Actin-membrane interface stress regulates Arp2/3-branched actin density during lamellipodial protrusion

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1. 

   eLife

   May 11, 2026

   eLife Assessment

   This study provides direct and compelling evidence that lamellipodial protrusions dynamically adjust Arp2/3 complex incorporation in response to mechanical counterforces, while also modulating cellular responsiveness to upstream signals like Rac GTPase. By combining endogenous labeling, live-cell imaging, and optogenetic signaling activation, the work demonstrates how adhesion state and physicochemical perturbations reproducibly alter branched actin organization, offering a fundamental advance over previous works. The findings deliver significant insights that will resonate broadly with cell biologists and biochemists studying actin dynamics and mechanotransduction.

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2. 

   eLife

   May 11, 2026

   Reviewer #1 (Public review):

   Summary:

   This is an interesting study describing intensity changes of lamellipodial Arp2/3 complex incorporation dependent on the substratum the cells are spreading on (PLL vs fibronectin), but also on manipulation of either contractility or osmotic pressure or even external mechanical load exerted onto cells, e.g., by increasing medium viscosity. The authors use quite fancy cell systems for their studies, first of all, a CRISPR-engineered fibroblast cell line in which both endogenous loci of the Arp2/3 complex subunit Arpc2 are tagged with mScarlet, but at the same time, conditionally removable using tamoxifen. These lines, optionally also harboring Pxn-GFP and Lifeact-miRFP670, have previously been described by the authors (Chandra et al, 2022, PMID: 34861242). In addition, they use cells allowing local …

   Reviewer #1 (Public review):

   Summary:

   This is an interesting study describing intensity changes of lamellipodial Arp2/3 complex incorporation dependent on the substratum the cells are spreading on (PLL vs fibronectin), but also on manipulation of either contractility or osmotic pressure or even external mechanical load exerted onto cells, e.g., by increasing medium viscosity. The authors use quite fancy cell systems for their studies, first of all, a CRISPR-engineered fibroblast cell line in which both endogenous loci of the Arp2/3 complex subunit Arpc2 are tagged with mScarlet, but at the same time, conditionally removable using tamoxifen. These lines, optionally also harboring Pxn-GFP and Lifeact-miRFP670, have previously been described by the authors (Chandra et al, 2022, PMID: 34861242). In addition, they use cells allowing local photoactivation of Rac signalling through a Tiam1 activation module combined with Halo-tagged Arpc2, apparently stably co-expressed in tamoxifen-treated Arpc2-KO fibroblasts. These cells may or may not have been published previously.

   Overall, the study provides convincing evidence that Arp2/3 complex accumulation in the lamellipodium negatively correlates with its width and perhaps the mechanical load these actin networks are exposed to at the leading edge membrane, shown initially through allowing cells to spread on substrates in which the formation of integrin-based adhesions is poor (PLL) or stimulated (through fibronectin). In the latter case, lamellipodia are comparably narrow, perhaps reasonably well clutched, and thus feel sufficient counter-force at the leading edge membrane to build a dense, Arp2/3-dependent actin network. Albeit interesting and important to show as the authors did, these results are not entirely surprising given the literature published on actin remodeling in cells in conditions similar to those used by the authors (i.e., on PLL). Thus, the results should be better embedded into the context of this previous literature to more precisely reveal which aspects are new and interesting and which ones are more or less intuitive and expected.

   However, the authors also show yet another result, which is quite spectacular indeed, revealing dramatic local protrusion of a Rac-dependent lamellipodium on PLL only in the presence of methylcellulose, but not on PLL alone. Although the authors cannot fully explain the mechanisms causing these results, they are thought-provoking and will certainly stimulate future, relevant research on this topic and new insights. Altogether, I think this is an interesting study that can be shared rapidly, given that the authors provide more experimental detail and transparency concerning their used cell model systems. Aside from a few other suggestions for amendments and corrections, I would also recommend citing classical literature that has provided the basis for the interpretation of the results shown here, as specified below.

