Ultrafast single-molecule imaging reveals focal adhesion nano-architecture and molecular dynamics

The ultrafast camera with single fluorescent-molecule sensitivities developed by Fujiwara et al. has greatly improved the time resolution of single-molecule localization microscopy, revealing the focal adhesion’s dynamic nano-architecture and leading to the model of compartmentalized archipelago of focal-adhesion protein islands.

brightfield imaging. Thus, the current study corroborate the model of plasma membrane/membrane cytoskeleton organization/compartmentalization, while describing a more generalizable experimental platform and a less perturbative tagging (fluorophores vs 40-nm nanoparticles). Next, the authors describe how the high-speed camera can also be used for Live-cell PALM, which was then applied to image caveolae and focal adhesions live cells. In the 2nd manuscript, the author used the imaging system thus developed for a more cell biological applications, with the goals of probing membrane organization in the ventral plasma membrane as well as in focal adhesions. Using Transferring Receptor and Cy3-DOPE, the authors showed that these labelled lipid and membrane proteins exhibit similar diffusion characteristics between dorsal and ventral plasma membrane. Similar kinetics is also observed for EGFR, suggesting that these kinetics are dependent on plasma membrane compartmentalization. Subsequently, live-cell PALM is applied to image focal adhesions revealing heterogeneous nanocluster organization of paxillin (so-called 'islands'), while TfR high-speed SPT is performed to probe membrane organization in focal adhesions. Analysis of TfR trajectory indicates that plasma membrane in focal adhesion is also compartmentalized but with smaller compartment and longer dwell time. High-speed SPT of integrin beta 3 was performed which reveal complex trajectory that can periodically be immobilized at paxillin-based 'islands'. In summary, this body of work is a technical tour de force study by established investigators in the field of single-molecule imaging/membrane biophysics that advance the state-of-the-art in fluorescence imaging capability by orders of magnitude. The experimental execution and supporting theories are sound and rigorous, while the technological advances presented should be generally useful and readily adaptable to other bioimaging modalities. The high-speed capability is likely to be game-changer in addressing a number of key biological/biophysical questions. Thus, I am in support of these works being eventually accepted for publication in JCB. That said, with the current structure of these two manuscripts, though the text itself is well-written, it is still quite challenging to digest such a large amount of information and a revision is strongly advised.
1. Of the two manuscripts, in the current organization, the first manuscript is clearly the strongest as it describes all of the novel results and major technical advances. In contrast, the key results on integrin diffusion in the 2nd manuscript is perhaps somewhat overshadowed by the authors's own previous work in 2018 Nature Chemical Biology Tsunoyama et al., that also describe similar trajectory characteristics of integrin.
As it seems that all the ground-breaking exciting results are all contained in 1st manuscript this has the side effect of depriving the 2nd manuscript of key results. At the same time, this also makes the 1st manuscript quite dense and challenging to digest.
My suggestion is to revise the 1st manuscript to focus on the camera, SPT, hop diffusion, and membrane organization. Then, the live-cell PALM sections can be moved from the 1st to the 2nd manuscript. This way, the first manuscript will be more nicely packaged as an ultrafast SPT & more biophysical membrane organization study, while the 2nd manuscript will be on fast live PALM super-resolution, their characterization, and more cell biological study. The content in both manuscripts may be better balanced and more digestible this way.
2. Some of the figure panels would benefit from more clear captions or sub-titles, so that readers do not need to go to the figure description for every panels. 3. While MINFLUX is another technique capable of high-speed fast single-molecule tracking and thus the authors may feel the need to differentiate their approach from MINFLUX, this reviewer is of the view that MINFLUX is much more of a niche technique compared to the more generalizable and modular capability presented by the fast camera in this study. Perhaps the MINFLUX discussion can be included as supplementary note instead so as not to detract from the main text. 4. One of the main weaknesses of 2nd manuscript starts with the abstract which is mostly descriptive. It is not clear from reading the abstract what is the key 'take-home' biological findings. As the authors appear to intend both manuscripts as a technological demonstration rather than a full-fledged mechanistic dissection, I would suggest to revise according to #1 above and also rewrite the abstract accordingly. Alternatively, additional biological perturbations may be needed for 2nd manuscript to dissect what factors are regulating membrane partitioning in focal adhesions. However, given that there is a vast amount of data in these two manuscripts already, this reviewer is of the view that a revision as in #1 is probably more advisable.
