Author response:
The following is the authors’ response to the original reviews
Public Reviews:
Reviewer #1 (Public Review):
Summary:
Axon growth is of course essential to the formation of neural connections. Adhesion is generally needed to anchor and rectify such motion, but whether the tenacity or forces of adhesion must be optimal for maximal axon extension is unknown. Measurements and contributing factors are generally lacking and are pursued here with a laser-induced shock wave approach near the axon growth cone. The authors claim to make measurements of the pressure required to detach axons from low to high matrix density. The results seem to support the authors' conclusions, and the work - with further support - is likely to impact the field of cell adhesion. In particular, there could be some utility of the methods for the adhesion and those interested in aspects of axon growth.
Strengths:
A potential ability to control the pressure simply via proximity of the laser spot is convenient and perhaps reasonable. The 0 to 1 scale for matrix density is a good and appropriate measure for comparing adhesion and other results. The attention to detachment speed, time, F-actin, and adhesion protein mutant provides key supporting evidence. Lastly, the final figure of traction force microscopy with matrix varied on a gel is reasonable and more physiological because neural tissue is soft (cite PMID: 16923388); an optimum in Fig.6 also perhaps aligns with axon length results in Fig.5.
We thank you for your many suggestions to improve the presentation to explain our experimental results obtained. We carefully reconsidered problems you pointed out and revised the manuscripts as follows.
Weaknesses:
The results seem incomplete and less than convincing. This is because the force calibration curve seems to be from a >10 yr old paper without any more recent checks or validating measurements.
As the force calibration data, although we have indicated by the experimental system over 10 years ago, we have used the same system under appropriate maintenance. The system performance has been checked regularly and maintained. Therefore, the calibration data displayed is suitable even in the present. There is no problem with the calibration data.
Secondly, the claimed effect of pressure on the detachment of the growth cone does not consider other effects such as cavitation or temperature, and certainly needs validation with additional methods that overcome such uncertainties.
The authors need to check whether the laser perturbs the matrix, particularly local density. A relation between traction stresses of ~20-50 pN/um2 in Fig.6 and the adhesion pressure of 3-5 kPa of FIg.3 needs to be carefully explained; the former units equate to 0.02-0.05 kPa, and would perhaps suggest cells cannot detach themselves and move forward.
We have previously reported that a single pulse from a Ti:sapphire femtosecond laser amplifier can effectively generate shockwave and stress waves with minimal thermal effects. Notably, during this process, the temperature elevation at the laser focal point is sufficiently suppressed, allowing efficient force generation without causing significant heating in the surrounding area. By applying this method, we have confirmed that cell have any damage after the force loading. Therefore, this approach enables cell detachment while minimizing thermal and cavitation-induced damage to the cell. This clarification has been incorporated into the revised results section (lines 119-120). We agree with the reviewer that the presented data was insufficient for supporting the proposed model. To this end, we have performed additional experiments and analyses, which are included in the revised version of the manuscript. To examine the impact of femtosecond laser irradiation on laminin, fluorescently labeled laminin was coated onto glass-bottom dishes, and the fluorescent intensity was analyzed before and after the impulsive force loading. The result indicates that the fluorescent intensity at the laser focal point remained unaffected by laser irradiation. This finding suggests that axon detachment results from the dissociation between L1 and laminin rather than the detachment of laminin from the substrate. These data have been incorporated into Supplementary Fig. 1 and page 5 (lines 113-120). In addition, explanation of the relationship between the adhesion pressure and the traction stress has been specified in page 8 (lines 253-258).
The authors need to measure axon length on gels (Fig.6) as more physiological because neural tissue is soft. The studies are also limited to a rudimentary in vitro model without clear relevance to in vivo.
In response to the reviewer’s request, we measured the axon length on the polyacrylamide gel with stiffness comparable to brain tissue (0.3kPa). The axon length was consistently shorter on the gel on the glass under our experimental conditions, in agreement with previous findings (Abe at al., 2021). Furthermore, a biphasic relationship between axon outgrowth and laminin concentration was observed. These results suggest that the biphasic behavior of axon outgrowth identified in this study is likely to occur in vivo. We have updated the Fig. 6 and specified the result (lines 224-225) in revised manuscript.
