{"id":"27a2f612-ece1-4692-8325-6ffaf4a08077","arxiv_id":"2505.01717","paper_version":1,"verdict":"CONDITIONAL","confidence":"MODERATE","novelty_score":6.0,"correctness_risk":"medium","formal_verification":"none","parameter_count":4,"one_line_summary":"Confined actomyosin gels take on the shape of their microwell, with actin polymerization controlling gel shape and myosin contractility controlling gel size.","lead":"This paper shows that actomyosin, a gel of actin and myosin motor proteins, contracts differently depending on the shape of the tiny container it is confined in, and that the container shape is imprinted on the final gel. It combines microscope experiments on frog-egg extracts with a fluid model to map how myosin force and actin polymerization tune the size and shape of the contracted gel, an idea for making shape-adaptive biomaterials.","discovery_kind":"extension","skeptic_critique":{"model":"deepseek-v4-flash","headline":"Shape transfer in the model is driven by the assumed boundary-enhanced polymerization k_surf^p/k_bulk^p=0.1, which the text calls necessary for wave generation but which is never measured in the microwells; the advection mechanism may be reproducing shape through this prescribed source rather than…","rationale":"In good faith, the paper presents a credible experimental phenomenon and a plausible active-fluid model: the cluster roundness data, the chemical perturbation experiments, and the complex confinement examples all support the empirical claim that microwell geometry affects gel shape. The reader's weakest assumption was the single-component treatment in Eq. (2), where myosin density is taken proportional to F-actin density. That is a legitimate concern, but the more load-bearing assumption for the mechanism is the boundary-enhanced polymerization rate: the text says k_surf^p/k_bulk^p=0.1 is necessary for wave generation, and this spatial source is what carries the boundary anisotropy into the bulk in the model. If this assumption is wrong for NOA81/PEG-PLL microwells, the advection mechanism in Fig. 3 may not transfer shape at all, even if myosin approximately follows actin. A uniform-kp simulation is a decisive internal check, and a FRAP or actin-incorporation experiment at the wall would test the assumption externally. These checks would either reinforce or weaken the causal story, but they do not change the overall verdict: the paper is still a CONDITIONAL acceptance because the empirical findings are strong while the mechanistic explanation depends on untested model assumptions and qualitative parameter matching.","tokens_in":9679,"tokens_out":8814,"duration_ms":97965,"concrete_test":"Run the simulation with k_surf^p = k_bulk^p (uniform polymerization) and otherwise identical parameters, including Pe=50 and the same phase-field confinement geometries. If the W/D-dependent roundness trend in Fig. 3(d) disappears, or if the periodic inward flow no longer forms, then boundary-enhanced polymerization is a necessary ingredient of the claimed advection mechanism, and an experimental measurement of the spatial polymerization profile near the microwell wall (e.g., FRAP or fluorescent actin-incorporation imaging) is required to substantiate the causal story. If the uniform-kp simulation still reproduces the shape-transfer trend, the concern is weakened.","verdict_should_be":"UNCHANGED","load_bearing_attack":"The central claim is that confinement shape is imprinted on the contracted actomyosin gel by myosin-driven actin flow. In the model, the shape information enters the dynamics primarily through the polymerization source in Eq. (2): kp is not uniform but is prescribed to be ten times larger near the boundary (k_surf^p/k_bulk^p=0.1). The text explicitly states this boundary-enhanced polymerization is a condition necessary for wave generation and holds for all the results, but no experiment in the NOA81/PEG-PLL microwell system measures the spatial profile of actin polymerization. If kp were actually uniform, the boundary would enter only through the no-flux condition and the phase-field geometry, and the simulation might fail to reproduce the roundness trend in Fig. 3(d). Thus the causal claim 'via myosin-driven actin flow' is not independently established: the model may be transferring the boundary shape through a prescribed, unmeasured source term rather than through active-flow mechanics alone. This concern does not undermine the empirical observation of shape transfer, but it makes the mechanistic explanation conditional on a specific boundary-polymerization assumption.","agreement_with_reader":"partial"},"referee_report":{"model":"deepseek-v4-flash","summary":"The manuscript reports an experimental and numerical study