2022B. Alric, C. Formosa-Dague, E. Dague, L.J. Holt and Delarue, Morgan. Macromolecular crowding limits growth under pressure. Nature Physics (2022).
Cells that grow in confined spaces eventually build up mechanical compressive stress. This growth-induced pressure (GIP) decreases cell growth. GIP is important in a multitude of contexts from cancer, to microbial infections, to biofouling, yet our understanding of its origin and molecular consequences remains limited. Here, we combine microfluidic confinement of the yeast Saccharomyces cerevisiae, with rheological measurements using genetically encoded multimeric nanoparticles (GEMs) to reveal that growth-induced pressure is accompanied with an increase in a key cellular physical property: macromolecular crowding. We develop a fully calibrated model that predicts how increased macromolecular crowding hinders protein expression and thus diminishes cell growth. This model is sufficient to explain the coupling of growth rate to pressure without the need for specific molecular sensors or signaling cascades. As molecular crowding is similar across all domains of life, this could be a deeply conserved mechanism of biomechanical feedback that allows environmental sensing originating from the fundamental physical properties of cells. (view pdf)
2021T. Szoradi, T. Shu, ..., M. Delarue and Holt, Liam. nucGEMs probe the biophysical properties of the nucleoplasm. bioRxiv (2021).
The cell interior is highly crowded and far from thermodynamic equilibrium. This environment can dramatically impact molecular motion and assembly, and therefore influence sub-cellular organization and biochemical reaction rates. These effects depend strongly on length-scale, with the least information available at the important mesoscale (10-100 nanometers), which corresponds to the size of crucial regulatory molecules such as RNA polymerase II. It has been challenging to study the mesoscale physical properties of the nucleoplasm because previous methods were labor-intensive and perturbative. Here, we report nuclear Genetically Encoded Multimeric nanoparticles (nucGEMs). Introduction of a single gene leads to continuous production and assembly of protein-based bright fluorescent nanoparticles of 40 nm diameter. We implemented nucGEMs in budding and fission yeasts and in mammalian cell lines. We found that the nucleus is more crowded than the cytosol at the mesoscale, that mitotic chromosome condensation ejects nucGEMs from the nucleus, and that nucGEMs are excluded from heterochromatin and the nucleolus. nucGEMs enable hundreds of nuclear rheology experiments per hour, and allow evolutionary comparison of the physical properties of the cytosol and nucleoplasm. (view pdf)
Mechanical stresses including tensile and compressive stresses are ubiquitous in nature, 10 and are now well-recognized as being inherent to the development of most cancers. They are integrated by mechanotransduction in cells. Tensile stress is largely associated with YAP/TAZ pathway activation. However, less is known about signaling induced by compressive stress, the latter arising from extracellular matrix remodeling and local tumor growth. In the present study, we used transcriptomic data obtained after unidirectional compression of wild-type and mutant PIK3CA breast cancer cells from Kim et al., 2019. We analyzed in unbiased manner signatures of cell signaling activation including phosphoinositide 3-kinases (PI3Ks) activity pathway in response to compressive stress. Because we found that PI3K activation occurred upon compression, we studied PI3K isoform-specific pathways using known transcriptional targets of PI3Kα inhibitor (BYL719) or of PI3Kβ inhibitor (AZD8186). Our study provides transcriptomic evidence for the role of PI3K pathway in compression-induced mechanotransduction, through the roles of isoform-specific class I PI3Ks and independently of PIK3CA alterations. In a compressive environment, the canonical pathways (YAP/TAZ and Piezo) were not increased, while other targetable mechanisms, such as PI3K signal or autophagy, may provide a proliferative advantage and increased cell resistance to chemotherapies. (view pdf)
Mammalian cells integrate different types of stimuli that govern their fate. These stimuli encompass biochemical (ligands, oxygen, pH) as well as biomechanical cues (shear, tensile, and compressive stresses) that are usually studied separately. The PI3K enzymes, producing signaling phosphoinositides at plasma and intracellular membranes, are key in intracellular signaling and vesicular trafficking pathways. Recent evidence in cancer research demonstrate that these enzymes are essential in mechanotransduction. Despite being a hub in mechanotransduction, the biological and clinical relevance of the integration of both biochemical and biomechanical cues by PI3K-driven signals, especially in cancer cells, is understudied and underestimated. In this Opinion article, we make the hypothesis that modelling biomechanical cues is critical to understand PI3K biochemical signals, highlight the first clues of their involvement in cell mechanobiology, identify missing knowledge in term of isoform specificity and molecular pathways of activation, and scrutinize the potential implication of such knowledge in cancer cells. (view pdf)
