Author response:
The following is the authors’ response to the original reviews.
We have addressed all the concerns and recommendations by the reviewers, in particular, the requested control experiments using alternative super-resolution microscopy approaches and analysis of the data using Voronoi tessellation in addition to DBSCAN, as requested by reviewer 3. We also provide additional data on tensin3 as suggested by reviewer 2. Finally, to provide a first insight on the role of mechanical forces in the distribution of integrin nanoclusters inside FAs as recommended by reviewer 1, we have performed experiments at different cell seeding times where it is known that FA maturation over time requires mechanical forces.
Public Reviews:
Reviewer #1 (Public review):
Summary:
In recent years, it has become increasingly evident how beautifully intricate IAC are at the nanoscale. Studies like the one presented here that shed light on the precise inner organisation of IAC are thus quite important and relevant in order to obtain a better in-depth understanding of IAC functioning and the contribution of different integrin subtypes to cell adhesive and mechanotransductive processes.
Interestingly, the authors found a distinct localisation of α5β1 and αvβ3 integrin nanoclusters within focal adhesion of human fibroblasts, with α5β1 integrin nanoclusters being at the periphery of IAC and αvβ3 integrin nanoclusters randomly distributed. Furthermore, a surprisingly high percentage of inactive integrins within IAC and relatively low spatial integrin colocalisation with adaptor proteins has been shown.
Strengths:
This is a very thoroughly performed STORM-based assessment of the nanodistribution of α5β1 and αvβ3 nanoclusters within IAC (and outside). The image quality is outstanding, and the authors have meticulously executed the experiments and the image analyses.
We are grateful to the reviewer for acknowledging the strengths of our study.
Weaknesses:
The only weakness is maybe that the manuscript remains descriptive. However, the high quality of the "description" of the nano-organisation of IAC by this scrupulous study is really important to better understand the inner workings of IAC. It provides a very solid foundation to look deeper into the (patho)physiological implications of this organisation, see recommendations (which are rather suggestions in this case).
We thank the reviewer for their feedback and have addressed their recommendations in our updated manuscript and accompanying reply (see recommendations to the authors). In summary, we have now performed experiments at different seeding times as FA maturation requires mechanical forces, and enquired whether forces might play a role in establishing the spatial distribution of the two different integrins within more mature IACs. The results are now shown as new Fig. 2 and discussed in pages 9 and 10. In addition, in order to get a first insight into the biological implications of our findings we performed dual-colour super-resolution experiments of tensin-3 and α<sub>5</sub>β<sub>1</sub> in FAs, as tensin-3 has been implicated in fibronectin fibrillogenesis. The results are now shown in Fig. S8 and we discuss their potential implications in pages 22 and 23 of the revised manuscript (see more details in the reply to the recommendation to the authors).
Reviewer #2 (Public review):
Summary:
In this study, dual-color super-resolution microscopy analysis was performed to study the co-operation between integrins and focal adhesion proteins in human fibroblast cells. The study focused on two integrins which have been previously found to be mainly responsible for focal adhesions, namely α5β1 and αvβ3.
Specifically, the study tried to shed light on the nanoclustering of integrins in focal adhesions.
In the current study, more integrin nanoclusters were observed in focal adhesions compared to other cell-matrix adhesion structures. The study revealed that both α5β1 and αvβ3 form nanoclusters, and those appear segregated from each other. While αvβ3 nanoclusters organize randomly inside focal adhesions regardless of their activation state, α5β1 nanoclusters, and particularly the nanoclusters containing β1-integrin in active conformation, preferentially organized at the edges of focal adhesions. The nanoclusters formed by each integrin were similar in size.
Cytoplasmic adapter proteins appeared less in nanocluster assemblies, suggesting that integrin nanoclusters are also forming without the studied cytoplasmic adapter proteins (talin, vinculin, paxillin). Active integrins were identified with the help of conformation-specific antibodies, and this enabled us to study the colocalization between integrins and their cytoplasmic adapter proteins. This analysis revealed that activated integrins are strongly engaged with adapter proteins.
Strengths:
The study stems from the thorough computational modelling of the nanoclusters, which enables quantification of the behavior of the clusters, including their mesoscale distribution.
The study strengthens the view that α5β1 and αvβ3 have specific functions in focal adhesions, α5β1 nanoclusters localizing preferentially on focal adhesion edges. The study also revealed that nanoclusters localized at the edges of focal adhesion were enriched for talin and paxillin but not for vinculin.