of actomyosin networks extracted from Xenopus laevis eggs and confined in microfabricated NOA81 microwells of controlled geometry. The authors show that as the microwell shape changes from circular to semicircular (increasing W/D), the contracted actomyosin cluster changes from circular to crescent-like, quantified by the roundness R = m/M (Figure 1h), while cluster area scales with microwell area (Figure 2). They model the system with an active fluid theory (Eqs. 1–3) that couples force balance, actomyosin mass conservation, and a phase-field boundary, assuming boundary-enhanced actin polymerization (k_bulk^p/k_surf^p = 0.1). With hand-tuned parameters Pe = 50 and k_surf^p = 2.5, the simulations reproduce the experimental roundness trends (Figure 3c,d). Drug perturbations with Cytochalasin D (CytoD) and Calyculin A (CalA) are used to test model predictions: CytoD increases roundness and CalA decreases cluster area (Figure 4). The study is extended to square and more complex microwells (Figure 5), where the model again qualitatively captures cluster shape. The central claim is that asymmetric confinement geometry is transferred to the contracted gel through myosin-driven actin advection, and that tuning contractility and polymerization rate gives control over gel size and shape.","tokens_in":9935,"tokens_out":3196,"duration_ms":35596,"significance":"If the mechanistic claim is established, the paper offers a useful bottom-up design principle for active, shape-adaptive biomaterials and adds to the understanding of geometric control of cytoskeletal organization in cell-sized confinement. The experiments are carefully quantified, including roundness and area measurements over multiple microwell shapes and sizes, and the drug perturbations provide an independent, if partially confounded, test of the model's qualitative predictions. The model is borrowed from the authors' prior work and is not introduced as new theory, but its application to asymmetric and complex confinements is new and generates falsifiable predictions. The phase diagrams in Figures 3f,g and 5e are a genuine strength because they connect model parameters to experimentally accessible perturbations. However, the model's central role in establishing the mechanism rests on two assumptions that are not directly measured in this system: the boundary-enhanced polymerization profile and the proportionality between myosin and F-actin densities.","major_comments":[{"comment":"The boundary-enhanced polymerization profile k_bulk^p/k_surf^p = 0.1 is prescribed in the model and stated to be 'a condition necessary for wave generation' that 'holds for all of the following results,' but no experiment in the NOA81/PEG-PLL microwell system measures the spatial profile of actin polymerization. Since the boundary shape enters the dynamics through this prescribed source term, the simulations could be reproducing the observed shape transfer because the model was given a shape-dependent source rather than because myosin-driven advection alone imprints the boundary geometry. Please provide a direct test of the polymerization profile in the microwells (for example, monomer incorporation or branching-density measurements near the wall), or a robustness check showing that the roundness trend in Figure 3d persists for a substantially smaller k_surf^p/k_bulk^p ratio.","section":"§3(b), Eq. (2)"},{"comment":"The two main simulation parameters, Pe = 50 and k_surf^p = 2.5, are chosen after seeing the control experimental roundness values, and the simulations are reported without error bars, number of independent runs, or convergence checks with respect to grid resolution and integration time. As these parameters set the contractile stress and the boundary polymerization rate, the agreement in Figure 3d does not by itself confirm the mechanism. Please report run-to-run variability, a small parameter sweep around Pe=50 and k_surf^p=2.5, and at least a qualitative convergence statement so the reader can judge whether the roundness and area trends are robust.","section":"§3(c,d), Figures 3(c)–3(g)"},{"comment":"The model assumes myosin density is proportional to F-actin density, reducing mass conservation to a single component. If myosin in the Xenopus extract redistributes independently of actin (for example, by differential binding, advection, or detachment under contraction), then the advective coupling that the model identifies as the shape-transfer mechanism would not necessarily hold in the experiments. The manuscript does not report any myosin labeling or otherwise justify this proportionality in the microwell geometry. Please add a direct justification or an experimental check (for example, myosin fluorescence in the