Reviewer #2 (Comments to the Authors (Required)): This well-documented manuscript provides a significant extension of prior research in the fields of single-molecule membrane motility patterns and focal adhesion research. It provides further support for the archipelago model of focal adhesion organization as reported by paxillin localization, as well as confirming that apical and basal plasma membranes show similar patterns of single-molecule trajectories, including 103-109 nm compartment size with hop diffusion of both protein and lipid. The new findings include evidence that EGFR occupancy-dimerization leads to longer confinement, that transferrin receptor hopdiffusion within focal adhesions is altered with a 2-fold reduction in compartment area and enhanced dwell lifetime, suggesting that the putative actin picket fence meshwork is finer/smaller, apparently due to fluid membrane compartmentalization rather than interference by the paxillin islands. Also interesting is quantification of beta-3 integrin immobilization, not surprisingly at the paxillin islands. The advances presented in this paper appear quite solid with impressive single-molecule tracking, but arguably often not surprising. Overall, it is not clear at present that this paper has enough truly novel content for JCB, i.e., with its present content, it appears borderline.
1. Although these findings add significantly to existing knowledge concerning membrane dynamics and focal adhesion structure, the amount of new knowledge presented in this paper seems somewhat borderline for JCB. Considering first the evaluation of dynamics at the apical versus the basal surfaces of these cells, even though the authors state at least four times that their findings were "surprising," this reviewer would have predicted that finding considering that there may be no evidence that these T4 epithelial cells have strong apical-basal polarity that might affect membrane organization. The authors should explain why they feel the results were surprising. This reviewer feels that unless the authors could use an epithelial cell with high polarity and distinct differences between apical and basal plasma membrane content, the results seem to be what would be predicted for a non-strongly polarized epithelial or fibroblastic cell in which apical and ventral membranes may not differ except at sites of focal adhesions.
2. Considering next the characterization of the fluid membrane in focal adhesions, the findings do provide this new information. However, it is not clear why the compartment size is changed. Do the authors see differences in membrane-associated actin (which might be obscured by the actin bundles inserting into focal adhesions, and thus might require TIRF microscopy)?
The characterization of paxillin islands by ultrafast PALM is interesting, with a useful quantification of island diameter. Although not absolutely essential, readers in the field will wonder whether these findings are specific for just this immortalized retinal epithelial cell line and not for other more-studied cell-type focal adhesions in the literature.
3. The demonstration of integrin hop diffusion is not surprising, nor is the immobilization at sites of integrin-based adhesion in focal adhesions. What would be useful to the field is a clarification of the nature, extent, and duration of the immobilization events, which seem to involve a range of effects. Immobilization for only 0.19 s in the example shown seems quite brief, and one wonders what it signifies. An in-depth consideration of findings for integrins seems needed, even if there cannot yet be any direct correlation with paxillin due to the lack of two cameras for tracking two fluorescent labels. Basically, the integrin data seem too limited for a JCB paper, since it has been known for decades by FRAP that integrins have immobilization in focal adhesions. A more in-depth analysis might enhance interest. A specific question is whether all integrin molecules studies have a short immobilization duration in the order of seconds, or whether some key anchoring integrin molecules have considerably longer periods, which should be possible to quantify. If longlived integrin adhesions exist, would they contribute to altering compartment size and the behavior of adjacent molecules? Re: JCB manuscript #202110162, retitled as, "Ultrafast single-molecule imaging reveals focal adhesion nano-architecture and molecular dynamics" Dear Joerg and Andrea, Thank you very much for critically reading and assessing our manuscript for publication as an Article in JCB. We would also like to thank you for obtaining the opinions of the two referees. Attached please find our revised manuscript. As we discussed with you, following your instructions, we have added significant amounts of new data and virtually rewrote this manuscript, and hereby submit a new manuscript. As we requested in our first submission, we hope that this paper can be published back-to-back with its companion Tools paper (#202110160, slightly retitled as "Development of ultrafast camera-based single fluorescent-molecule imaging for cell biology"). We are submitting these two companion manuscripts at the same time.
We have addressed all of the points raised by you and your referees in the revised manuscript, and have basically complied with all of the recommendations.
