Mechanical interaction enables a collective mode of protocell proliferation
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eLife Assessment
This study presents a key finding: self-generated mechanical stresses enable collective protocell proliferation without dedicated division machinery, offering insight into primitive life's population growth. While quantitative imaging, membrane tension measurements, and computational modeling support the mechanism, establishing causal links between deformation and division and testing sensitivity assumptions would strengthen the work. Overall, the work reports important findings, and although the evidence in support of the conclusions is largely solid, some incomplete elements need to be addressed.
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Abstract
The proliferation of primitive life-forms (known as protocells) without sophisticated cell-division molecular machineries has been an intriguing question in evolutionary biology, synthetic biology, and living matter physics. While various modes of protocell proliferation at the individual level have been proposed, the growth dynamics of protocell colonies (i.e., protocolonies) received less attention. Here we chose to study this question in protocolonies consisting of densely packed protocells derived from wall-deficient L-form bacteria. We discovered that protocolonies proliferated robustly under spatial confinement, while isolated protocells failed to divide and eventually experienced membrane rupture due to imbalance of surface and volume growth. Combining results from quantitative imaging and computational modeling, we attributed this unexpected finding to mechanical shearing between densely packed protocells driven by their growth activities; such mechanical shearing enhances cell deformation, thereby enabling cell division and sustaining population growth in a protocolony. Our study reveals a unique role of self-generated mechanical stresses in the lifestyle of primitive life-forms. The findings may help to understand and control the collective growth dynamics of synthetic protocells or active droplets.
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eLife Assessment
This study presents a key finding: self-generated mechanical stresses enable collective protocell proliferation without dedicated division machinery, offering insight into primitive life's population growth. While quantitative imaging, membrane tension measurements, and computational modeling support the mechanism, establishing causal links between deformation and division and testing sensitivity assumptions would strengthen the work. Overall, the work reports important findings, and although the evidence in support of the conclusions is largely solid, some incomplete elements need to be addressed.
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Reviewer #1 (Public review):
Li and Wu, in this article, explore the proliferation of wall-less L-forms derived from Bacillus subtilis as mimics for protocells and report an interesting new mechanism for their proliferation. The authors carry out live-cell imaging of the L-forms and find that the clusters of cells forming proto-colonies proliferate better than the isolated single cells of L-forms. They further examine the causes for this indefinite proliferation of proto-colonies of L-forms, as compared to the isolated cells, which lyse and die out sooner. The authors show that when L-forms exist as isolated single cells, the growth in volume exceeds the rates at which surface area increases, leading to lysis. The authors further quantify the circularity and effective radius in growing proto-colonies, qualitatively estimate membrane …
Reviewer #1 (Public review):
Li and Wu, in this article, explore the proliferation of wall-less L-forms derived from Bacillus subtilis as mimics for protocells and report an interesting new mechanism for their proliferation. The authors carry out live-cell imaging of the L-forms and find that the clusters of cells forming proto-colonies proliferate better than the isolated single cells of L-forms. They further examine the causes for this indefinite proliferation of proto-colonies of L-forms, as compared to the isolated cells, which lyse and die out sooner. The authors show that when L-forms exist as isolated single cells, the growth in volume exceeds the rates at which surface area increases, leading to lysis. The authors further quantify the circularity and effective radius in growing proto-colonies, qualitatively estimate membrane tension and suggest that the confined space allows for mechanical shear in these cells. They propose that the mechanical stress on the membranes from adjacent cells in confined spaces deforms membranes and supports cell division to keep the population growing. These findings are also supported by modelling the proto-colonies in quasi-2D planes.
The study is quite interesting and significant as it has implications for both evolutionary aspects as well as clinical importance, given the proliferation of certain pathogens as L-forms. The aspect of carrying out long-term imaging of colonies of L-forms as spatially constrained entities and the findings are fascinating. While the conclusions presented are backed by experiments, I only have a few questions concerning the proposed mechanism of division and proliferation of these proto-colonies.
(1) The authors propose that the growth of neighbours leads to shearing forces in membranes and show that membrane tension increases at the periphery of the proto-colonies. They suggest that the increased membrane tension leads to a greater chance of deformation, enabling cell division. However, it is not quite clear how greater membrane tension could lead to cell division. Studies have suggested that membrane fluidisation is important for the cytokinesis event, which includes FtsZ-based division (Ramirez-Diaz, 2025).
(2) Thus, it becomes quite important to rule out any role for the cytoskeletal proteins in the observed division with an increase in membrane tension. The authors note in line 188 that the division in protocells is independent of FtsZ, but this independence is for protocells that divide by extrusions and resolution, where the membrane is highly fluidised (Mercier et al., 2012).
(3) The authors may use the L-form derivative where the FtsZ protein can be depleted and assess the proliferation of the proto-colonies. Likewise, authors should rule out the role of MreB as well.
