Author response:
The following is the authors’ response to the original reviews.
General comments:
You will see that many of the reviewers’ comments overlap. From our discussion with them, we agree that several of these comments should be addressed in this study, particularly comments related to the interpretation of the effect of drugs acting on the cellular cytoskeleton (reviewers #1 and #2). We also agree that the comparison of isogenic cell lines such as the mcf10a series or the 4T1 series should address some of the concerns regarding the interpretation of the mechanical fingerprint (reviewer #3). Also, certain methodological aspects should be easily clarified (reviewers #1 and #3).
We also agreed that other comments may be more difficult to address in the context of this study. This is the case for comments related to establishing a link between different mechanical signatures and different cellular functions/outcomes (Reviewers #1 and #3). One could test whether migration or proliferation is altered by changing the mechanical fingerprint, or you could simply discuss these aspects by carefully reviewing the literature to corroborate mechanical signatures with known cellular phenotypes (e.g. migration speed, adhesion, cell size...). This is also the case for comments on the influence of other cellular parameters such as molecular crowding and energy metabolism, which could be left for future work or where you could use a low dose of cycloheximide (below the level of deleterious effects) to address the effect of cytoplasmic proteins (reviewer #3).
We thank the editor for providing this helpful overview of the requested revisions. We have carefully addressed these points throughout the revised manuscript. The only difficulty was to establish the isogenic cell lines as requested. It took us over 18 months to find a source of these cells in Europe, and since then we are trying hard, but not successful to get these cells stably growing in the condition necessary for the optical tweezers experiments. As we have now spent more than 2 years on this without success, we decided to resubmit the paper without this part to not further delay this manuscript. The additional experiments and revisions have substantially strengthened the manuscript. Especially, the addition of Latrunculin A as suggested was an excellent request, as now the results regarding actin depolymerization and mechanical properties are in excellent agreement with the expected effects, as Latrunculin A is much more efficient in depolymerizing actin than cytochalasin B. The major changes are summarized below, followed by a detailed point-by-point response to all reviewer comments.
General changes
(1) Repeated measurements on wild-type HeLa cells.
(2) Repeated all Cytochalasin B and Nocodazole experiments and increased the number of analyzed cells to approximately 60 per condition.
(3) Performed additional experiments using Latrunculin A and combined Latrunculin A + Nocodazole treatment.
(4) Performed immunostainings for all cytoskeletal perturbation conditions (WT, Cytochalasin B, Latrunculin A, Nocodazole, Cytochalasin B + Nocodazole, and Latrunculin A + Nocodazole).
(5) Refined the rheological analysis procedure and expanded the methodological description.
(6) Revised the manuscript text throughout and expanded the discussion of limitations and biological interpretation.
Public Reviews:
Reviewer #1 (Public Review):
A limit of the paper is that the biological mechanisms by which intracellular mechanics is modulated (e.g. among cell types) remains unexplored and only briefly discussed. Yet this limit is greatly offset by the rigor of the approach.
We thank the reviewer for this positive assessment and agree that a more extensive discussion of the biological mechanisms underlying the observed mechanical fingerprints strengthens the manuscript. We have substantially expanded the Discussion and Conclusion sections to address potential contributions of cytoskeletal organization, intracellular transport, molecular crowding, and metabolic state. In addition, we now discuss the relationship between the identified mechanical phase space and known cellular phenotypes where appropriate, while explicitly outlining the limitations of the current study and the need for future investigations linking intracellular mechanics to cellular function.
Reviewer #2 (Public Review):
The most difficult part of the method is the part with actin polymerization inhibition with cytochalasin B. The data shows that viscoelastic parameters as well as active energy parameters are unaffected by cytochalasin B. It is reasonable to expect that elasticity will reduce and fluidity will increase upon application of such a drug. The stiffness-reducing effect was observed only when CB was used with nocodazole most likely because of phagocytosis of the bead, which is governed by microtubule. The use of other actin-depolymerizing drugs such as latrunculin A would be needed to test actin’s role in mechanical fingerprints. If actin’s role is only explained by accompanying microtubule inhibition, it is not a convenient system to directly test the mechano-adaptation process.
We thank the reviewer for this important suggestion. To strengthen the interpretation of the actin perturbation experiments, we repeated the Cytochalasin B measurements with an increased number of cells and performed additional experiments using Latrunculin A, a mechanistically distinct and more potent actin-depolymerizing compound. Together with complementary immunostaining experiments, these additional data reveal distinct contributions of the two major cytoskeletal systems to the intracellular mechanical fingerprint. Whereas actin depolymerization primarily affects intracellular stiffness and fluidity, microtubule depolymerization has the strongest effect on intracellular activity while also contributing to cellular softening. These additional experiments provide a substantially clearer interpretation of the respective roles of actin filaments and microtubules in shaping the intracellular mechanical fingerprint.