Microdevices composed of microwell arrays integrating nanoelectrodes (OptoElecWell) were developed to achieve dual high-resolution optical and electrochemical detections on single Saccharomyces cerevisiae budding yeast cells. Each array consists of 1.6 × 105 microwells of 8 µm diameter and 5 µm height, with a platinum nanoring electrode for in-situ electrochemistry, all integrated on a transparent thin wafer for further high-resolution live-cell imaging. After optimizing the filling rate, 32% of cells were effectively trapped within microwells. This allowed analyzing S. cerevisiae metabolisms associated with basal respiration while simultaneously measuring optically other cellular parameters. In this study, we focused on the impact of glucose concentration on respiration and intracellular rheology. We found that while oxygen uptake rate decreased with increasing glucose concentration, diffusion of tracer nanoparticles increased. Our OptoElecWell based respiration methodology provided similar results compared to the commercial gold-standard Seahorse XF analyzer, while using 20 times lesser biological samples, paving the way to achieve single cell metabolomics. In addition, it facilitates an optical route to monitor the contents within single cells. The proposed device, in combination with the dual detection analysis, opens up new avenues for measuring cellular metabolism and relating it to various cellular physiological and rheological indicators at single cell level. (view pdf)
2020Rizzuti, I. F., P. Mascheroni, S. Arcucci, Z. Ben-Mériem, A. Prunet, C. Barentin, C. Rivière, H. Delanoë-Ayari, H. Hatzikirou, J. Guillermet-Guibert and Delarue, Morgan. Mechanical control of cell proliferation increases resistance to chemotherapeutic agents. Physical Review Letter (2020).
While many cellular mechanisms leading to chemotherapeutic resistance have been identified, there is an increasing realization that tumor-stroma interactions also play an important role. In particular, mechanical alterations are inherent to solid cancer progression and profoundly impact cell physiology. Here, we explore the impact of compressive stress on the efficacy of chemotherapeutics in pancreatic cancer spheroids. We find that increased compressive stress leads to decreased drug efficacy. Theoretical modeling and experiments suggest that mechanical stress leads to decreased cell proliferation which in turn reduces the efficacy of chemotherapeutics that target proliferating cells. Our work highlights a mechanical-form of drug resistance, and suggests new strategies for therapy. (view pdf)
2018Holt, L. J., O. Hallatschek, and Delarue, Morgan. Chapter 12 - Mechano-chemostats to study the effects of compressive stress on yeast. Methods in Cell Biology (2018).
Cells need to act upon the elastic extracellular matrix and against steric constraints when proliferating in a confined environment, leading to the build-up, at the population level, of a compressive, growth-induced, mechanical stress. Compressive mechanical stresses are ubiquitous to any cell population growing in a spatially-constrained environment, such as microbes or most solid tumors. They remain understudied, in particular in microbial populations, due to the lack of tools available to researchers. Here, we present various mechano-chemostats: microfluidic devices developed to study microbes under pressure. A mechano-chemostat permits researchers to control the intensity of growth-induced pressure through the control of cell confinement, while keeping cells in a defined chemical environment. These versatile devices enable the interrogation of physiological parameters influenced by mechanical compression at the single cell level and set a standard for the study of growth-induced compressive stress. (view pdf)
Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed genetically encoded multimeric nanoparticles (GEMs) to dissect these mechanisms. GEMs are homomultimeric scaffolds fused to a fluorescent protein that self- assemble into bright, stable particles of defined size and shape. By combining tracking of GEMs with genetic and pharmacological approaches, we discovered that the mTORC1 pathway can modulate the effective diffusion coefficient of particles R20 nm in diameter more than 2-fold by tuning ribosome concentration, without any discernable effect on the motion of molecules 5 nm. This change in ribosome concentration affected phase separation both in vitro and in vivo. Together, these results establish a role for mTORC1 in controlling both the mesoscale biophysical properties of the cytoplasm and biomolecular condensation. (view pdf)
2017Delarue, Morgan, Gregory Poterewicz, Ori Hoxha, Jessica Choi, Wonjung Yoo, Jona Kayser, Liam Holt, and Oskar Hallatschek. SCWISh network is essential for survival under mechanical pressure. Proceedings of the National Academy of Sciences 114, no. 51 (2017): 13465-13470.