(4) Although the growth rates have been shown to be similar for proto-cells and the proto-colonies, and only the membrane tension has been shown to be higher at the periphery, it is also important that the authors rule out any increased lipid synthesis in the fraction of dividing cells in these proto-colonies. Without this, one could also envisage a model where membranes are fluidised due to an increase in lipid biosynthesis in a fraction of cells in these confined spaces, leading to increased vesiculations which experience membrane shear and deform. The authors can also consider examining proto-colonies of L-forms of branched-chain fatty acid-deficient strains.
(5) Lastly, why does CellROX stain the proto-colonies? Are these tightly packed cells experiencing higher oxidative stress, and could that also contribute to membrane tension? This should at least be discussed.
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Reviewer #2 (Public review):
Summary:
The manuscript "Mechanical interaction enables a collective mode of protocell proliferation" addresses an interesting and potentially high-impact question about protocell proliferation in prebiotic environments. The central observation that wall-deficient B. Subtilis proliferate in dense colonies but die by membrane rupture in isolation is striking and a fundamental contribution to the field. However, the data and the mechanistic explanation offered for this observation are incomplete. The measurement and analyses used to build the mechanistic case raise methodological questions that may be difficult to fully resolve with the existing data and approach, and the authors should therefore consider whether additional independent experiments are needed to support the mechanical shearing hypothesis.
Streng…
Reviewer #2 (Public review):
Summary:
The manuscript "Mechanical interaction enables a collective mode of protocell proliferation" addresses an interesting and potentially high-impact question about protocell proliferation in prebiotic environments. The central observation that wall-deficient B. Subtilis proliferate in dense colonies but die by membrane rupture in isolation is striking and a fundamental contribution to the field. However, the data and the mechanistic explanation offered for this observation are incomplete. The measurement and analyses used to build the mechanistic case raise methodological questions that may be difficult to fully resolve with the existing data and approach, and the authors should therefore consider whether additional independent experiments are needed to support the mechanical shearing hypothesis.
Strengths:
The central observation that wall-deficient B. Subtilis proliferate in dense colonies but die by membrane rupture in isolation is convincing and a significant contribution to the field interested in the growth of protocells. This adds an important aspect of collective growth that is different from individual dynamics.
Weaknesses:
(1) The surface-volume balance ratio η is an elegant concept and provides an intuitively reasonable framework for understanding why isolated cells lyse. However, its application here rests on treating cells as flat discs of uniform thickness, and Figure S4 makes clear that the cells are highly irregular and lobulated in ways that make this approximation questionable. The authors should clarify whether they have validated this assumption, for instance, through direct thickness measurements or sensitivity analysis. However, even with such validation, the modest quantitative differences between aggregated and isolated η trajectories, combined with the inherent difficulty of accurate perimeter measurement in these morphologically complex cells, mean that η measurements are unlikely to provide robust quantitative support for the mechanism. The authors should therefore consider whether η is better presented as a motivating conceptual framework rather than primary quantitative evidence and seek more direct experimental support for the surface-volume balance argument through independent means. For instance, osmotic pressure manipulation to test whether reducing volume expansion pressure preferentially rescues isolated cells.
(2) The comparison of circularity between colony and isolated cells is complicated by the fact that the segmentation approach is fundamentally different in the two conditions; isolated cell boundaries are detected against a clear background, while colony boundaries are detected from inter-cell fluorescence gradients. The authors should address whether this introduces systematic bias. However, this may be difficult to fully resolve given the inherent complexity of the system, and that the deformation-division correlation in Figure 3C, while suggestive, would be substantially strengthened by a more direct perturbative approach. Specifically, can cell deformation be mechanically induced in isolated cells, for instance, using micromanipulation, external flow, or confinement in fabricated microstructures, to test whether artificially deformed isolated cells gain the ability to divide? Such an experiment would provide direct evidence for the deformation-division link that the correlational analysis cannot.
(3) The interpretation of FliptR lifetime as a direct membrane tension readout is complicated in this system because cell-cell interfaces contain two apposed bilayers in proximity, potentially altering FliptR photophysics through changes in local membrane density and dielectric environment independently of tension. The authors should address whether they have considered this possibility and what controls were performed. Disambiguating tension-dependent from environment-dependent lifetime changes is technically challenging and suggests that the membrane tension argument would be more convincingly supported by an independent measurement approach. For instance, tether-pulling experiments using optical tweezers on isolated versus colony cell membranes, or testing whether membrane tension-modulating interventions such as osmotic shifts produce the predicted changes in cell fate, would provide more direct evidence. The current FLIM data should be regarded as suggestive rather than conclusive.