Analysis of adaptor protein nanoclusters (paxillin, talin, and vinculin) revealed that all adapter protein nanoclusters studied here close to active β1 nanoclusters are enriched on the focal adhesion edge region, whereas integrin adaptor nanoclusters far from active β1 appear to be more uniformly distributed.
Importantly, the current study suggests that integrin subtype-specific nanoclusters are not only present at an early stage of adhesion formation, but integrin nanoclusters remain segregated from each other also in mature focal adhesions, maintaining their sizes and number of molecules.
Interestingly, the study revealed that selected cytoplasmic adaptors (paxillin, talin, and vinculin), also form nanoclusters of similar size and number of single molecule localizations as the integrins, regardless of whether they locate inside or outside focal adhesions. The adapter nanoclusters are enriched in the focal adhesion "belt", colocalizing with the active α5β1 integrin nanoclusters.
We are grateful to the reviewer for acknowledging the strengths of our study.
Weaknesses:
The current study is highly dependent on the antibodies. It is possible that antibodies containing two binding sites for antigen influence the nanoscale organization (and also activation) of the receptors. Control experiments to study the possible contribution of antibodies to the measured outcome should be performed to verify the main findings. One possible approach could be to use fluorescently tagged integrins available. Alternatively, integrins (or adapter proteins) could be tagged with a small ligand and detected using a monovalent binder.
We understand the concern of the reviewer regarding the use of antibodies for imaging. Nevertheless, we would like to clarify that antibody labelling has always been performed after cell fixation, precluding potential cross-linking artefacts due to protein mobility and avoiding unwanted receptor activation.
Nevertheless, and although it is highly unlikely to happen in fixed cells, there could be two potential sources of antibody (Ab) labelling artefacts. As the reviewer noted, a primary Ab containing two binding sites could bind to two adjacent proteins (within ~10 nm from each other), potentially underestimating the stoichiometry of the nanoclusters, i.e., number of receptors or proteins per nanocluster. However, in our manuscript we never attempted to provide an estimation of the nanocluster stoichiometry, as it is highly challenging (and prone to artefacts) to provide quantification of the number of proteins using super-resolution-based single-molecule localisation methods which rely on the stochastic blinking of individual fluorophores.
A second source for potential artefacts comes from the use of the secondary Ab, which (albeit unlikely) could bind to two different primary Abs. To exclude this potential artefact, we performed super-resolution imaging using DNA-PAINT as a different imaging strategy. In this case, the DNA docking site is site-specifically coupled to one camelid single-domain Ab (sdAB), having a much smaller size as compared to a secondary Ab, reducing therefore linkage error and increasing the accessibility of primary Ab-labelled proteins. These new data are included now in Fig. S4. As can be observed, no differences in terms of nanocluster sizes and/or compositions were observed for any of the proteins investigated using DNA-PAINT as compared to our initial STORM data. These control experiments thus rule out any potential artefacts introduced by the secondary Ab (for more details, please see the reply to the recommendations for authors section).
Only a limited number of integrin adapter proteins were investigated. Given the high number of identified adapter proteins, this is an understandable choice. However, it would be fascinating to understand if the nanoclusters of inactive integrins are dominantly bound with a certain adapter protein, such as tensin.
We fully agree with the reviewer and have now performed dual-colour super-resolution STED microscopy of α<sub>5</sub>β<sub>1</sub> and tensin-3 on HFF cells seeded for 24 hours. Interestingly, instead of being an integrin inactivator, we found that tensin-3 is also highly enriched at the FA periphery where a large fraction of active β<sub>1</sub> integrins are located, suggesting that at these particular regions, active β<sub>1</sub> could be either engaged to talin (as shown in our original data) or to tensin-3 (our new data shown in Fig. S8). We provide more details of our answer in the section of “recommendation to the authors”. Additional experiments, which in our opinion fall outside of the scope of this work, would be necessary to identify other potential integrin inactivator partners, but certainly a topic of future interest to our group.