Reviewer #1 (Recommendations For The Authors):
The force calibration curve seems to be from a >10 yr old paper without any more recent checks or validating measurements - which are essential. Effects of cavitation and temperature must be checked, and validated with additional methods that overcome such uncertainties. The authors need to check whether the laser perturbs the matrix, particularly local density. A relation between traction stresses of ~20-50 pN/um2 in Fig.6 and the adhesion pressure of 3-5 kPa of FIg.3 needs to be carefully explained; the former units equate to 0.02-0.05 kPa, and would perhaps suggest cells cannot detach themselves and move forward. The authors need to measure axon length on gels (Fig.6) as more physiological because neural tissue is soft. The studies are also limited to a rudimentary in vitro model without clear relevance to in vivo.
Thank you this reviewer for the recommendations on our manuscript. For this, we have answered above comments. Please find our response there.
Reviewer #2 (Public Review):
Summary:
The authors measure axon outgrowth rate, laminin adhesion strength, and actin rearward flow rate. They find that the axon outgrowth rate has a biphasic dependence on adhesion strength. In interpreting the results, they suggest that the results "imply that adhesion modulation is key to the regulation of axon guidance"; however, they measure elongation rate, not guidance.
Strengths:
The measurements of adhesion strength by laser-induced shock waves are reasonable as is the measurement of actin flow rates by speckle microscopy.
Weaknesses:
They only measure the length of the axons after 3 days and have no measurements of the actual rate of growth cone movements when they are moving. They do not measure the rate of actin growth at the leading edge to know its contribution to the extension rate. This is inadequate.
These studies are unlikely to have an impact on the field because the measurement of axon growth rate at short times is missing.
We thank the reviewer for understanding novelty of our study. We agree with the reviewer’s comment. Following the comment, we performed time-lapse imaging of growth cone movements and quantified the migration rate. Consistent with the length of axons, the migration rate did not exhibit a monotonic increase with increased L1CAM-laminin binding but rather displayed biphasic behavior, where excessive L1CAM-laminin binding led to a reduction in the migration rate. Notably, the biphasic migration behavior was abolished in the L1CAM knockdown neurons. We believe these results provide further support for our proposed model. This has been incorporated into new Fig.5 and page 7 (lines 209-218) of the revised manuscript. In addition, the experimental method has been added in page 13 (lines 385-391).
Reviewer #2 (Recommendations For The Authors):
This is a very weak paper because of the lack of relevant measurements to enable correlations between actual extension rate, traction force, and rates of speckle movement.
Thank you this reviewer for the critical comment on our model. we performed time-lapse imaging of growth cone movements and quantified the migration rate. From this reviewer and reviewer #3 comments, we recognized the importance of prior studies that the measurement of adhesion strength in the growth cone, traction force, the correlation between retrograde flow and outgrowth, and biphasic dependence of substrate concentration of neurite outgrowth (Please also find our response to recommendations from reviewer #3).
Reviewer #3 (Public Review):
Summary:
Yamada et al. build on classic and more recent studies (Chen et al., 2023; Lemmon et al., 1992; Nichol et al., 2016; Zheng et al., 1994; Schense and Hubbell, 2000) to better understand the relationship between substrate adhesion and neurite outgrowth.
Strengths:
The primary strength of the manuscript lies in developing a method for investigating the role of adhesion in axon outgrowth and traction force generation using a femtosecond laser technique. The most exciting finding is that both outgrowth and traction force generation have a biphasic relationship with laminin concentration.
Weaknesses:
The primary weaknesses are a lack of discussion of prior studies that have directly measured the strength of growth cone adhesions to the substrate (Zheng et al., 1994) and traction forces (Koch et al., 2012), the inverse correlation between retrograde flow rate and outgrowth (Nichol et al., 2016), and prior studies noting a biphasic effect of substrate concentration of neurite outgrowth (Schense and Hubbell, 2000).
Overall, the claims and conclusions are well justified by the data. The main exception is that the data is more relevant to how the rate of neurite outgrowth is controlled rather than axonal guidance.
This manuscript will help foster interest in the interrelationship between neurite outgrowth, traction forces, and substrate adhesion, and the use of a novel method to study this problem.