We have made large organizational changes to these two manuscripts, as recommended by Reviewer 1. Specifically, we have moved the sections describing the application of the ultrafast camera to ultrafast PALM imaging, from the revised Tools paper (companion manuscript) to the revised Article paper (this manuscript). At the same time, we have moved the sections reporting the compartmentalization of the basal plasma membrane (PM), its characteristics, and the effect of compartmentalization on EGFR diffusion before and after the ligand EGF addition, which were previously described in this Article manuscript, to the companion Tools manuscript. Instead, as recommended by Reviewer 1, this Article manuscript now includes the application of the ultrafast camera to ultrafast PALM (moved from the original Tools manuscript). Furthermore, this revised Article manuscript now includes new important experimental results about the applications of the ultrafast camera to ultrafast dSTORM and simultaneous two-color ultrafast PALM-dSTORM, as well as the results about the nano-scale architecture and dynamics of focal adhesions (FAs) obtained by these new methods. This was also motivated by the recommendations from Reviewer 2, who encouraged us to extend our studies of the focal adhesion.
As a result, we believe that the manuscript has been considerably strengthened. We would like to thank you and your reviewers again for critically reading our manuscript and providing constructive comments and recommendations. We hope that this manuscript is now acceptable for publication in The Journal of Cell Biology.
The main text has been comprehensively rewritten and, therefore, no highlighting was done.

Reviewer #1 (Comments to the Authors (Required)):
Thank you very much for carefully reading our manuscripts and for your kind and constructive comments. We addressed all your comments in our rebuttal for the revised Tools manuscript, and thus will refrain from repeating them here.
Please note the following ways in which we highlighted the new results and the results moved from the previous Tools manuscript to this revised Article manuscript.
The main text has been comprehensively rewritten and, therefore, no highlighting was done.

Reviewer #2 (Comments to the Authors (Required)):
This well-documented manuscript provides a significant extension of prior research in the fields of singlemolecule membrane motility patterns and focal adhesion research. It provides further support for the archipelago model of focal adhesion organization as reported by paxillin localization, as well as confirming that apical and basal plasma membranes show similar patterns of single-molecule trajectories, including 103-109 nm compartment size with hop diffusion of both protein and lipid. The new findings include evidence that EGFR occupancy-dimerization leads to longer confinement, that transferrin receptor hop-diffusion within focal adhesions is altered with a 2-fold reduction in compartment area and enhanced dwell lifetime, suggesting that the putative actin picket fence meshwork is finer/smaller, apparently due to fluid membrane compartmentalization rather than interference by the paxillin islands. Also interesting is quantification of beta-3 integrin immobilization, not surprisingly at the paxillin islands. The advances presented in this paper appear quite solid with impressive single-molecule tracking, but arguably often not surprising. Overall, it is not clear at present that this paper has enough truly novel content for JCB, i.e., with its present content, it appears borderline.
Thank you very much for critically reading our manuscript and for your kind and constructive feedback.
For strengthening this Article manuscript, we developed new methodologies for ultrafast live-cell dSTORM and simultaneous two-color ultrafast live-cell PALM and dSTORM, based on the developed ultrafast camera (Fig. S3), and applied these techniques to reveal the molecular organization and dynamics of the focal adhesion (FA) (Figs. 4 G, H, 5, 6, 7, and 9 A-b and Figs. S2, S4, and S5 and related text).
Following the recommendations by Reviewer 2, we extensively applied the developed ultrafast techniques, especially the simultaneous two-color PALM-dSTORM imaging, to further investigations of the FA, and found that FA proteins, including paxillin, integrins β1 and β3, talin, FAK, and vinculin, often assemble into nano-clusters. These clusters are frequently ≥13 nm in diameter and contain ≥6 copies of one of the FAprotein species, and we refer to these greater nano-clusters as FA-protein islands. The mean island diameter is 29~32 nm in mouse embryonic fibroblasts (MEFs) (Figs. 5 D, F, and 6 C; Figs. S2 and S4 A), which are the values obtained after the correction for single-molecule localization accuracies (Fig. S2). This correction has never been conducted previously. These estimates are generally consistent with the diameters of adhesion particles found by cryo-electron microscopy (Patla et al., 2010) and integrin nanoclusters identified by super-resolution microscopy (Changede et al., 2019). However, the broad distribution of the FA-protein-island diameters, ranging from 13 to 100 nm, is quite new and we believe this is a characteristic feature of the FA-protein islands. The broad diversities were not only found in the sizes but also implied in the protein compositions and molecular stoichiometries of the FA-protein islands. Such diversities might be important for the functions of FAs, as they must respond to various force levels, force loading rates, and the diverse characteristics of the extracellular matrix.