Reviewer #3 (Public review):
Summary:
In their study, the authors reveal using dual-color super-resolution STORM microscopy modality and immunolabeling in fixed adherent cells, that β1 and β3 integrins as well as adaptors (paxillin, talin and vinculin) are all organized in nanoclusters of similar size (50nm) and molecular density (20 copy number) inside FAs but also outside. Using activityspecific immunolabeling of β1 and β3 integrins, they revealed that active integrin subpopulations were both clustered but in distinct exclusive nano-aggregates in agreement with Spiess et al. (2018). Once more, the "active" integrin nanoclusters displayed similar properties in terms of size and molecular density, suggesting that molecular organization in nanoclusters is an intrinsic property of integrins in plasma membrane multimerizing independently of their location (inside or outside FAs), their level of activation, or their connection to the cytoskeleton. Then the authors followed up by analyzing at the mesoscale how these "universal" nanoclustered adhesive units are distributed spatially. Inspecting the surface density of nanoclusters revealed that the density of integrin nanoclusters in FAs was 5x larger, compared to integrin nanoclusters outside adhesions. Interestingly, whereas the density of total integrin nanoclusters was 2-4x larger than adaptor nanoclusters, the density of "active" integrin nanoclusters stoichiometrically matches that of talin and vinculin nanoclusters, and was slightly outnumbered by paxillin nanoclusters. These findings suggest that inside FAs, among the total number of integrin nanoclusters, the subset of "active" integrin nanoclusters could be engaged with "adaptor" nanoclusters on a 1:1 ratio. Using analysis of the nearest neighbor distance (NND) between distinct integrin clusters and each of the adaptors, the authors report that they found negligible spatial colocalization of integrins with these adaptor proteins and that spatial segregation is essentially determined by the density of nanoclusters within the FAs. As authors reported that α5β1 and αvβ3 do not intermix at the nanoscale, the authors finally highlighted how α5β1 and αvβ3 distinct nanoclusters are differently organized and segregated inside FAs. Adapting the NND analysis in order to inspect how far the nanoclusters are from the edges of FAs they are located in, authors revealed that α5β1 but not αvβ3 integrin nanoclusters are enriched on FA edges and that similar FA edge-enriched distribution for "active" α5β1 and adaptor protein nanoclusters was found for talin and paxillin but not vinculin. The latter results suggest that FA edges could constitute multiprotein hubs for enhanced colocalization and activation for α5β1 integrin nanoclusters and adaptors such as talin and paxillin. Unfortunately NND analysis could not confirm this enhanced colocalization hypothesis.
General Assessment:
While the study presents some valuable findings, it reads currently as a compilation of intriguing but preliminary observations derived primarily from a single methodology (dual-color STORM and DBSCAN clustering analysis). As the initial findings often lack confirmation through additional data analysis (such as the NND analysis the authors used), there's a critical necessity to bolster the methodological approach. This should involve replicating the main findings using alternative single-molecule super-resolution techniques (such as quantitative DNA-PAINT) or employing different clustering analytical tools (such as voronoi-tessellation). Furthermore, the manuscript feels incomplete, focusing solely on describing molecular organization without offering substantial insights into how these observations correlate with the regulation, activation, and functionality of integrins at the cellular level.
We appreciate the comment of the reviewer and have taken their recommendation to heart in order to validate our methodology. In summary, we have now performed extensive DNA-PAINT to replicate most of our initial findings obtained by STORM, as requested by the reviewer. In addition, as a different super-resolution imaging strategy, we have also used STED microscopy to confirm the nanoclustering of integrins and some of the adaptors demonstrating now, by means of three different super-resolution techniques, that both integrins and their adaptors form nanoclusters of similar size and composition, regardless of whether they are inside or outside FAs. We have included these data as Figs. S3 and S4 and discussed the results in pages 8-9 of the main manuscript.
Regarding the use of an alternative analysis for the data, we have now used the Voronoi tessellation algorithm to re-analyse our STORM data, as requested by the reviewer. The results of the analysis, which render similar sizes and number of localizations as obtained by DBSCAN, are now included in Fig. S5 and mentioned in page 8 of the main manuscript.
The manuscript presents extensive datasets and utilizes methodologies in which the investigators demonstrate expertise. Nevertheless, there's uncertainty regarding the novelty and broad appeal of the findings. For instance, the observation of integrin nanoclustering has been previously reported in several publications (e.g., Changede et al., Dev Cell 2015; Spiess et al., JCB 2018; Fujiwara et al., JCB 2023). Similarly, the accumulation of specific proteins at the periphery of FAs has been documented elsewhere (e.g., Sun et al., NCB 2016; Stubb et al., NatComm 2019; Nunes-Vicente TCB 2023), as well as the differential dynamic organization of α5β1 and αvβ3 integrins inside FAs (e.g., Rossier et al., NCB 2012). Beyond the universal organization of adhesive proteins, there's a need to identify novel insights that significantly advance the field. One potential avenue could involve pinpointing the molecular determinant controlling the FA edge enrichment of active α5β1 integrins and talin nanoclusters. For instance, could there be an interplay between α5β1 and αvβ3 integrin nanoclusters visible on one's organisation when suppressing the other using deletion (KO) or depletion (SiRNA)? Also, could KANK, which also exhibits enrichment and regulates talin activity (e.g., Sun et al., NCB 2016), play a role in this process? Identifying the molecular players that regulate even partially the mesoscale organization of nanoclusters of proteins would really benefit the breadth of this manuscript.