Cells that proliferate within a confined environment build up mechanical compressive stress. For example, mechanical pressure emerges in the naturally space-limited tumor environment. However, little is known about how cells sense and respond to mechanical compression. We developed microfluidic bioreactors to enable the investigation of the effects of compressive stress on the growth of the genetically tractable model organism Saccharomyces cerevisiae. We used this system to determine that compressive stress is partly sensed through a module consisting of the mucin Msb2 and the cell wall protein Sho1, which act together as a sensor module in one of the two major osmosensing pathways in budding yeast. This signal is transmitted via the MAPKKK kinase Ste11. Thus, we term this mechanosensitive pathway the SMuSh pathway, for Ste11 through Mucin/Sho1 pathway. The SMuSh pathway delays cells in the G1 phase of the cell cycle and improves cell survival in response to growth-induced pressure. We also found that the cell wall integrity (CWI) pathway contributes to the response to mechanical compressive stress. These latter results are confirmed in complimentary experiments in Mishra et al. When both the SMuSh and the CWI pathways are deleted, cells fail to adapt to compressive stress, and all cells lyse at relatively low pressure when grown in confinement. Thus, we define a network that is essential for cell survival during growth under pressure. We term this mechanosensory system the SCWISh (survival through the CWI and SMuSh) network. (view pdf)
Increasingly accurate and massive data have recently shed light on the fundamental question of how cells maintain a stable size trajectory as they progress through the cell cycle. Microbes seem to use strategies ranging from a pure sizer, where the end of a given phase is triggered when the cell reaches a critical size, to pure adder, where the cell adds a constant size during a phase. Yet the biological origins of the observed spectrum of behavior remain elusive. We analyze a molecular size-control mechanism, based on experimental data from the yeast S. cerevisiae, that gives rise to behaviors smoothly interpolating between adder and sizer. The size-control is obtained from the accumulation of an activator protein that titrates an inhibitor protein. Strikingly, the size-control is composed of two different regimes: for small initial cell size, the size-control is a sizer, whereas for larger initial cell size, it is an imperfect adder, in agreement with recent experiments. Our model thus indicates that the adder and critical size behaviors may just be different dynamical regimes of a single simple biophysical mechanism. (view pdf)
During tumor progression, cancer cells acquire the ability to escape the primary tumor and invade adjacent tissues. They migrate through the stroma to reach blood or lymphatics vessels that will allow them to disseminate throughout the body and form metastasis at distant organs. To assay invasion capacity of cells in vitro, multicellular spheroids of cancer cells, mimicking primary tumor, are commonly embedded in collagen I extracellular matrix, which mimics the stroma. However, due to their higher density, spheroids tend to sink at the bottom of the collagen droplets, resulting in the spreading of the cells on two dimensions. We developed an innovative method based on droplet microfluidics to embed and control the position of multicellular spheroids inside spherical droplets of collagen. In this method cancer cells are exposed to a uniform three-dimensional (3D) collagen environment resulting in 3D cell invasion. (view pdf)
The surrounding microenvironment limits tumour expansion, imposing a compressive stress on the tumour, but little is known how pressure propagates inside the tumour. Here we present non-destructive cell-like microsensors to locally quantify mechanical stress distribution in three-dimensional tissue. Our sensors are polyacrylamide microbeads of well-defined elasticity, size and surface coating to enable internalization within the cellular environment. By isotropically compressing multicellular spheroids (MCS), which are spherical aggregates of cells mimicking a tumour, we show that the pressure is transmitted in a non-trivial manner inside the MCS, with a pressure rise towards the core. This observed pressure profile is explained by the anisotropic arrangement of cells and our results suggest that such anisotropy alone is sufficient to explain the pressure rise inside MCS composed of a single cell type. Furthermore, such pressure distribution suggests a direct link between increased mechanical stress and previously observed lack of proliferation within the spheroids core. (view pdf)
2016Delarue, Morgan, Jörn Hartung, Carl Schreck, Pawel Gniewek, Lucy Hu, Stephan Herminghaus, and Oskar Hallatschek. Self-driven jamming in growing microbial populations. Nature physics 12, no. 8 (2016): 762.