contracted cluster versus the flow region).","section":"Eq. (2) and accompanying text"},{"comment":"The CytoD and CalA perturbations are presented as clean tests of decreasing k_surf^p and increasing Pe, respectively, but CytoD is known to reduce effective actomyosin contractility, and the text itself acknowledges that the absence of a cluster-size reduction under CytoD 'could be due to the reduction in effective actomyosin contractility upon inhibition of actin polymerization.' This confound weakens the inference that the increased roundness in Figure 4c is specifically a k_surf^p effect. Similarly, the CalA roundness change is described as 'minute (or negligible),' yet Figure S6 shows significant decreases for W/D = 0.0, 0.5, and 1.0; the text should reconcile these statements. Please discuss the confound explicitly and, if possible, add a measurement of myosin activity or actin filament length that separates the two effects.","section":"§4, Figures 4(c), 4(d), and S6"}],"minor_comments":[{"comment":"The text reports n = 4 microwells for some averages in Figure 1(i) and Figure 2(c), which is small; please state explicitly in the figure caption or text whether these data points come from independent experiments or repeated fields of view, and consider showing individual data points in the boxplots.","section":"Figure 1(h) and Figure 2(b,c)"},{"comment":"Cluster identification used thresholding and, in some cases, manual identification; please describe the criteria for manual selection and whether the roundness results are sensitive to the threshold choice, as this could affect the quantitative claims.","section":"Materials and Methods, Image analysis"},{"comment":"The quartic parameter is defined in the Methods but the relationship between its magnitude and the visually distinct star-like shape is not intuitive; a short explanation of why the control value 0.069 and CalA value 0.088 correspond to square versus star-like would improve readability.","section":"Figure 5(b) and 5(d)"},{"comment":"The symbols k_surf^p, k_bulk^p, Pe, and lambda are used with inconsistent formatting (subscripts and superscripts) in the text and equations; please unify the notation, particularly in the caption of Figure 3.","section":"General notation"},{"comment":"The text says 'roundness decreased as k_surf^p increased' and 'slightly dependent on Pe,' but the phase diagram in Figure 3(f) appears to show a non-monotonic or weak dependence; please state whether the quoted dependence is the average trend over the plotted region or a qualitative summary.","section":"§3(d), Figure 3(f)"}],"recommendation":"major_revision","confidential_remarks":"The paper is a solid combination of a clean microfabrication experiment and a borrowed active-fluid model, but the mechanistic conclusion depends on two unmeasured assumptions (boundary-enhanced polymerization and myosin–actin proportionality) and on hand-tuned parameters. I do not see a fatal flaw, but the authors need to add robustness checks or direct measurements to make the central claim convincing. The scope fits cond-mat.soft and the experimental findings themselves are likely citable even if the modeling is weakened."},"author_rebuttal":null,"desk_editor":{"model":"deepseek-v4-flash","letter":"Colleague,\n\nThis is a paper you'll want to know about: Negi et al. confine Xenopus extract actomyosin in NOA81 microwells of controlled shape (circular segments with W/D from 0 to 1, plus squares and letters). The punchline is that the final contracted gel adopts the confinement shape—roundness decreases smoothly with W/D—and the square and letter shapes are imprinted on the cluster. They also show a clean separation of controls: CytoD (lower polymerization) makes clusters rounder, CalA (higher contractility) makes them smaller, matching their active-fluid phase diagrams. That is a genuinely new dataset and a useful design handle.\n\nWhat's well done: the W/D mapping is systematic, the main trend has reasonable sample sizes, and the drug perturbations are an independent test of the model's qualitative predictions. The square and letter confinements are a nice generalization. The model is borrowed from the authors' earlier PRR work, but the phase diagrams generate predictions that the experiments then confirm—you don't often see that clean a loop.\n\nWhere I'd press: (1) The model's shape transfer leans on boundary-enhanced actin polymerization (k_surf^p/k_bulk^p=0.1), which is assumed, not measured in this system. The stress-test note is onto something: the text says this assumption holds for all results, and without it the simulation might not reproduce the roundness trend. So the mechanistic claim \"via myosin-driven actin flow\" is really \"via advection plus a prescribed surface polymerization source.