We could not agree more with the reviewer and in fact, we are currently investigating the mechanisms that control the enrichment of α<sub>5</sub>β<sub>1</sub> and adaptors at the edges of FAs. However, considering the amount of work needed to determine the spatiotemporal organization of other molecular players using super-resolution imaging constitutes a major tour de force.
To get a first insight into the process of active α<sub>5</sub>β<sub>1</sub> enrichment at the FA edges, we hypothesised that mechanical forces exerted by the actomyosin machinery could influence the lateral distribution of both integrin subsets (α<sub>5</sub>β<sub>1</sub> and α<sub>v</sub>β<sub>3</sub>) inside FAs. Since FA maturation and strengthening over time requires mechanical forces, we performed experiments at different cell seeding times (90 min, 3 hours and 24 hours) and used STORM imaging to follow the evolution of integrin nanoclustering in time as well as their spatial distributions inside FAs. Interestingly, while nanoclustering of both integrin sub-sets inside FAs is not influenced by seeding times, their lateral distribution was markedly different, with α<sub>5</sub>β<sub>1</sub> nanocluster distribution being already established at earlier seeding times, while α<sub>v</sub>β<sub>3</sub> nanocluster distribution appeared as rather random at earlier seeding times and progressively organized reaching a well-defined lateral spacing at 24 hours of spreading time. These initial data strongly suggest that mechanical forces might play a role in the distinct lateral distribution of both subsets of integrin nanoclusters over time. We have now included these data as new Fig. 2 of the revised manuscript and discuss the results in the associated text (pages 9 and 10). We also discuss potential avenues for further research along the directions suggested by the reviewer.
In addition, since it has been recently shown that tensin-3 interaction with talin drives the formation of fibronectin-associated fibrillar adhesions (Atherton et al, J Cell Biol 2022) which are enriched in β<sub>1</sub> integrins, we performed dual-colour super-resolution STED microscopy of β<sub>1</sub> and tensin-3 on HFF cells seeded for 24 hours. Interestingly, our initial data show co-enrichment of both tensin-3 and active β<sub>1</sub> nanoclusters at the FA periphery, suggesting that at these particular regions, active β<sub>1</sub> could be either engaged to talin (as shown in our original manuscript) or to tensin. Our current working hypothesis is that α<sub>5</sub>β<sub>1</sub> enrichment at the FA periphery serves to facilitate the translocation of α<sub>5</sub>β<sub>1</sub> integrins from FAs to fibrillar adhesions, most probably in a talin-tensin-dependent manner. We have now included these data as Fig. S8 and accompanying discussion in pages 22 and 23 of the revised manuscript.
Echoing the previous concern, the manuscript described a novel and rather surprising finding related to molecular clustering of adhesion proteins. Indeed, the fact that nanoclusters exhibit uniform size and molecular density regardless of the protein type, location, or activation level is indeed surprising and raises many questions about the methodology used to assess molecular clustering. I feel that the description and characterization of integrin nanoclusters appear incomplete and need to be expanded by comparing different analytical strategies for protein clustering. Furthermore, a lack of the manuscript in its actual form concerns the quantification of integrin numbers inside the observed nanoclusters. I agree that the path from optical microscopy to protein stoichiometry quantification is hard and full of drawbacks. But the authors do not fully address these issues that are extremely important when discussing protein nanoclustering. This quantitative aspect should be discussed.
We appreciate the comment of the reviewer as indeed, the existence of “universal” nanoclusters is intriguing. Recently, together with Prof. S. Mayor we have written a short review in Curr. Opin. Cell Biol 2024 proposing that nanoclustering constitutes a molecular-scale organisation principle that governs cellular information flow at the plasma membrane. Our proposal is supported by an extensive number of recent papers showing that most cell membrane receptors and downstream signalling components are organized as pre-assembled nanoclusters. We posit that these nanoclusters serve as modular units whose concatenation in a specific spatiotemporal sequence leads to distinct signalling outputs. Thus, the existence of universal nanoclusters of integrin receptors and adaptors is indeed intriguing but not surprising to us.