   Specific criticism and comments:

   (1) I feel the paper is interesting for actin remodeling and Arp2/3 complex aficionados, but quite difficult to read and to understand in places for non-experts in the field, so I think the text requires more detailed explanation of specific terms, model systems used, and overall correction of either grammatical or semantic errors, or colloquial language.

   (2) In general, I think the characterization of Arp2/3 complex incorporation into the lamellipodia of cells spreading on PLL versus FN is interesting, as it has not been done previously in such a systematic fashion to my knowledge. However, I think the authors could emphasize better how this relates to previously established structural features of actin filament networks, published on PLL. So more than 3 decades ago, Hotchin & Hall published clear evidence that starved fibroblasts can only form focal complexes or adhesions downstream of PDGF or LPA-stimulation if seeded on FN, but not on PLL (see Figure 1 in PMID: 8557752). Around the same time, Flinn and Ridley showed this virtual absence of classical, Rac-dependent focal complexes to be accompanied by the formation of beautiful, broad lamellipodia (see Fig. 1A in PMID: 8743960), which only formed in the absence of excess RhoA activity and thus contractility by the way (see also below). A few years later, Small et al summarized all these phenotypes in a comprehensive review and also showed that cells on PLL (similar to the rapidly migrating keratocytes) combined large, flat lamellipodia with tiny, nascent adhesions scattered throughout these structures (see Figure 2 in PMID: 10047522). These authors also noted that the sole inhibitor-mediated reduction of contractility could switch FN-phenotypes with narrow, ruffling lamellipodia and peripheral focal complexes back to a PLL-type phenotype of broad lamellipodia (see Figure 1 in PMID: 10047522). In the following decade then, different labs (Verkhovsky, Bershadsky, Vavylonis, Watanabe et al) showed beautiful phase contrast or fluorescence movies illustrating that the broad lamellipodial phenotype of cells plated on PLL was accompanied by low frequency membrane ruffling and instead a rapid, continuous rearward flow of continuously assembling actin filament networks, partly also directly shown with actin networks labeled with both LifeAct and Arp2/3 complex subunits (see e.g. PMIDs 18800171 and 22500749). In Alexandrova et al, 2008 (PMID 18800171), authors showed that the formation of adhesions in spreading cells triggers the transition from fast to slow flow (which is of course relevant to the current study and conclusions), whereas Ryan et al, 2012 (PMID 22500749) already established the broad incorporation of actin and Arp2/3 complex into the very broad lamellipodia formed on PLL by Xenopus fibroblasts and the rapid flow of both components from distal to proximal lamellipodial regions. None of these seminal studies has been cited, although they are highly relevant for the interpretation and conclusions of the results presented. I would strongly recommend specifically referring to these studies, as this will actually support the conclusions and interpretations drawn.

   (3) On the subject of literature, on the second page of the intro, end of 2nd paragraph, the authors describe Rac signaling to Arp2/3 complex through WRC considered essential for Arp2/3-mediated actin assembly at lamellipodial leading edges, but aside from one of their own papers cite none of the seminal studies by Insall, Scita, Stradal, Rottner, Bogdan labs having published seminal aspects on this pathway.

   Considering the rapid F-actin flow in lamellipodia, obviously accompanied by admittedly sparse but continuous Arp2/3 complex incorporation, it is not so surprising that the latter will be obligatory here, and also the accumulation of its prominent activator WRC, as well as the branch stabilizer cortactin. Thus, the data described on page 3 of the Results section could also be framed in the context of all this previously published knowledge, providing a more comprehensive and realistic view of the relevance and novelty of the described data.

   (4) In the abstract, the authors state in the context of the force-feedback mechanism established in vitro for the formation of Arp2/3 complex-dependent actin networks that "this phenomenon has not been explored through the examination of real-time responses of endogenous actin networks in cells". In my view, this is not correct, as in their prominent Cell paper, the Sixt laboratory has done exactly that (Mueller et al, 2017, PMID: 18800171). Although Mueller et al have not looked at Arp2/3 complex dynamics as far as I recall, they have still connected the extent and hence intensity of actin networks at the leading edges of keratocyte lamellipodia with the forces exerted onto them, including direct experimental manipulation of those forces. Although the study has been cited in an independent context, this point should be made clear, and the corresponding sentence in the abstract should be amended.