\" That doesn't sink the paper—the experimental shape transfer is robust—but it makes the mechanism conditional on an unmeasured parameter. (2) Simulation results have no error bars or convergence checks, and Pe and ksurf_p are hand-tuned. (3) No code or data are shipped, and cluster segmentation includes a manual step. These are referee-level requests, not fatal flaws.\n\nBottom line: this is a serious paper worth refereeing. The empirical mapping is solid and the design-principle claim is useful. The modeling is qualitative but clearly labeled; the missing measurement of ksurf should be acknowledged and ideally tested (e.g., by varying surface chemistry or imaging polymerization near the wall). I'd send it to review and ask for code/data plus a more careful statement about the boundary-polymerization assumption.\n\nRecommendation: send to peer review.","headline":"Solid experimental mapping of confinement-shape transfer to contracted actomyosin gels, with a plausible but partly assumed model mechanism; deserves peer review.","tokens_in":10446,"tokens_out":2327,"would_cite":true,"duration_ms":24197,"reading_group":"yes","serious_thinker":"yes","would_accept_peer_review":true},"rs_alignment":null,"lean_confirmation":null,"pith_extraction":{"msc":[],"pacs":[],"model":"deepseek-v4-flash","headline":"Confined frog-egg actin gels take on the shape of the microwell, with myosin-driven flow and polymerization rate controlling size and shape.","keywords":["actomyosin gel","active fluid model","geometric confinement","microwell","myosin contractility","actin polymerization","cytoskeleton","Péclet number"],"falsifier":"Dual-color fluorescence imaging of myosin and F-actin during contraction in a semicircular microwell, quantifying local densities along the flat side and the curved wall, would settle the mechanism: if myosin and F-actin densities drift out of proportion before or during cluster formation, the single-component active fluid model is not the right description. A complementary check is to reconstruct the final cluster by advecting the measured initial density field $\\rho$ under the measured velocity field; if the predicted shape disagrees with the observed cluster, then myosin-driven advection alone does not transfer the wall shape.","tokens_in":9492,"feed_emoji":"📐","tokens_out":11350,"duration_ms":105967,"temperature":0.7,"pith_summary":"By confining frog-egg actomyosin extracts in microfabricated microwells shaped as circular segments, squares, and letters, this paper shows that the boundary shape is not merely a container: it is transferred to the contracted gel that forms inside. High-speed imaging and particle image velocimetry reveal an inward, myosin-driven actin flow, and the final cluster inherits the well's asymmetry — crescents in semicircular wells, square and even star-like shapes in square wells. The paper argues, with an active fluid model, that this shape transfer is an advective process: myosin contractile stress drives an inward flow that carries actin toward the cluster, while enhanced actin polymerization near the boundary determines how faithfully the wall shape is copied. Because myosin contractility and actin polymerization can be tuned chemically, the authors propose that gel size and shape in confined architectures can be controlled by two parameters: effective contractility and surface polymerization rate. If right, this gives a design rule for making shape-adaptive biomaterials from cytoskeletal motors.","feed_headline":"Myosin flow imprints microwell shape on actin gels","feed_subtitle":"Frog-egg actin networks shrink into crescents, squares, or letters; force and polymerization tune the gel.","key_machinery":"The load-bearing object is an active fluid model of the actomyosin gel coupled to a phase field for the microwell boundary. The gel is treated as a viscous fluid whose stress has a passive part and an active contractile part proportional to F-actin density, with myosin density assumed proportional to F-actin density so that mass conservation reduces to a single component. The dynamics are three coupled equations: a Stokes-like force balance for the velocity field, a reaction-diffusion-advection equation for actomyosin density with polymerization source and depolymerization sink, and a phase-field equation for the boundary. Two dimensionless parameters carry the argument: the Péclet number, the ratio of myosin-driven advection to diffusion, and the surface polymerization rate, which is taken to be higher near the wall than in the bulk. This model converts the geometric question — does boundary shape reach the cluster? — into a testable statement: the inward