Depolymerization of MT with nocodazole did not reduce the solid-like property A. Adding discussion and comparison with other papers in the literature using nocodazole will be helpful in understanding why.
We thank the reviewer for this suggestion. We have expanded the discussion and now compare our observations with previous AFM studies investigating Nocodazole treatment. While AFM measurements of cortical mechanics often report little change or even increased stiffness after microtubule depolymerization, our intracellular measurements reveal pronounced softening and strongly reduced intracellular activity. We now discuss that this difference likely reflects the distinct intracellular mechanical compartment probed by intracellular microrheology compared with cortical AFM measurements.
Overall, the usefulness of the concept of mechanical fingerprints and comparisons with other cell mechanics studies (from other groups) will make this manuscript stronger.
We thank the reviewer for this suggestion. Throughout the revised manuscript we have strengthened the comparison of the mechanical fingerprint with previous literature. In particular, we now discuss the cytoskeletal perturbation experiments in the context of published AFM studies, compare the observed mechanical differences between cell types with previous measurements where available, and expand the discussion of the biological interpretation and limitations of the proposed mechanical fingerprint.
Reviewer #3 (Public Review):
The importance of the mechanical fingerprint is diluted due to some missing controls needed for biological relevance.
We thank the reviewer for raising this important point. To strengthen the biological interpretation of the mechanical fingerprint, we performed substantial additional experiments, including repeated cytoskeletal perturbation measurements with increased sample sizes, additional Latrunculin A experiments, and complementary immunostaining analyses. We also expanded the discussion to address the influence of factors beyond the cytoskeleton, including molecular crowding and metabolic state, and explored possible relationships between the proposed mechanical phase space and cellular phenotypes. While we agree that future studies using well-controlled isogenic model systems will be required to establish direct links between intracellular mechanics and biological function, we believe that the additional experiments and expanded discussion substantially strengthen the biological relevance of the present study.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
A caveat of the general methodology, which is partially acknowledged in the MS is that beads are endocytosed and likely end up in specific lysosomal compartments. Therefore, it is not clear whether the mechanical fingerprint fully represent the material properties of bulk cytoplasm, and not something more specific to lysosomal organelles. For instance, lysosome motion may be largely driven by motors moving along MT cytoskeletal track, and the extracted effective energy may as such not fully represent the crowding and effective active temperature of the cytoplasm. This limit certainly affect the interpretation of the results in other cell types, in which membrane trafficking and cytoskeletal organization may vary largely. I believe it would be very important to outline this limitation of the work and discuss it in light of the results obtained throughout.
We thank the reviewer for pointing out this important limitation, which was not sufficiently addressed in the original manuscript. We have now acknowledged this issue throughout the manuscript and added a limitation section to the conclusion to clarify that our findings specifically relate to internalized objects surrounded by a membrane and therefore primarily reflect the properties of membrane-bound organelles in the 1 µm size regime, rather than the bulk cytoplasm as a whole.
We consider this focus on membrane-enclosed intracellular objects to be biologically relevant and interesting in its own right. Alternative approaches for introducing tracer particles, such as microinjection or particle guns, are generally more invasive and less reproducible. We therefore deliberately focused on phagocytosed beads as a minimally perturbative and robust experimental system in this study.
The evolution of the mechanics in Hela Cells using cytoskeletal drugs in interesting, but I was confused by the fact that authors interpret the effect of cytochalasin solely on the cortex. As they are probing intracellular rheology, variations (or lack thereof) may rather reflect bulk F-actin networks? Also the compensation mechanism is interested, but it would need to be strengthened by immunostaining for instance, to support the claim, that microtubule depolymerization enhances F-actin networks.
We thank the reviewer for this important comment. To elaborate on the effect of cytoskeletal filaments, we extended our analysis by repeating the experiments, increasing the number of samples, and investigating the effect of an additional drug, Latrunculin A. Additionally, we conducted immunostaining with subsequent confocal imaging to deepen our understanding of the effect of the respective drugs. The additional experiments reveal that actin and microtubules contribute differently to the fingerprint. Actin depolymerization primarily affects intracellular stiffness and fluidity, whereas microtubule depolymerization has the strongest effect on both mechanics and intracellular activity. Combined perturbation produces the largest overall effect. Based on these additional data, we no longer invoke the compensation mechanism proposed in the original manuscript. While interactions between the actin and microtubule cytoskeleton have been reported previously, our immunostaining experiments do not provide evidence for a compensatory increase in actin organization following microtubule depolymerization. We have therefore removed this interpretation from the revised manuscript and replaced it with a discussion based on the newly acquired perturbation and imaging data.