Furthermore, we discovered that the FA-protein islands are not distributed homogeneously within the FA, but rather form loose island clusters of ≈320 nm in diameter ( Fig. 1 D-

d, archipelago model of FAprotein island clusters and oligomers; Figs. 5 F-H, and 6 C, D).
Our analysis, using paxillin, indicated that these 320-nm loose island clusters function as the units for recruiting paxillin to the FA (Fig. 7 A-D). The dynamics of the recruitment and exchange of paxillin molecules could never have been observed without applying ultrafast dSTORM to live cells (Fig. 7 A, B).
Please note that, as per the recommendations by Reviewer 1, we have moved the sections describing the compartmentalization of the basal plasma membrane (PM) and the hop diffusion of Cy3-DOPE, transferrin receptor, and EGF receptor before and after the addition of the ligand EGF, from the Article manuscript (this manuscript) to the Tools manuscript. Now, all of the sections including PALM observations have been moved from the original Tools manuscript to this revised Article manuscript.
The compartmentalization of the FA's fluid membrane region and the hop diffusion of transferrin and integrin molecules there remain in the Article manuscript. Thus, virtually all the sections related to FA organization and molecular dynamics are now included in this Article manuscript.
1. Although these findings add significantly to existing knowledge concerning membrane dynamics and focal adhesion structure, the amount of new knowledge presented in this paper seems somewhat borderline for JCB. Considering first the evaluation of dynamics at the apical versus the basal surfaces of these cells, even though the authors state at least four times that their findings were "surprising," this reviewer would have predicted that finding considering that there may be no evidence that these T4 epithelial cells have strong apical-basal polarity that might affect membrane organization. The authors should explain why they feel the results were surprising. This reviewer feels that unless the authors could use an epithelial cell with high polarity and distinct differences between apical and basal plasma membrane content, the results seem to be what would be predicted for a nonstrongly polarized epithelial or fibroblastic cell in which apical and ventral membranes may not differ except at sites of focal adhesions.
We have moved the sections describing the compartmentalization of the basal PM and the hop diffusion of Cy3-DOPE, transferrin receptor, and EGF receptor before and after the addition of the ligand EGF, from the original Article manuscript to the revised Tools manuscript. However, we will address this point raised by Reviewer 2 here.
Prior to this study, we had not been able to perform ultrafast observations of PM molecules in the basal PM, even using 40-nm gold particles (using ultrafast bright-field microscopy; this method using 40-nm gold particles as probes was the only way to perform ultrafast single-particle tracking at ≥10 kHz), because these gold particles cannot enter the space between the basal PM and the coverslip. With the development of ultrafast single fluorescent-molecule imaging (SFMI) using the <1 nm fluorescent probes described in this study (Tools manuscript), scientists can now perform ultrafast observations of molecular dynamics in the basal PM. The question of whether the compartmentalization and hop diffusion in the basal PM are different from those in the apical PM was frequently raised when we presented our ultrafast single-particle tracking data of hop diffusion in the apical PM at various meetings. Therefore, we would say that the questions of whether the basal PM is compartmentalized in the same way as the apical PM and whether molecules in the basal PM undergo hop diffusion similar to those in the apical PM have been long-term enigmas, even in non-polarized cells.
Indeed, we were surprised to find the almost identical characteristics of the basal and apical PMs because, even in non-polarized cells, the architecture of the basal PM facing the substrate (coverslip) within distances less than 40 nm might be quite different from that of the apical PM, which only faces the cell culture medium (the statement about the gap size less than 40 nm is based on the observations that gold particles of 40 nm in diameter do not bind to transferrin receptors and phospholipids located in the basal PM, whereas they bind to those in the apical PM, and that when colloidal-gold-bound membrane molecules in the apical PM reach the cell edge, which is the interface between the apical and basal PMs, they generally cannot enter the basal PM or they stop diffusing after they slightly enter the basal PM). This point is now described in the Discussion of the revised Tools manuscript (the last paragraph on p. 22). Meanwhile, we deleted the expression "surprising" from the manuscript except for one instance, where we want to explain why we used another method to confirm the results of the direct hop diffusion observations (the last paragraph on p. 18).