advective flux transfers wall asymmetry to the contracting cluster, while the balance of polymerization and contractility decides how round or how faithful to the boundary the final gel is.","core_discovery":"The central claim is that the final shape of a contracting actomyosin gel inside a closed confinement is set by the shape of the confinement itself, transmitted through myosin-driven advection rather than by passive diffusion or spontaneous symmetry breaking alone. In circular microwells the network contracts to a circular cluster, while in semicircular wells it forms a crescent whose roundness decreases monotonically as the flat-side-to-diameter ratio rises from 0 to 1, independent of well diameter. A phase-field active fluid model reproduces the trend: the gel is an active viscous fluid carrying myosin-generated contractile stress, mass conservation is a reaction-diffusion-advection equation for F-actin density with surface-enhanced polymerization, and the phase field imposes the boundary. The paper also claims that the same mechanism works in square and letter-shaped wells, where the cluster copies the boundary, and that two parameters — the Péclet number (advective transport relative to diffusion) and the surface polymerization rate — separately set size and shape: higher contractility shrinks the cluster, higher surface polymerization makes it more asymmetric. Chemical perturbations (cytochalasin D inhibiting polymerization, calyculin A increasing myosin contractility) shift the observed clusters in the direction the model predicts.","pith_inferences":["A test the paper does not run: reconstruct the final cluster by advecting the measured initial F-actin field with the measured velocity field; agreement would directly confirm the advective shape-transfer mechanism, and disagreement would challenge it.","If myosin density is truly slaved to F-actin, then interventions that decouple myosin from actin filaments, such as disabling myosin's actin-binding domain, should abolish shape transfer even when the gel still contracts; dual-color imaging of myosin and F-actin during contraction could test this prediction.","The corner outgrowths that appear in squares and letters suggest a criterion the model does not spell out: fidelity of shape transfer should depend on local boundary curvature, with sharper corners concentrating advective flux; measuring cluster shape as a function of corner angle would quantify that dependence.","The paper's two-parameter description implies a master-curve collapse: plotting roundness and area ratio from many chemical doses against an inferred Péclet number and surface polymerization rate should collapse onto the simulation phase diagrams; this is a quantitative extension the authors do not report."],"forward_implications":["In any confined actomyosin gel with advection-dominated transport, the contracted cluster should inherit the global asymmetry of the wall rather than relaxing to a circle, as seen for crescents, squares, and letter-shaped wells.","The two-parameter phase diagram implies that a myosin activator shrinks the gel while leaving shape nearly unchanged, whereas an actin polymerization inhibitor makes the gel rounder without reliably shrinking it.","Microwell diameter at fixed shape does not change roundness, so the shape transfer is a geometric effect of boundary asymmetry rather than a size-dependent instability.","Because the model reproduces square and letter-shaped compartments, the same design rule should apply to arbitrary polygonal and patterned boundaries: the wall acts as a template whenever advection dominates diffusion.","Chemical inhibition or activation of the two molecular processes gives an experimental route to resize and reshape confined cytoskeletal gels on demand."],"supporting_citations":[{"why":"It supplies the earlier demonstration that confining boundaries steer the contraction of active gels, which this paper extends to asymmetric and complex microwells.","marker":"[10]"},{"why":"It provides the droplet-confined actomyosin baseline — inward actin flow, cluster formation, and positioning — against which the circular microwell measurements are compared.","marker":"[13]"},{"why":"It provides the active fluid model and the Péclet-number and surface-polymerization parameter picture that the paper adapts to asymmetric boundaries.","marker":"[15]"},{"why":"It establishes the frog-egg extract as a system whose bulk cytoplasm polymerizes actin and supports myosin-driven gelation-contraction.","marker":"[21]"},{"why":"It supplies evidence that confinement induces actin flow in meiotic cytoplasm, the basis for assuming enhanced actin polymerization near the boundary.","marker":"[25]"},{"why":"It