The final figure using principal component analysis is very interesting, but it would be important to link this to phenotypic signatures of the different cells. Could the authors try to link resistance, fluidity and activity to the different functions/behavior of cells? For instance, some of these cells are migratory but some may move much faster than others, and it would be very interesting to correlate the degree of activity or fluidity with speed of migration, or cell shape/size/contractile state for example.
Indeed, this is an important point. Establishing direct links between the mechanical fingerprint and functional cellular properties such as migration, contractility, proliferation, or morphology would substantially strengthen the biological interpretation of the identified phase space. We carefully considered this suggestion and explored several approaches to relate the measured mechanical parameters to cellular phenotype. However, obtaining directly comparable quantitative functional data across all investigated cell types proved challenging. Parameters such as migration speed, adhesion, and contractility depend strongly on experimental conditions, including substrate properties, assay design, and culture conditions, making literature values difficult to compare across studies. To address the reviewer’s concern, we expanded the discussion and incorporated comparisons to available literature where appropriate. For example, previous studies have reported higher migration rates for HeLa cells compared with MCF7 cells, which is qualitatively consistent with the higher intracellular activity observed in HeLa cells. However, due to the limited comparability and availability of quantitative functional data across the investigated cell types, we refrained from performing a formal correlation analysis. In addition, we grouped the investigated cell lines according to several broad phenotypic classifications, including epithelial/mesenchymal character, cancer status, metastatic potential, and migratory potential, and examined their distribution within the proposed phase space. While this exploratory analysis provides additional biological context, it did not reveal robust relationships that could support definitive conclusions regarding structure–function relationships. We therefore agree with the reviewer that establishing direct links between intracellular mechanical fingerprints and cellular function represents an important next step. To this end, future studies will combine intracellular rheological measurements with independently quantified functional assays, ideally in well-controlled isogenic model systems.
Reviewer #2 (Recommendations For The Authors):
The study needs more thorough validation against known technology (such as AFM) or literature, e.g., rheological change upon the same drugs used in the current study.
We thank the reviewer for this suggestion. We have expanded the discussion of the cytoskeletal perturbation experiments and now compare our observations to previous AFM studies and related literature on cytoskeletal mechanics. Consistent with AFM measurements of cortical mechanics, actin depolymerization using Cytochalasin B or Latrunculin A resulted in a reduction of cellular stiffness. In contrast, microtubule depolymerization produced effects that differ from many AFM studies, which report either no change or an increase in cortical stiffness following Nocodazole treatment. We now explicitly discuss that this discrepancy likely reflects the different mechanical compartments probed by the two techniques. AFM predominantly measures the actin-rich cell cortex, whereas our intracellular microrheology measurements probe the mechanical environment experienced by membrane-bound intracellular particles. We therefore interpret the differing response to microtubule depolymerization as evidence that intracellular active mechanics and cortical mechanics can be influenced by distinct physical mechanisms. These comparisons have been incorporated into the Results and Discussion sections of the revised manuscript.
Page 8: Citation to Fig. 3a is missing before mentioning Fig. 3b.
We revised the manuscript to ensure that all references are given in an appropriate order.
Proper uses of hyphens are recommended to avoid confusion. For example, ’a yet not understood change’ can be written as ’ a yet-not-understood change’.
We thank the reviewer for this suggestion. We carefully revised the manuscript to improve the use of hyphenation and compound modifiers throughout the text. The specific example highlighted by the reviewer, as well as similar constructions, have been corrected to improve readability and avoid ambiguity.
Reviewer #3 (Recommendations For The Authors):
As it reads, sinusoidal waves are applied sequentially from 1- 1024Hz. Please clarify if amplitude is the same for each frequency, also how many frequencies are used? On this point, due to perturbations due to alterations in pre-stress, are the orders of frequencies randomized?
We thank the reviewer for pointing out this ambiguity. We have revised the manuscript to provide a more detailed description of the active microrheology protocol. Specifically, we now state that all measurements were performed using a constant trapping-laser oscillation amplitude of 200 nm and that the applied frequencies were 1, 2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 Hz. The frequencies were applied sequentially in increasing order and were not randomized. This information has now been added to the manuscript.