In any case, the concern of the reviewer is well-taken, and as mentioned above, we have used a different algorithm to detect and quantify nanoclustering, obtaining similar values using either Voronoi tessellation or DBSCAN approaches. These data are now included as Fig. S5 in the manuscript.
Regarding the quantification of integrin numbers inside the observed nanoclusters, we agree with the reviewer that determining protein stoichiometry using single-molecule localization microscopy or STED remains a major technical challenge and is highly prone to artefacts. For this reason, we refrain from making claims about absolute protein numbers per nanocluster. Our relative comparison of nanoclustering among the different proteins investigated is thus exclusively based on the number of single-molecule localisations contained in each nanocluster which is a fair approach since we always use the same reporter fluorophore and maintain similar excitation conditions throughout our experiments. We have now included a few lines on page 9 regarding quantification of the absolute protein numbers inside the nanoclusters and further discuss in the revised manuscript the limitations of single-molecule localisation methods towards the stoichiometry determination of the nanoclusters (see page 20 of the revised manuscript).
First, it is crucial for the authors to carefully examine and discuss in their manuscript whether there are any potential biases or limitations in the experimental techniques (dual-color STORM) or data analysis methods employed (DBSCAN). Second, the authors did not in the current manuscript, but should provide control samples to demonstrate the sensitivity and dynamic range of their experimental strategy.
As already mentioned, we have validated the STORM data using both DNA-PAINT and STED and, validated our data analysis obtained with DBSCAN using the Voronoi tessellation algorithm. See Figs. S3, S4 and S5. In terms of sensitivity and dynamic range of our methodology: our set-up has single-molecule detection sensitivity which is demonstrated by the fact that we observe and detect discrete blinking events, a property of single-molecule fluorescence emission and key ingredient to super-resolution single-molecule localisation microscopy. The dynamic range (if we understand correctly the question of the reviewer) is given by the number of frames used to accumulate single-molecule localisations. In our case, we stop acquisition after we deplete most of the single-molecule spots in the imaging view, which typically occurred after 70,000 frames acquisition, as correctly mentioned in the material & methods section.
In STORM images displayed in Figure S1, the authors highlighted localization clusters detected by DBSCAN as a signature for integrin nanoclusters. But the authors do not discuss the localization spots that were not detected by DBSCAN. Could they be individual integrins? And if so, they should also be considered as useful information? This brings me to another related technical question about how DBSCAN handles the case where fluorescent molecules are blinking. This is important as multiple emissions by a single fluorophore could be detected as a nanocluster of several molecules where it would be an artefact due to the photophysics of the fluorophore. Could the authors comment on these points?
As mentioned in the original manuscript, between 20-30% of the localizations were not assigned to nanoclusters (Fig. S1H, I) since we imposed a minimum of ten localizations within the radius defined by DBSCAN to be considered as a true nanocluster. This essentially means that regions with less than 10 localizations were not considered in our nanoclustering analysis. However, we cannot be certain as to whether these lower number of localizations correspond to individual integrins, stochastic blinking of the fluorophore or small aggregates containing only a couple of integrins, for the same reasons that we cannot provide quantification of the absolute number of proteins included in each nanocluster: stoichiometry determination by means of single-molecule super-resolution methods is highly prone to artefacts.
Regarding the concern of how DBSCAN handles fluorophore blinking, the reviewer is completely right as the photophysics of the fluorophore can influence the analysis of the data and the identification of true nanoclusters. To decouple the photophysics of the fluorophore we first assess the number of blinking events within the DBSCAN radius, i.e., number of localizations corresponding to individual antibodies sparsely distributed on the glass surface. In our case, the median values for the two activator-reporter pairs corresponded to 5 localizations for Alexa 405-Alexa647-conjugated Abs and 3 localizations for Cy3-Alexa 647-conjugated Abs (see Fig. 1E). Yet, despite these median values, the number of localizations per individual Ab naturally shows a distribution. Thus, to avoid any overestimation in the degree of nanoclustering, we impose an additional constrain to our analysis and consider true nanoclusters only those ones containing at least 10 localizations. We have now significantly extended the explanation in the main text (see page 6) as well as materials & methods so that it becomes clearer to the reader.