We thank the reviewer for appropriate comments and recognition of the strength to our manuscript. Regarding to these comments, we recognized the importance of prior studies that the measurement of adhesion strength in the growth cone, traction force, the correlation between retrograde flow and outgrowth, and biphasic dependence of substrate concentration of neurite outgrowth. With respecting the prior studies, we revised the introduction (lines 38-44, 61-65) and discussion (lines 272-281) in the manuscript. The references suggested by the reviewer have been added (Ref. 17, 26, 27, 31, and 35) (see also below responses).
Reviewer #3 (Recommendations For The Authors):
Overall, I found the experiments discussed in the manuscript to be excellent. My primary suggestion is to slightly expand the introduction and discussion to put this work in context better. Additionally, the writing is unclear in places and would be helped by a careful edit.
We appreciate the reviewer’s constructive critiques and would like to thank him/her for the experimental suggestions, which we have taken into account in the revised version of the manuscript. We trust that the additional modification of the text will satisfactorily address the reviewer’s concerns.
In more detail:
The introduction is well-written but could be improved by discussing how these studies build earlier work. Through the 1980s and 90s, an important question was whether growth cone guidance occurred as the result of chemical cues that altered the activity of signaling pathways or differences in the adhesion between growth cones and substrates. While there was some clear evidence that growth cones were steered to more adhesive substrates (Hammarback and Letourneau, 1986), there were also important exceptions. For example, (Calof and Lander, 1991) examined the biophysical relationship between neuronal migration and substrate adhesion and found that laminin, which tends to support rapid migration and neurite outgrowth, tended to decrease adhesion.
Thank you for critical comments to our manuscript. We have modified the introduction to discuss our understanding of the growth cone guidance, particularly regarding the role of neurite migration and substrate adhesion into introduction (line 38-40, 42-44) in revised manuscript.
To better understand the relationship between substrate adhesion and outgrowth, Heidemann's group (Zheng et al., 1994) was, to the best of my knowledge, the first paper to directly measure the force required to detach growth cones from substrates; including laminin and L1. For DRG neurons, this was ~ 1000 - 3000 dynes (i.e., 10 to 30 nN) and they noted that traction force generation is 3 to 15 times less than the force needed to dislodge growth cones. Additionally, that manuscript goes on to suggest, "These data argue against the differential adhesion mechanism for growth cone guidance preferences in culture." With the rising development of powerful molecular genetic tools and a growing appreciation of the importance of signaling pathways in neurite outgrowth (Huber et al., 2003), the field as the whole has focused on the molecular aspects of growth cone guidance, leaving many aspects of the physical process of neurite outgrowth unanswered. The strength of this manuscript is that it develops a new method for measuring growth cone adhesion forces, which reassuringly generates similar results to classic studies. In turn, it combines this with molecular genetic analysis to determine the contribution L1-LN interaction makes to the overall adhesion strength.
We will ensure that the manuscript explicitly acknowledges the significance of Zheng et al. (1994) in shaping the field and clarifies how our study expands upon these foundational findings. Following the reviewer’s suggestion we have added Zheng et al. (1994) in reference and modified discussion (line 272-281, Ref. 17) in revised manuscript.
There are also a couple of other papers directly relevant to this work. In particular, (Koch et al., 2012) measured the traction forces generated by hippocampal neurons on polyacrylamide gels. They estimated it to be ~ 5 to 10 Pa. While the overall results are similar, in this manuscript, it is reported that the forces generated by hippocampal neurons are significantly higher, in the range of 25-75 Pa. I don't have an issue with this difference, but please look at the Koch paper and see if there is some technical reason for the different estimates of traction forces. Along these lines, please note the Young's modulus of the gels used in the experiments.
As you mentioned, the traction force measured in our experiments is more than 5 times stronger than that reported by Koch et al., While the exact reason remains unclear, difference in gel-coating may have influenced the result. In the study by Koch et al., pre-coating was performed using Cell-Tak before laminin coating. in contrast, our study used poly-lysin for pre-coating. This methodological difference may have affected the measurement of traction force. However, at least, our experiments have consistently yielded reproducible results.