   (5) One point that struck me a little bit was the authors' detailed description of cell spreading on PLL and the quite strong variability of Arp2/3 incorporation dependent on the timing after spreading (as for instance the very strong and quite narrow Arp2/3 leading edge intensity at 2 hours post-seeding in Figure 3S2D). In the authors' view, they have worked with a very clean system, as they emphasized to even have eliminated the FN-locus in their cells, excluding the secretion of endogenous FN (PMID: 34861242), but how about ECM components potentially present in serum, such as, for instance, vitronectin? Indeed, it looks like the authors have done all experiments in the presence of 10% serum as far as I can see, although most of the classical PLL-experiments mentioned above have been performed with starved cells in the absence of serum. I think it would generate a more complete picture of the phenotypes and results as compared to the literature if the authors performed a subset of the key experiments on PLL without serum. I don't think the starving of cells as such is important and could be counteracted by simply lamellipodia-inducing growth factors adding into the spreading medium, traditionally perhaps PDGF or EGF (dependent on the receptor distribution of those fibroblasts), but the absence of serum would have two advantages: it would not only exclude any potential impact of serum-containing ECM components, but also alleviate the hyperstimulation of the Rho-pathway through LPA-bound BSA, the major serum-protein, which has previously been shown to counteract the "undisturbed" formation of PLL-type lamellipodia (see Figure 1B in Flinn & Ridley, PMID: 8743960).

   (6) Regarding the scanning EM-images shown in the Supplement, currently called Figure 3S2A and -B (in the text erroneously termed Figures 3S1A and-B, see above). I am not sure how representative these individual EM-images of the cell plated on PLL are, given the data of rapid rearward flow of actin and Arp2/3 complex subunits, at least at early stages of spreading. Again, the classical literature on PLL-type lamellipodia and, in particular, previously published movies of such lamellipodia suggest broad lamellipodia with few ruffles, and the opposite with cells plated on FN. So in this context, the scanning EM-data shown on both PLL and FN do neither fit the authors' own data very well nor the literature, and I would recommend making sure that the individual cells selected were (i) correctly annotated and (ii) representative of a specific time point of spreading actually fitting the previously described data.

   (7) It also surprised me to see that the authors describe the spreading process on PLL to actually be much slower than on FN (see Figure 3S2C - in the text Figure 3S1C). It is tempting to speculate that this might change if plating the cells in serum-free medium, as traditionally, full spreading and lamellipodia formation downstream of PDGF-stimulation (at least in 3T3 fibroblasts) is described to occur in the range of 10-30 minutes at maximum, and not several hours as shown here. This point could also be considered, or at least discussed.

   (8) The movies are of very high quality and beautiful to look at, but it would help the reader to get a bit more information in the legends (like the meaning of the time-stamps, which will display elapsed time in minutes:seconds I assume, but this info is missing from the legends as far as I can see. Also, it would help the reader to better mark in the movies when a specific treatment kicks in. For instance, in movie 10, the legend states treatment starts at 10:00 (minutes:seconds?), but it would help very much if the authors could paste the term "blebbistatin" directly into the movie, beginning with the frame of treatment start.

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3. 

   eLife

   May 11, 2026

   Reviewer #2 (Public review):

   The authors work with endogenously labeled Arp2/3 complexes in mouse fibroblast cell lines plated on surfaces coated with fibronectin or poly-L-lysine. They observe increased retrograde flow, but decreased actin and Arp2/3 densities, in the absence of integrin-based adhesions. Interestingly, they further find that an increase in branching density can be achieved in the absence of adhesion by a diverse set of perturbations, including blebbistatin, physical compression under agarose, and methylcellulose-mediated increases in extracellular viscosity. Although all of these conditions are likely to have pleiotropic effects on cell physiology and signaling, one plausible common denominator is that they promote cell spreading and may thereby increase membrane tension.