Also, using isolated and stochastically physisorbed fluorophores (Ab coupled with activator /reporter pairs used in this study) on glass helped define the signature in STORM of a single isolated molecule. To obtain the signature of clustered fluorophores, the authors could use anti-donkey antibodies to cross-link those STORM-specifically labeled Ab as a means to artificially obtain clustered fluorophores. Ultimately, to avoid the bias effect of the glass surfaces on the photophysics of fluorophores and be in the same imaging conditions as for the described nanoclusters, the authors should use model systems composed of multimers of GFP vs. single GFP, immunolabeled with a GFP-binding monoclonal antibody. This will permit evaluation of the cluster signature obtained with DBSCAN analysis of STORM data for single vs. multimers of known stoichiometry. This would constitute an undisputable molecular stoichiometry ruler.
We appreciate the suggestions of the reviewer. Regarding the potential bias effect of the glass surface on the photophysics of the fluorophores we would like to clarify that the “calibration” for the number of blinking events per individual Ab on glass were performed on the same sample containing the cells that we image, so that we maintain exactly the same experimental and imaging conditions avoiding any potential artefacts. To our understanding this approach is more accurate than performing the calibration on glass substrates and then moving to samples containing the cells. This information is now contained in page 6 of the revised manuscript and in the materials and method section. Once the number of blinking events from individual Abs on glass within the DBSCAN radius are determined, one can then determine the number of localizations within the same DBSCAN radius on other parts of the sample. More localizations within the same DBSCAN radius basically means more molecules, and thus nanoclusters. This approach has been extensively used by other experts in the field as we properly acknowledge in our manuscript (Pageon et al, Mol. Cell. Biol 2016; Spiess et al. J. Cell Biol 2022).
Using anti-donkey antibodies to cross-link those STORM-specifically labelled Ab in order to artificially obtain clustered fluorophores, as suggested by the reviewer, is indeed a sound approach to retrieve signatures of clustering. Nevertheless, we have preferred not to use this approach because those artificially induced clusters would have very little resemblance to the real nanoclusters and would only allow us to validate the performance of DBSCAN for cluster recognition. As mentioned above, DBSCAN is a well-established algorithm and used by many different experts in the field and thus can be trusted by the community. Instead, and following the recommendation of the reviewer, we now provide results using an alternative cluster analysis algorithm (Voronoi tessellation) reaching similar conclusions regarding the existence of integrin and adaptor nanoclustering inside FAs.
Finally, the suggestion of using monomeric vs multimeric GFPs to determine the stoichiometry of the nanoclusters is highly appreciated. Indeed, we have used this approach in the past to identify nanoclustering of the chemokine receptor CXCR4 in living T cells (Mol. Cell 2018 and PNAS 2022). However, these experiments are best performed at sub-labelling conditions, which inherently underestimate the degree of nanoclustering. Combining GFPs with PALM to enable super-resolution is another approach but also subject to artefacts regarding the photo-conversion efficiency of GFPs as we reported earlier (Nature Methods 2017) and leading to underestimation of nanocluster stoichiometry.
In summary, providing nanocluster stoichiometry from single-molecule localisation images remains a major technical challenge and is highly sensitive to methodological assumptions. We have therefore focused here on providing robust evidence for the existence of integrin and adaptor nanoclustering, using three different superresolution approaches and two independent analytical methods for cluster determination.
Due to the surprising finding of the nanoclusters' "universality", it is imperative for the authors to validate the findings through complementary methodologies and analytical tools. This should involve replication of results using alternative super-resolution techniques (quantitative DNA-PAINT) and exploring different clustering algorithms (VoronoïTesselation) to ensure the robustness and reliability of the observations.
As already mentioned, we have now performed extensive DNA-PAINT to replicate most of our initial findings obtained by STORM, as requested by the reviewer. In addition, as a different super-resolution imaging strategy, we have also used STED microscopy to confirm the nanoclustering of integrins and some of the adaptors demonstrating now, by means of three different super-resolution techniques, that both integrins and their adaptors form nanoclusters of similar size and composition, regardless of whether they are inside or outside FAs. We have included these data as Figs. S3 and S4 and discussed the results in pages 8-9 of the main manuscript.
Regarding the use of an alternative analysis for the data, we have now used the Voronoi tessellation algorithm to re-analyse our STORM data, as requested by the reviewer. The results of the analysis, which render similar sizes and number of localizations as obtained by DBSCAN, are now included in Fig. S5 and mentioned in page 8 of the main manuscript.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
This work already, as is, provides significant and novel information on IAC.