(4) The Cellular Potts Model reproduces the experimental observations, but since its key parameters, particularly the substrate-pinning energy, were calibrated against those same observations, this demonstrates internal consistency rather than independent validation. The η-based lysis criterion is implemented as a model input, meaning the model cannot independently confirm the η hypothesis. The authors should clarify the extent to which model parameters were fitted to data versus independently motivated and be explicit that the model is best understood as a mechanistic illustration rather than independent evidence.
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Reviewer #3 (Public review):
Summary
This manuscript reports that protocells derived from wall-deficient B. subtilis proliferate well when densely packed but fail to divide and eventually lyse when isolated. The authors attribute this density-dependent proliferation to mechanical shearing between growing neighbors, which deforms cells and increases the likelihood of membrane stalk formation and subsequent scission, enabling division without any dedicated molecular machinery. Through a combination of quantitative imaging, membrane tension measurements, and Cellular Potts Model simulations, the authors make a compelling case that self-generated mechanical stresses are critical for sustaining population growth in protocolonies. The findings have implications for understanding the lifestyles of primitive life forms, L-form bacterial …
Reviewer #3 (Public review):
Summary
This manuscript reports that protocells derived from wall-deficient B. subtilis proliferate well when densely packed but fail to divide and eventually lyse when isolated. The authors attribute this density-dependent proliferation to mechanical shearing between growing neighbors, which deforms cells and increases the likelihood of membrane stalk formation and subsequent scission, enabling division without any dedicated molecular machinery. Through a combination of quantitative imaging, membrane tension measurements, and Cellular Potts Model simulations, the authors make a compelling case that self-generated mechanical stresses are critical for sustaining population growth in protocolonies. The findings have implications for understanding the lifestyles of primitive life forms, L-form bacterial pathogenesis, and the design of synthetic cells.
Strengths
The central finding is both surprising and counterintuitive: crowding is not just tolerated by protocells but is required for sustained population growth. The mechanism the authors propose is interesting: mechanical shearing between growing neighbors deforms cells, increasing the likelihood of membrane stalk formation and thus division, all without dedicated molecular machinery. Conceptually, this is a type of biophysical "scaffold" (Jacobeen et al. 2018, Nat. Phys.; Day et al. 2022, Biophys. Rev.) in which key elements of a Darwinian loop, namely a life cycle involving growth and reproduction, are provided "for free" by physics, enabling open-ended Darwinian evolution that can eventually bring these life cycle components under developmental control. Such scaffolds, both biophysical and ecological (Black et al. 2020, Nat. Ecol. Evol.; Libby & Rainey 2013, Phys. Biol.), are likely key mechanisms in the origin of life and in evolutionary transitions in individuality, and this paper provides a nice example of how they can work in a protocell context.
The combination of experiments and modeling works well. The membrane tension measurements are the strongest piece of evidence for the proposed mechanism, showing directly that tension is elevated in protocolonies and concentrated at cell-cell interfaces. The Cellular Potts Model captures the key experimental features. The discussion is nicely balanced, particularly the note about Gram-negative L-forms, whose rigid outer membrane may preclude this mechanism, which is a testable prediction for future work. I would suggest the authors also discuss the connection to biophysical scaffolding, as I think this is conceptually important and would help situate their work within a broader framework for understanding how primitive life cycles can arise from physical processes (see also Zamani-Dahaj et al. 2023, Genes; Hammerschmidt et al. 2014, Nature).
Weaknesses
The surface-volume balance analysis is central to the argument, and it depends on the assumption that cells have a fixed thickness of 0.8 µm, taken from the width of walled cells. But these are wall-deficient cells, which are mechanically quite different, and their thickness could plausibly vary during growth or under compression. I think the paper would benefit from either a direct measurement of cell thickness or a sensitivity analysis showing how η responds to plausible variation in this parameter. If the results are robust, that would put the analysis on much firmer ground.
The positive correlation between cell shape deformation and division rate (Figure 3C) is central to the proposed mechanism, but I think the paper needs to be more careful about the jump from correlation to causation. The authors propose that deformation increases the likelihood of membrane stalk formation, leading to scission. That is plausible, but an alternative is that cells with higher local growth rates both deform more and divide more frequently, with the two outcomes driven independently by the same underlying cause. The paper does show that average volume growth rates are indistinguishable between aggregated and isolated cells, which argues against a simple "faster growth explains everything" interpretation, but this does not rule out local variation within protocolonies driving the correlation. I think the most convincing experiment would be to apply external mechanical stress to isolated cells and see if that alone can drive division, decoupling deformation from growth. I realize that this may be technically very difficult, but at a minimum, the paper should acknowledge this as an alternative hypothesis.
The Cellular Potts Model has quite a few free parameters (Table S1), and it is not clear how tightly these are constrained by the data. A sensitivity analysis would go a long way toward showing that the results are robust and not overly dependent on specific parameter choices.
In any case, this is a strong paper with a cool finding and an interesting mechanistic explanation. I think it will be of broad interest, particularly to people thinking about the origins of life and synthetic cell design.
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