How many beads are probed in a given cell?
We thank the reviewer for this question. We have clarified this point in the Methods section and now explicitly state that only a single phagocytosed probe particle was analyzed per cell. Of course, many different cells, and hence beads, have been analyzed per cell type.
Is the graph in 1 c G’, G” per cell or average of many cells?
We thank the reviewer for pointing out this ambiguity. In the original version of the manuscript, Figure 1b showed data from a representative cell, whereas Figure 1c displayed an average over multiple cells. To avoid confusion, we revised Figure 1 and now show representative data from a single measurement throughout the analysis workflow (Figure 1c,e,f).
Figure 1e is quite nice, however, is there an equivalent performed in a nonlinear ECM such as collagen for comparison, in a similar vein can the equivalent be calculated for cells with/without treatment with low doses of cycloheximide to reduce protein synthesis? Yes, cytoskeletal elements are important for cell mechanics, but cytoplasm crowding is often an overlooked factor.
We thank the reviewer for this important suggestion, and we are glad that the reviewer likes figure 1e. Regarding non-linear ECM, we have not done such experiments using optical tweezers. Collagen is a highly heterogeneous material and using the small deformations that we can obtain using the optical tweezers, our access to the non-linear contributions is rather limited.
However, we agree that factors beyond the cytoskeleton, including molecular crowding and protein content, can make important contributions to intracellular mechanics. While investigating these effects experimentally, for example through cycloheximide treatment, would be highly interesting, such studies were beyond the scope of the present work.
The primary focus of this study was to establish and validate a mechanical fingerprint for intracellular active microrheology and to investigate how this fingerprint responds to perturbations of the cytoskeleton. The additional experiments performed during revision therefore concentrated on strengthening the interpretation of the cytoskeletal contributions.
At the same time, we agree that molecular crowding represents an important alternative mechanism influencing intracellular mechanics. We have therefore expanded the Discussion and Conclusion sections to explicitly acknowledge this limitation and now cite recent studies demonstrating strong effects of molecular crowding on intracellular rheology (Umeda et al,. 2023, Ebata et al., 2023). We further discuss that, besides cytoskeletal organization, metabolic state, intracellular transport, and molecular crowding are likely contributors to the observed mechanical fingerprint.
The biggest issue is the interpretation of the different factors as each of these cells have different energetic needs. The comparison between cancer cells with different aggressiveness, immune and epithelial cells. For example, some types of cancer cells will be dominated by glycolysis vs oxphos, which will influence both the cytoplasmic and nuclear mechanics? It would be useful to carefully assess factors not restricted to
(a) Cytoskeleton
(b) Protein synthesis
(c) Metabolic state
For similar lines and/or cells where there are lineages that are either more metastatic in cancer, normal counterpart or drug resistant in an effort to link the fingerprint to a biological output. Specifically, is migration, proliferation, survival correlated with the measurements. The reviewer is sensitive to the technical difficulties of the experiments. However, the interpretation and importance of the mechanical fingerprinting requires additional work as mentioned above.
We thank the reviewer for this thoughtful comment. We agree that intracellular mechanics is likely influenced by a broad range of biological factors beyond the cytoskeleton, including metabolic state, molecular crowding, intracellular transport processes, and protein synthesis. We also agree that the biological significance of the mechanical fingerprint would be strengthened by establishing direct links to functional cellular outputs such as migration, proliferation, or survival. To address the first point, we have expanded the Discussion and Conclusion sections of the manuscript to explicitly acknowledge that the observed fingerprint is unlikely to be determined solely by cytoskeletal organization. In particular, we now discuss the potential contributions of metabolic state, intracellular transport, and molecular crowding, and cite recent studies demonstrating the importance of these factors for intracellular mechanics. To address the second point, we explored several strategies to relate the measured mechanical fingerprints to cellular phenotype. We expanded the discussion of available literature, including examples where mechanical properties and migratory behavior appear qualitatively consistent. In addition, we grouped the investigated cell lines according to broad biological characteristics, including epithelial/mesenchymal character, cancer status, metastatic potential, and migratory potential, and examined their distribution within the proposed phase space. While this exploratory analysis provides additional biological context, it did not reveal robust relationships that would support definitive conclusions regarding structure–function relationships. We therefore agree that establishing direct links between intracellular mechanics and cellular function represents an important next step. Such studies will require quantitative functional assays performed under controlled and directly comparable conditions, ideally using well-defined isogenic model systems. We now discuss these limitations and future directions explicitly in the revised manuscript.