   This study addresses a question of broad …

   Reviewer #2 (Public review):

   The authors work with endogenously labeled Arp2/3 complexes in mouse fibroblast cell lines plated on surfaces coated with fibronectin or poly-L-lysine. They observe increased retrograde flow, but decreased actin and Arp2/3 densities, in the absence of integrin-based adhesions. Interestingly, they further find that an increase in branching density can be achieved in the absence of adhesion by a diverse set of perturbations, including blebbistatin, physical compression under agarose, and methylcellulose-mediated increases in extracellular viscosity. Although all of these conditions are likely to have pleiotropic effects on cell physiology and signaling, one plausible common denominator is that they promote cell spreading and may thereby increase membrane tension.

   This study addresses a question of broad interest. The relationship between protrusive actin assembly, resisting forces, and membrane tension has received considerable attention in recent years (for a recent overview, see PMID: 38991476). Earlier work established that branched actin networks can respond to force by increasing network density in vitro (PMID: 26771487; PMID: 35748355), and pioneering work from the Sixt laboratory showed that keratocyte lamellipodia adapt to resisting forces by increasing actin density in cells (PMID: 28867286). Against that background, the manuscript contains novel and insightful observations. At the same time, the current version would be strengthened by a more rigorous mechanistic analysis and by clearer reporting of experimental systems and statistics.

   Major points:

   (1) Engagement with prior work on membrane tension and protrusion.

   The relationship between protrusive actin assembly and membrane tension is a subject of major current interest (PMID: 38991476), and it is unfortunate that the authors do not engage more fully with seminal prior work on this subject. In particular, work from the Weiner laboratory showed that membrane tension can act as an inhibitor of cell protrusion and branched actin assembly, at least in some cell types (PMID: 22265410; PMID: 37311454). In addition, a membrane-tension-sensitive signaling pathway involving PLD2 and mTORC2 has been proposed to mediate this negative feedback (PMID: 27280401). These findings appear, at least at first glance, to contrast with the model advanced here, in which elevated membrane tension is associated with increased branching density. A more explicit discussion of these findings and of the apparent differences between systems would be essential. Testing the relevance of some of the proposed negative-feedback regulators, for example, mTORC2 or PLD2, under at least some conditions expected to increase membrane tension would substantially strengthen the manuscript.

   (2) The central assumption regarding membrane tension should be tested directly.

   Part of the model put forward by the authors rests on the assumption that most of the perturbations used to promote cell spreading, with the exception of hyperosmotic treatment, also increase membrane tension. This is a testable hypothesis. Multiple mechanical and optical methods have been established for this purpose, including tether pulling, micropipette aspiration, and fluorescent membrane-tension probes. Directly measuring membrane tension under at least a subset of the key perturbations would significantly strengthen the manuscript.

   (3) WAVE and cortactin localization should be quantified.

   The claim that WAVE and cortactin localization are independent of fibronectin-integrin engagement (Figure 2A-B) deserves to be established quantitatively. I appreciate that some variability is expected because these experiments use exogenous fluorescently tagged constructs, but the current presentation relies too heavily on representative kymographs. Quantitative analysis would make this conclusion more convincing.

   (4) The interpretation of the increased-viscosity experiments needs stronger physical justification.

   I am aware of the recent high-profile work showing that elevated extracellular viscosity can promote migration (PMID: 36323783), and the present manuscript is clearly supporting this. However, the physical basis for this perturbation is neither well reasoned nor explained clearly enough here. The authors use 0.6% methylcellulose of the 1500 cP grade (the relevant viscosity of the final medium should be stated explicitly btw!). Estimating the added viscosity at 7 cP = 0.007 Pa·s (up from 1 to 8 cP), one can formulate the rough back-of-the-envelope calculation for the added viscous stress:

   delta τ = delta η v/h

   where τ= viscous stress (Pa = pN/µm²), η = viscosity, v= protrusion speed, h = characteristic shear length scale. For cells protruding at 1 um/min, this resistance will be 0.00001-0.001 Pa. Even if the cells would protrude 100 times faster, the resistance would not exceed one pascal! Hence, the added bulk viscous stress opposing protrusion at this viscosity appears negligible relative to the known force-generating capacity of lamellipodia. This does not invalidate the biological phenotype, but it does suggest that the interpretation should be much more careful.