contributes the active-fluid force-balance and mass-conservation equations for pulsatory patterns that the numerical model is built on.","marker":"[31]"},{"why":"It identifies cytochalasin D as an actin polymerization inhibitor, the perturbation used to lower the effective surface polymerization rate.","marker":"[33]"},{"why":"It establishes calyculin A as a myosin phosphatase inhibitor that increases myosin contractility, the perturbation used to raise the effective Péclet number.","marker":"[34]"}],"fun_headline_variants":["Myosin flow imprints microwell shape on actin gels","Actomyosin gels copy boundary shape via myosin drift","Confined actin gels take well shape from myosin advection","Geometric confinement directs actomyosin gel contraction","Myosin and actin polymerization tune gel shape in wells"],"cache_read_input_tokens":3200,"weakest_assumption_plain":"The load-bearing premise is that myosin density stays proportional to F-actin density throughout contraction, so one concentration variable represents the whole network; if myosin redistributes independently of the actin filaments in the extract, the advection-based shape transfer predicted by the model would not necessarily occur.","fun_headline_variants_meta":{"raw":{"variants":["Myosin flow imprints microwell shape on actin gels","Actomyosin gels copy boundary shape via myosin drift","Confined actin gels take well shape from myosin advection","Geometric confinement directs actomyosin gel contraction","Myosin and actin polymerization tune gel shape in wells"]},"model":"deepseek-v4-flash","effort":"low","cost_usd":0.000288,"raw_usage":{"total_tokens":1698,"prompt_tokens":963,"completion_tokens":735,"prompt_tokens_details":{"cached_tokens":384},"prompt_cache_hit_tokens":384,"prompt_cache_miss_tokens":579,"completion_tokens_details":{"reasoning_tokens":656}},"tokens_in":579,"tokens_out":735,"duration_ms":7645,"temperature":1.0,"reasoning_tokens":656,"cache_read_input_tokens":384,"cache_creation_input_tokens":0},"cache_creation_input_tokens":0},"created_at":"2026-08-16T04:11:10.272280+00:00","model_set":{"reader":"deepseek-v4-flash"},"falsifier":"Dual-color fluorescence imaging of myosin and F-actin during contraction in a semicircular microwell, quantifying local densities along the flat side and the curved wall, would settle the mechanism: if myosin and F-actin densities drift out of proportion before or during cluster formation, the single-component active fluid model is not the right description. A complementary check is to reconstruct the final cluster by advecting the measured initial density field $\\rho$ under the measured velocity field; if the predicted shape disagrees with the observed cluster, then myosin-driven advection alone does not transfer the wall shape.","supporting_citations":[{"cited_title":"C.; Kr \\\"o ger, M.; Bausch, A","cited_arxiv_id":null,"evidence_quote":"It supplies the earlier demonstration that confining boundaries steer the contraction of active gels, which this paper extends to asymmetric and complex microwells."},{"cited_title":"T.; Miyazaki, M","cited_arxiv_id":null,"evidence_quote":"It provides the droplet-confined actomyosin baseline — inward actin flow, cluster formation, and positioning — against which the circular microwell measurements are compared."},{"cited_title":null,"cited_arxiv_id":null,"evidence_quote":"It provides the active fluid model and the Péclet-number and surface-polymerization parameter picture that the paper adapts to asymmetric boundaries."},{"cited_title":"M.; Wühr, M.; Anderson, G","cited_arxiv_id":null,"evidence_quote":"It establishes the frog-egg extract as a system whose bulk cytoplasm polymerizes actin and supports myosin-driven gelation-contraction."},{"cited_title":"Confinement induces actin flow in a meiotic cytoplasm","cited_arxiv_id":null,"evidence_quote":"It supplies evidence that confinement induces actin flow in meiotic cytoplasm, the basis for assuming enhanced actin polymerization near the boundary."},{"cited_title":"V.; Bois, J","cited_arxiv_id":null,"evidence_quote":"It contributes the active-fluid force-balance and mass-conservation equations for pulsatory patterns that the numerical model is built on."},{"cited_title":"F.; Flanagan, M","cited_arxiv_id":null,"evidence_quote":"It identifies cytochalasin D as an actin polymerization inhibitor, the perturbation used to lower the effective surface polymerization rate."},{"cited_title":"Calyculin A, an enhancer of myosin, speeds up anaphase chromosome movement","cited_arxiv_id":null,"evidence_quote":"It establishes calyculin A as a myosin phosphatase inhibitor that increases myosin contractility, the perturbation used to raise the effective Péclet number."}],"review_version":1}