The organization of the apical PM of strongly polarized epithelial cells would be entirely different from that of the basal PM or that of non-polarized cells, due to the presence of dense microvilli. Therefore, although the dynamics of PM molecules there would be quite interesting, it is beyond the scope of the present research. Here, we believe we should concentrate on the basal PM, which is our main subject matter.
2. Considering next the characterization of the fluid membrane in focal adhesions, the findings do provide this new information. However, it is not clear why the compartment size is changed. Do the authors see differences in membrane-associated actin (which might be obscured by the actin bundles inserting into focal adhesions, and thus might require TIRF microscopy)?
Unfortunately, we cannot experimentally show that the compartment boundaries in the FAs' fluid membrane regions are produced by the actin filaments as in the bulk PM, as described in the third paragraph of "TfR undergoes hop diffusion inside the FA" (p. 17); i.e., "attempts to directly observe the effects of actin depolymerizing drugs failed, because at the concentrations where their effects were detectable, the cells became round and some did not survive".
The observation of the PM-associated actin filaments using optical microscopy such as TIRF microscopy, has been difficult (in the literature, one can find papers stating that they observed individual actin filaments underlying the PM, but the evidence was insufficient; by the way, throughout the present research, we used TIRF microscopy, particularly for the observations described in this Article manuscript, and so this is not an issue for us at all). The largest problem is that cortical actin filaments densely exist three-dimensionally near the PM and by just observing the images (even using EM images), it is impossible to tell which actin filaments (and which parts of single actin filaments) are associated with the PM. This is why we needed to use electron tomography to perform the 3D reconstruction of the structures on the PM cytoplasmic surface, in order to identify the actin filaments that are located within ≈8 nm from the PM inner surface (Morone et al., JCB, 2006). We tried to use this method for identifying actin filaments on the FA cytoplasmic surface, but due to the presence of very dense actin filament bundles, we could not obtain the actin structures there.
Therefore, we only speculate that the actin filament meshes on the FA cytoplasmic surface are finer (p.

18, the second paragraph from the bottom).
The characterization of paxillin islands by ultrafast PALM is interesting, with a useful quantification of island diameter. Although not absolutely essential, readers in the field will wonder whether these findings are specific for just this immortalized retinal epithelial cell line and not for other more-studied cell-type focal adhesions in the literature.
To address this point raised by Reviewer 2, we performed new investigations using mouse embryonic fibroblasts (MEFs), which have been widely employed in FA studies. Specifically, we extensively used paxillin knock-out MEFs rescued with mEos3.2-paxillin (cloned; pp. 10-11). The new results are described in Figs. 5-7 and related text. In terms of the diameters of the paxillin islands, they are quite similar between the human T24 epithelial cells and MEFs (Figs. 4 G and 5 D).
The nature, extent, and duration of the immobilization events for both integrins β3 and β1 have already been described in our previous paper (Tsunoyama et al., Nat. Chem. Biol., 2018), but in the previous work, we could not determine where the immobilization events occurred in the FA. Therefore, we performed these experiments in the present study, and found that the integrins are anchored (immobilized) on paxillin islands (and so, probably the FA-protein islands). This issue was pointed out by Reviewer 1 in her/his point 1, "the key results on integrin diffusion in the 2nd manuscript is perhaps somewhat overshadowed by the authors's own previous work in 2018 Nature Chemical Biology Tsunoyama et al." Immobilization for only 0.19 s in the example shown seems quite brief, and one wonders what it signifies. An indepth consideration of findings for integrins seems needed, even if there cannot yet be any direct correlation with paxillin due to the lack of two cameras for tracking two fluorescent labels.
Basically, the integrin data seem too limited for a JCB paper, since it has been known for decades by FRAP that integrins have immobilization in focal adhesions. A more in-depth analysis might enhance interest.