   (5) Cell lines and experimental systems are insufficiently described.

   Most biological experiments in this manuscript appear to have been performed in engineered mouse fibroblast lines, but the Methods do not provide sufficient clarity about which specific cell lines were used in which experiments. More concerning, the manuscript refers inconsistently to the base model as both a mouse dermal fibroblast line and MEFs, while the only clearly distinct named line appears to be JR20 fibroblasts used for traction-force microscopy. Along similar lines, the Arp2/3 knockout cells in Figure 2 are not adequately explained in the Results, Methods, or figure legends, regarding how these cells were generated or how the knockout was validated. The authors only later note in the Discussion that these conditional knockouts were described in an earlier paper. In general, the manuscript would benefit from much more explicit reporting of which cell line or derivative was used in each experiment.

   (6) Some experiments and quantifications appear to suffer from limited replication.

   For example, the optogenetic Rac activation experiment in Figure 2E appears to have been performed possibly only for a single cell per condition, since the raw intensity traces are shown without clear indicators of variability. If that reading is correct, this is below the standard typically expected for mechanistic support and seriously reduces confidence in the strength of this particular conclusion.

   (7) Statistical reporting needs clarification.

   Although the Methods state that the graphs show 95% confidence intervals, the manuscript does not clearly define the underlying statistical unit for many quantified datasets. In several figures, sample sizes are reported as numbers of cells pooled across only two or three independent experiments, but it is not clear whether the authors performed statistical analyses on pooled single-cell measurements or on experiment-level means. The authors should explicitly state for each quantified panel what n represents, what the error bars denote, which statistical test was used, and whether the analyses were performed on per-cell values or on independent experimental replicates.

   (8) The Discussion is rather expansive relative to the amount of experimental evidence presented.

   Parts of the Discussion feel more speculative and interpretive than necessary, and the manuscript would be strengthened by focusing the Discussion more tightly on the principal findings, limitations, and immediate implications of the work.

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4. 

   eLife

   May 11, 2026

   Reviewer #3 (Public review):

   Summary:

   Butler et al. investigated how different force mechanisms influence Arp2/3-related branched actin networks at the leading edge of lamellipodial protrusions in mouse dermal fibroblasts. In particular, their study aimed at characterizing the specific contribution and interplay between load force and adhesion signaling on the regulation of branched actin networks in live-cell experiments using endogenously one-labeled Arp2/3 subunit. A key finding of their work is that by plating fibroblasts on two different substrates supporting or not integrin engagement, they observe striking differences in branched network architectures that cannot be explained solely by integrin signaling. Instead, several of their results point to mechanical feedback resulting from changes in membrane tension during spreading, …

   Reviewer #3 (Public review):

   Summary:

   Butler et al. investigated how different force mechanisms influence Arp2/3-related branched actin networks at the leading edge of lamellipodial protrusions in mouse dermal fibroblasts. In particular, their study aimed at characterizing the specific contribution and interplay between load force and adhesion signaling on the regulation of branched actin networks in live-cell experiments using endogenously one-labeled Arp2/3 subunit. A key finding of their work is that by plating fibroblasts on two different substrates supporting or not integrin engagement, they observe striking differences in branched network architectures that cannot be explained solely by integrin signaling. Instead, several of their results point to mechanical feedback resulting from changes in membrane tension during spreading, regulating the density of branched actin networks. Finally, by modifying the extracellular viscosity, the authors suggest that the stress generated at the actin-membrane interface would play a key role in regulating branched actin density in protrusions.

   Major Strengths:

   (1) The combination of methods used in this paper (endogenous labeling of Arp2/3, Arp2/3 genetic knockout, optogenetic activation of Rac) provides a unique opportunity to monitor spatial and temporal reorganization of endogenous branched networks generated by Arp2/3 in live cells in response to different biochemical and mechanical manipulations.