The unexpectedly low spatial colocalisation of integrins with adaptor proteins might indeed be caused by the potentially quite long extension of talin upon force exposure and the ample zones of activity of IAC proteins, imaging the involved proteins in scale, as can be seen in Barnett and Goult (2022, doi: 10.3389/fncel.2022.1014629)? In super-resolution microscopy, this spatial separation might, in fact, become apparent. It would be interesting to see whether lowering the actomyosin contraction by different concentrations of blebbistatin lowers this separation. In general, it would also be interesting to understand whether lowering the forces can disrupt the nano-organisation and the strong separation of the two analysed integrin subtypes. It is true that nascent adhesion formation is force-independent, but maybe the forces play a role in establishing the particular integrin subtype nano-organisation within more mature IAC. I am also aware that a lot of work has already gone into the conclusion of this project.
We thank the reviewer for these thoughtful comments and suggestions. Our most recent preliminary data (not yet included in this manuscript) indeed indicate that the physical separation of integrin nanoclusters (and adaptors) inside focal adhesions (FAs) is force-dependent. We are currently reproducing these experiments using lipid bilayers of varying viscosities and controlled ligand density to explore how ligand mobility (i.e., equivalent to force exerted from the extracellular side) controls the degree of IAC nanoclustering and their spatial segregation in FAs. This approach is more amenable to super-resolution microscopy as lipid bilayers are quite thin and optically transparent, yet the experiments are still challenging, time-consuming, and thus ongoing.
To obtain a first hint as to whether forces might play a role in establishing the spatial distribution of the two different integrins within more mature IACs as the reviewer suggests, we have performed experiments at different seeding times (90 min, 3 hours and 24 hours). Our results show that even at earlier times (90 min), when a lower number of mature FAs are established, nanoclustering of integrins and main adaptors are similar to 24 hours. In contrast, and as suggested by the reviewer, the spatial distribution of the different subsets of integrin nanoclusters inside FAs is markedly different as a function of seeding time, with α<sub>5</sub>β<sub>1</sub> nanocluster distribution being already established at 90 min, while α<sub>v</sub>β<sub>3</sub> nanocluster distribution appears rather random at earlier seeding times and progressively organizes reaching a well-defined lateral spacing at 24 hours of spreading time. As FA strengthening over time requires mechanical forces, and α<sub>v</sub>β<sub>3</sub> is preferentially involved in FA strengthening (Roca-Cusachs et al PNAS 2009), these data strongly suggest that forces play a differential role in the lateral distribution of both integrin nanoclusters over time. We have now included these data as a new Fig. 2 in the revised manuscript and discuss the results in the associated text (pages 9 and 10). We also mention in the discussion additional experiments, as suggested by the reviewer, to further substantiate this hypothesis.
Considering the high quality of the work and the new insight about the inner organisation of IAC, maybe the summary Figure 5 should be elaborated a bit, taking into account e.g. different lengths of extended talin proteins and also the various positions of vinculins on talin proteins (depending on opened cryptic binding sites), as well as the possibility that various actin filaments might be associated with single talins. What I mean is, the authors impressively demonstrate the complexity of IAC nano-organisation, which should be paid more tribute in the concluding figure. The quality of the figure should be adapted to the quality of the work.
We have adapted Figure 5 (now Figure 6) as suggested by the reviewer.
I would be curious to hear a bit more about the further speculations of the authors in the discussion, e.g., about why the integrin subunits are organised in this way. Why might the α<sub>5</sub>β<sub>1</sub> be preferentially located in the periphery? What is the potential physiological relevance of this organisation? Is this organisation different in other cell types (have the authors looked at other cells)? Is the organisation lost in pathophysiological situations, such as cancer?
Although we do not know yet what drives the preferential location of α<sub>5</sub>β<sub>1</sub> nanoclusters to the FA periphery, it is known that Kank2 also exhibits enrichment at the FA periphery, regulates talin activity and it is involved in the formation of α<sub>5</sub>β<sub>1</sub>-enriched fibrillar adhesions (Sun et al, Nature Cell Biol 2016). Thus, it is highly probable that α<sub>5</sub>β<sub>1</sub> enrichment at the FA periphery is a necessary step for their translocation from mature FAs to fibrillar adhesions to then assemble fibronectin into the fibrillar networks as found and needed in connective tissues. Consistent with this idea, we have observed similar α<sub>5</sub>β<sub>1</sub> distribution on other fibroblast cell lines (MEFS), which are the primary cells that produce fibrillar adhesions. Thus, α<sub>5</sub>β<sub>1</sub> nanocluster distribution inside FAs might be physiologically important for the process of fibronectin fibrillogenesis.