A specific question is whether all integrin molecules studies have a short immobilization duration in the order of seconds, or whether some key anchoring integrin molecules have considerably longer periods, which should be possible to quantify. If long-lived integrin adhesions exist, would they contribute to altering compartment size and the behavior of adjacent molecules? Immobilization for 0.19 s is just one typical example (the image sequence is reproduced here in Fig. 9 A-a in the revised manuscript). We selected this image sequence because it nicely shows a typical sequence of events for an integrin β3 molecule, starting from the entry from the bulk PM into the FA region, to immobilization, resuming diffusion, and finally moving away from the FA. To avoid confusion, in the new Fig. 6 A-b in the revised manuscript, we now show another typical case where integrin β3 is immobilized in the FA at time 0, and remained immobilized till the end of the recording.
In addition, in the Discussion section (second paragraph on p. 24), we added the following sentences to clarify this point and its significance. "Previously, using the video-rate SFMI, Tsunoyama et al. (2018) found that both integrins β3 and β1 undergo temporary (< 80-s) immobilizations in the FA, but perform distinct functions in the FA formation, maintenance, and disintegration (also refer to the following publications: Roca-Cusachs et al., 2009;Rossier et al., 2012;Schiller et al., 2013). In the growing phase, integrin β1 initially exhibited longer immobilizations, while integrin β3 did so when the FA was in the mature steady phase. In the process of FA disintegration, the prolonged immobilizations of integrin β1 were reduced first, while integrin β3 continued exhibiting longer immobilizations for some time. In the present research, we unequivocally demonstrated that these immobilizations occur at the FA-protein islands, clearly demonstrating the functional importance of the FA-protein islands (Fig. 9)." Furthermore, in the Results section directly describing our observations, to avoid confusion, we added the following paragraph (first paragraph on p. 20). " Tsunoyama et al. (2018) previously found that integrins β3 and β1 undergo temporary immobilizations in the FA in the time scales of 0.66 s ~ 79 s and 0.5 ~ 43 s, respectively (exponential lifetimes), and by using integrin β3 we have shown here that 68% of these temporary immobilizations occur on the paxillin-enriched islands. The remaining 32% of the temporary immobilization might occur in other FA-protein islands containing <6 paxillin copies. Therefore, we conclude that the cell linkage to the extracellular matrix primarily occurs through the integrin molecules mediating the linkage of the FA-protein islands to the extracellular matrix. Although each integrin molecule might contribute to the binding for periods on the order of one to several 10s of seconds, multiple integrin molecules would be dynamically and continually recruited to the FA-protein islands, exchanging with those located outside the FA-protein islands. As a result, the FA-protein islands would remain linked to the extracellular matrix for much longer durations. The dynamic linkage of the integrins via the paxillin islands would facilitate the rapid control of FA formation and disintegration (Shibata et al., 2012;Rossier et al., 2012;Tsunoyama et al., 2018;Orré et al., 2021)." Taken together, we believe that these results represent useful advances in our understanding of integrin dynamics compared to previous studies that relied on FRAP, due to the higher spatial resolution and direct observations of single-molecule behaviors (their distributions), rather than simple descriptions of the average behavior (which might be confounded by many diverse events).
To contribute further to FA research, we have generated new data that are presented in Figs. 5-7 and related text (we already gave some descriptions of these new data in our response to Reviewer 2's general comment).
Please note the following way in which we highlighted the new results and the results moved from the previous Tool's manuscript to this revised Article manuscript.
The main text has been comprehensively rewritten and, therefore, no highlighting was done. Figs. 2, 3, and 4 A-F are the figures moved from the original Tools manuscript. They are surrounded by blue rectangles. The titles of their captions are highlighted in cyan. Newly produced figures (mostly by performing more experiments) are indicated by green rectangles. They are Figs. 1 D, 4 H, 5, 6, 7, and 9 A-b and Figs. S2 -S4, and in their captions, only their titles are highlighted in green. Videos 1, 2, and 5 are new, and in their captions, their titles are highlighted in green. Fig. 4 G is a revised figure using the newly-developed method to correct for the effect of the singlemolecule localization error on the diameter estimation of paxillin-enriched islands, and thus is shown by an orange rectangle. Fig. 1's caption had to be rewritten entirely due to the organizational changes of these two manuscripts, and so it is highlighted in yellow.  Thank you for submitting your revised manuscript entitled "Ultrafast single-molecule imaging reveals focal adhesion nanoarchitecture and molecular dynamics". The reviewers all now support publication so we would be happy to publish your paper in JCB pending final revisions necessary to meet our formatting guidelines (see details below). In your final revision, please be sure to address reviewer #1's final minor concerns.