   (2) The authors provide a deep characterization of the actin-network organization and dynamics observed when plating cells on different substrates, engaging or not integrins (Figure 1 and associated supplementary: intensity and width of the signal in protrusions, retrograde flow, incorporation of actin to the edge, nascent focal adhesions), which serves as a strong basis to build the rest of the paper. They also offer a comprehensive analysis of the different parameters that could explain the lack of dense branched actin network at the leading edge of fibroblasts grown on PLL-coated surfaces (they exclude the contribution of reduced branch nucleation by NPF or insufficient branch stabilization in Figure 2, the insufficient integrin-mediated signaling activating NPF in Figure 2).

   (3) After having ruled out the influence of adhesion signaling in the regulation of branched actin-network density at the leading edge of the cells, the authors demonstrate that the enrichment of Arp2/3 at the leading edge is evolving together with cell spreading, suggesting a possible role of membrane tension in the process (Figure 3 and associated supplementary). To prove their point, they tested numerous methods to promote adhesion-independent cell spreading (Figures 4 to 6), while describing well the limitations of each of these techniques. These methods included promoting rapid spreading on PLL-coated substrate using blebbistatin or physical compression under agarose, and finally increasing extracellular viscosity by treating cells with methylcellulose. All of these treatments led to very consistent results upon the increase in membrane tension, supporting the idea of membrane tension controlling the branched actin organization of cells. This conclusion was further supported by an experiment (Figure 4 S1) in which a hyper-osmotic shock was performed, increasing the actin-membrane interface stress while keeping the spreading area of cells, which led to a drastic increase in Arp2/3 density at the protrusions.

   (4) By activating Rac optogenetically in cells plated on PLL treated with methylcellulose (Figure 8), the authors observe the formation of robust protrusions enriched in Arp2/3, showing that increased extracellular viscosity can bypass the requirement for ECM proteins to activate protrusion driven by signaling.

   Weaknesses:

   (1) Although the lamellipodial architecture in cells plated on PLL appears very different from the one developed by cells grown on fibronectin (Figure 1, wider and less homogenous), the branched network is still present, and one may wonder how these differences can affect the functionality of the lamellipodia (for example, by measuring the impact on migration in 2D and 3D systems).

   (2) To explain the differences observed in the branched actin networks developed by cells on PLL and FN, the authors envision several hypotheses, among which signaling factors or branched-promoting factors would be decreased in the absence of integrin adhesions. They could have, in addition, assessed actin network dynamics and turnover (we could imagine that competition between Arp2/3- and non-Arp2/3- driven structures could be different in the presence or absence of adhesions, the competition being nicely visible from Figure 2B and 2C, where, in the absence of Arp2/3, cells form prominent filopodia).

   (3) All of the methods used to apply physical forces on barbed ends have their own caveats and alter not only membrane tension (but the limitations are discussed in the paper). The paper may have benefited from micropatterning the cells to either reduce or force the spreading of cells in a controlled fashion. In addition, the conclusions on levels of interface stress between plasma-membrane and the barbed-ends of actin lamellipodial networks rely on an estimate of the effect of perturbations rather than on actual measurements of these stress levels.

   Likely impact of the work on the field, and the utility of the methods and data to the community:

   Although the finding that branched actin networks respond to the application of physical force by increasing their density was already known from previous in vitro studies, this paper offers a detailed and compelling characterization of the reorganization of endogenously labelled branched actin networks upon different mechanical perturbations. In addition to showing the effect of increased extracellular viscosity on promoting branched actin network densification in the absence of ECM, this paper sheds new light on the interplay between signaling and mechanics in regulating protrusion and spreading. While the authors show that both signaling and mechanical feedback are important regulators of branched actin regulation and cell spreading, they demonstrate that optogenetic Rac activation is not sufficient to trigger branch network formation in the absence of sufficient mechanical support. They thus propose that biochemical signaling would act at a different level than mechanics by promoting protrusion persistence and coherence. This work will therefore impact the field of cell biology in offering a new perspective to understand the interplay between mechanical and biochemical feedback in 2D and 3D migration. It may also have broader implications as the formation of branched actin networks under the regulation by mechanical loads has been shown to be involved in other processes such as endocytosis.

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5. 

   Version published to 10.64898/2026.03.06.710140 on bioRxiv

   Mar 9, 2026