(Nichol et al., 2016) nicely shows an inverse relationship between RF rate and LN density at low concentrations. While the results reported here are similar, a strength of this paper is that it extends the work to higher LN concentrations.
Thank you for pointing out the relevance of Nichol et al., 2016 to our study. We agree that their study provides important insights into the relationship between RF rate and LN density at low concentrations. The novelty our study lies not only in extending the analysis to higher LN concentrations, but also performed analysis that include adhesion strength, traction force, and migration rate in the growth cone. We have included this discussion (line 259-261, Ref. 26) in revised manuscript.
My understanding is that the biphasic effect of LN in neurite outgrowth was previously established. For example, Buetter and Pittman, 1991 note a biphasic effect of LN conc on some parameters of neurite outgrowth, such as RMS, a measure of growth cone velocity, but not others, such as total neurite length. Likewise, (Schense and Hubbell, 2000) noted a biphasic effect of RGB peptides on outgrowth. In light of this, it would seem the main contribution of this paper is the finding that traction force generation has a bi-phasic relationship with LN concentration.
Thank you for your thoughtful comment. We agree that the main contribution of this study is demonstrating that the biphasic behavior of axon migration arises from the biphasic dependence of the traction force on laminin concentration. We have included this discussion (line 272-281, Ref. 31) in the revised manuscript.
Please appreciate that I'm not asking the authors to copy-paste the text above into the manuscript. Instead, the references provide a starting point for better explaining the novel contributions here. The interaction of adhesions, traction force generation, the rate of neurite outgrowth, and biophysics of growth cone guidance is a classic problem in neuronal mechanics but is far from solved. My hope is that this manuscript might inspire more interest in this problem.
Thank you for your thoughtful feedback and for highlighting the importance of better contextualizing our novel contributions within the broader field of neuronal mechanics. We appreciate your emphasis on the classic yet unresolved nature of the interactions between adhesions, traction force generation, axon outgrowth rate, and the biophysics of growth cone guidance.
We hope these revisions help strengthen the manuscript’s impact and inspire further investigation into this important problem. We appreciate your insightful comments and the opportunity to improve our work.
The text would be improved with a careful copy edit, for example:
The last sentence of the introduction currently reads, "We suggested mechanism of the axon outgrowth which depends on the density of laminin on the substrate, revealing L1CAM-laminin binding as a mechanism for the regulation of axon outgrowth." which is challenging to understand.
We appreciate the reviewer’s comment pointing out the lack of clarity in the final sentence of the introduction. To improve readability and clarity, we have revised the sentence as follows:
“In this study, we suggested mechanism of the axon outgrowth that depends on the density of laminin on the substrate, i.e. the L1CAM-laminin binding is key to the regulation of axon outgrowth..” We believe this revised version better conveys our main finding in a more concise and comprehensible manner.
Line 224 needs to be F-actin and the next sentence is difficult to understand.
Thank you for pointing this out. We have corrected "F-action" to "F-actin" to ensure accuracy (line 256). Additionally, we have revised the following sentence to improve clarity (line 256-258).
Line 232 instead of "traction force slows", did you mean the rate of retrograde flow slows?
Thank you for pointing this out. We mean to refer to the rate of retrograde flow, not the traction force itself. We have revised the wording accordingly to avoid confusion (line 266).
Line 242, shear-stress instead of share-stress.
We have corrected the typo into "shear-stress" (line 282).
Lines 255, 267, and the abstract. The paper doesn't directly address axonal guidance. It would be more accurate to replace axonal guidance with neurite outgrowth.
Thank you for your insightful comment. We agree that the term "neurite outgrowth" more accurately reflects the scope of our study, as we do not directly examine the mechanisms of axonal guidance. Accordingly, we have revised the text in Lines 273, 275, and the abstract to replace "axonal guidance" with "neurite outgrowth" to better align with the presented data and experimental focus.
Line 362, perhaps reference (Minegishi et al., 2021) here as it provides a nice explanation of the technique.
Thank you for the helpful suggestion. We have now added a reference to Minegishi et al., 2021 (line 416, Ref.35) in revised manuscript, as it indeed provides a clear explanation of the method.