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Thank you for this interesting contribution, we look forward to publishing your paper in Journal of Cell Biology.

Comments
In this revision the author has substantially revised and reorganized their manuscript, presented additional technical characterizations and analysis, and expanded the investigations to additional adhesion proteins. In particular, in addition to demonstrating the impressive super-resolution imaging capability enabled by their camera, they also perform systematic characterization of the camera performance with respect to fluorophore lifetime, excitation intensity, and localization accuracy analysis which I believe will help serve as a useful instructive example for the field. The expanded analysis included the investigation into the nanocluster organization of important focal adhesion proteins including paxillin, talin, FAK, and integrin b1 and b3. These results in a better definition of the nanocluster organization of these proteins, as well as potential differences between proteins. Although the analysis was performed in the presence of endogenous proteins which complicate stoichiometric analysis, they carefully characterize the expression level and take the necessary precaution in the interpretation. Intriguing observation that they made include the potential hierarchical organization of nanoclusters, i.e. sub-micron scale (300 nm) of the ~30 nm nanoclusters which they infer from the correlation analysis of proteins such as talin, FAK, and paxillin. These additional data augmented the previous data on TfR diffusion in the membrane which further corroborate granular organizations of focal adhesions. Taken together, in my opinion, that manuscript is a tour de force body of work in cellular biophysics, which advance technical ability, and present new conceptual advances on how adhesions are organized at the molecular scale. As the notion of nanocluster organization as subunits of adhesive structures are becoming appreciated in various systems beyond integrins, and are now being reported in cadherins, notch receptors etc., this study is timely. While I would like to see additional molecular insights into the observed nanoscale organization, given the lengths and the highly technical scope of the current study, I believe further molecular dissection may best be left for follow-up studies. On the whole, I am largely satisfied with the revision and support its acceptance to the Journal of Cell Biology. I believe this study will prove foundational in the long run and encourage the authors to disseminate this capability and expand their studies to explore a broader range of proteins in the future.
Minor comments: Figure 2A, and similar. It may help to put the Laser power on the left rather than on the right of the figures The supplementary movies mostly show close up view of adhesions. Since the authors discuss large field-of-view live PALM imaging as one of the major capabilites, it would be helpful to include example movies to demonstrate this.
Reviewer #2 (Comments to the Authors (Required)): The authors of this resubmitted manuscript have responded by revisions that have substantially expanded the data, specific conclusions, and overall interest of this companion paper to a Tools submission. The findings provide unprecedented detail and understanding of the nano-scale organization and dynamics of focal adhesions. Although various publications over recent years have been providing more and more insight into the complex substructure and internal dynamics of focal adhesions, this study represents a next step in understanding, with intriguing details about the heterogeneity of paxillin and other focal adhesion protein islands, their organization into groupings ("archipelagos") restricted by putative actin fences that are smaller than bulk plasma membrane actin barriers, and insight into a range of integrin associations that are remarkably short and variable. Overall, the substantial additions to this study, including the use of ultra-fast versions of PALM and dSTORM with dual color information in combination with single-molecule tracking make this paper a major new contribution to the field. Unusually, this normally very critical reviewer could not identify any remaining points of concern. Publication is recommended with high priority. Re: JCB manuscript #202110162RR, entitled, "Ultrafast single-molecule imaging reveals focal adhesion nano-architecture and molecular dynamics" Dear Joerg and Andrea, Thank you very much for accepting our manuscripts (this manuscript and #202110160RR). We are extremely glad to know that these manuscripts have been accepted. We now strongly hope that these manuscripts will be published back-to-back in the Journal of Cell Biology.
We have addressed the minor points raised by Reviewer 1 in the revised manuscript (#202110162RR) as follows.
Minor comments: Figure 2A, and similar. It may help to put the Laser power on the left rather than on the right of the figures.
Done for Figures 2A and S3B.
The supplementary movies mostly show close up view of adhesions. Since the authors discuss large fieldof-view live PALM imaging as one of the major capabilites, it would be helpful to include example movies to demonstrate this.
The new video has been produced addressing this point. Please see new Video 1.
We have not placed the raw data on this platform, but will seriously consider doing this.
Thank you very much again for accepting our manuscripts.