figure S10 (a) and (c) can clearly refer to the effect of higher PTX content in samples. However, I suggest the authors show both cases in the same graph, i.e., particle trajectories for both cases in one graph and time-displacements for both cases in one graph. This will ensure the same scale range for both instances and give a direct comparison. In regard to the time-displacement plot, the authors seem to have misunderstood my comment. I expect the graph to show a continuous increment in the displacement of the particle from its starting point. For slower particle, the slope of the linear increment is less steep. The pauses or stops of the particle should be indicated by horizontal traces parallel to the x-axis. This should be possible to elucidate from the data already provided. The starting point should be (0,0), next simply add up the displacement with previous y-values. Such modification would ensure easier understanding of the concept.

Overall, my concerns have been adequately addressed. Therefore, it is my pleasure to recommend this manuscript as acceptable for the journal Crystals.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

This revised paper was significantly improved in clarity for readers and also had extraordinary amount of data. I recommend it for publication in Crystals.

Author Response

Reviewer 2 was satisfied with Round 1 revisions.