Since it has been documented that tensin is important for fibronectin fibrillogenesis (Pankov et al J Cell Biol 2000) and more recently, it has been shown that tensin-3 interaction with talin drives the formation of fibronectin-associated fibrillar adhesions (Atherton et al, J Cell Biol 2022), we thought to investigate the spatial distribution of tensin-3 and its relationship with α<sub>5</sub>β<sub>1</sub> inside FAs by means of dual colour super-resolution STED microscopy. Interestingly, our initial data on HFF cells seeded for 24 hours show both enrichment of tensin-3 and α<sub>5</sub>β<sub>1</sub> nanoclusters at the edges of mature FAs, supporting our working hypothesis that α<sub>5</sub>β<sub>1</sub> enrichment at the FA periphery serves to translocate α<sub>5</sub>β<sub>1</sub> integrins from FAs to fibrillar adhesions, probably in a talin-tensin-dependent manner. While these initial data are quite exciting, many more experiments that include simultaneous super-resolution mapping of α<sub>5</sub>β<sub>1</sub>, talin and tensin in mature FAs are required to fully validate our hypothesis. Yet, because of their relevance we consider it appropriate to include these data as Fig. S8 and discussing their potential implications in pages 22 and 23 of the revised manuscript.
Reviewer #2 (Recommendations for the authors):
(1) Perform control experiments to confirm that the nanocluster size/composition is not affected by the antibodies used.
As explained in the response to the public reviews, antibody labelling has always been performed after cell fixation, precluding potential cross-linking artefacts due to protein mobility and avoiding unwanted receptor activation. In addition, we have performed super-resolution imaging using DNA-PAINT as a different imaging strategy. In this case, the DNA docking site is site-specifically coupled to one camelid single-domain Ab (sdAB), having a much smaller size as compared to a secondary Ab, reducing therefore linkage error and increasing the accessibility of primary Ab-labelled proteins. As can be observed in new Fig S4, no differences in terms of nanocluster sizes and/or compositions were observed for any of the proteins investigated using DNA-PAINT as compared to our initial STORM data. These control experiments thus rule out any potential artefacts introduced by the secondary Ab. Finally, we would like to highlight that our results on the nanoclustering of integrins in terms of their size and number of localizations is consistent with previous results obtained by other groups around the world using similar labelling protocols as us (Spies et al, J. Cell Biol 2022), or relying on halo-tag strategies, as suggested by the reviewer (see Fujiwara et al, J. Cell Biol. 2023). The consistency of these results amongst different groups gives us further confidence that the nanocluster size/composition are not affected by the antibodies used.
(2) Extend the study by inspecting a set of integrin adapter proteins for their association with inactive integrins, focusing on adapters associated with the maintenance of the inactive state. Possible candidates would be tensin and filamin, for example.
We thank the reviewer for the suggestion and have now performed dual-colour super-resolution STED microscopy of α<sub>5</sub>β<sub>1</sub> and tensin-3 on HFF cells seeded for 24 hours. Interestingly, instead of being an integrin inactivator, we found that tensin-3 is also highly enriched at the FA periphery where a large fraction of active β<sub>1</sub> integrins are located, suggesting that at these particular regions, active β<sub>1</sub> could be either engaged to talin (as shown in our original data) or to tensin-3 (our new data shown in Fig. S8). These results might be surprising at first, since tensin competes with talin for the same binding site to the cytoplasmic β-tail of integrins, and thus believed to act as integrin inactivator, as the reviewer indicates. Nevertheless, recent data has shown that tensin is capable to activate integrins (in particular if β<sub>1</sub> is phosphorylated) by interacting with the actin cytoskeleton, providing mechanical coupling for integrin activation (Georgiadou & Ivaska, Trends Cell Biol. 2017). We have now included these new data as Fig. S8 in the revised manuscript. Additional experiments, which in our opinion fall outside of the scope of this work, would be necessary to identify other potential integrin inactivator partners, but certainly a topic of future interest to our group.
(3) While the methods are described in sufficient detail, it is important to ask if the findings are based on sufficient data. Table S5 provides detailed information about the number of samples studied, and it appears that only small numbers of samples were investigated for certain protein pairs. This should be discussed, and perhaps more data should be obtained to strengthen the data.
We have now performed additional experiments using DNA-PAINT as alternative super-resolution imaging technique (as also requested by reviewer 3) which adds additional data to the whole manuscript.