In natural settings, microbes tend to grow in dense populations where they need to push against their surroundings to accommodate space for new cells. The associated contact forces play a critical role in a variety of population-level processes, including biofilm formation, the colonization of porous media, and the invasion of biological tissues. Although mechanical forces have been characterized at the single cell level, it remains elusive how collective pushing forces result from the combination of single cell forces. Here, we reveal a collective mechanism of confinement, which we call self-driven jamming, that promotes the build-up of large mechanical pressures in microbial populations. Microfluidic experiments on budding yeast populations in space-limited environments show that self-driven jamming arises from the gradual formation and sudden collapse of force chains driven by microbial proliferation, extending the framework of driven granular matter. The resulting contact pressures can become large enough to slow down cell growth, to delay the cell cycle in the G1 phase, and to strain or even destroy the microenvironment through crack propagation. Our results suggest that self-driven jamming and build-up of large mechanical pressures is a natural tendency of microbes growing in confined spaces, contributing to microbial pathogenesis and biofouling. (view pdf)
There is increasing evidence that multicellular structures respond to mechanical cues, such as the confinement and compression exerted by the surrounding environment. In order to understand the response of tissues to stress, we investigate the effect of an isotropic stress on different biological systems. The stress is generated using the osmotic pressure induced by a biocompatible polymer. We compare the response of multicellular spheroids, individual cells and matrigel to the same osmotic perturbation. Our findings indicate that the osmotic pressure occasioned by polymers acts on these systems like an isotropic mechanical stress. When submitted to this pressure, the volume of multicellular spheroids decreases much more than one could expect from the behavior of individual cells.(view pdf)
2015Podewitz, Nils, M. Delarue, and J. Elgeti. Tissue homeostasis: A tensile state. EPL (Europhysics Letters) 109, no. 5 (2015): 58005.
Mechanics play a significant role during tissue development. One of the key characteristics that underlie this mechanical role is the homeostatic pressure, which is the pressure stalling growth. In this work, we explore the possibility of a negative bulk homeostatic pressure by means of a mesoscale simulation approach and experimental data of several cell lines. We show how different cell properties change the bulk homeostatic pressure, which could explain the benefit of some observed morphological changes during cancer progression. Furthermore, we study the dependence of growth on pressure and estimate the bulk homeostatic pressure of five cell lines. Four out of five results in a bulk homeostatic pressure in the order of minus one or two kPa. (view pdf)
2014Delarue, Morgan, Jean-François Joanny, Frank Jülicher, and Jacques Prost. Stress distributions and cell flows in a growing cell aggregate. Interface focus 4, no. 6 (2014): 20140033.
We discuss the short-time response of a multicellular spheroid to an external pressure jump. Our experiments show that 5 min after the pressure jump, the cell density increases in the centre of the spheroid but does not change appreciably close to the surface of the spheroid. This result can be explained if the cells are polarized which we show to be the case. Motivated by the experimental results, we develop a theory for polarized spheroids where the cell polarity is radial (except in a thin shell close to the spheroid surface). The theory takes into account the dependence of cell division and apoptosis rates on the local stress, the cell polarity and active stress generated by the cells and the dependence of active stress on the local pressure. We find a short-time increase of the cell density after a pressure jump that decays as a power law from the spheroid centre, which is in reasonable agreement with the experimental results. By comparing our theory to experiments, we can estimate the isotropic compression modulus of the tissue. (view pdf)
In most instances, the growth of solid tumors occurs in constrained environments and requires a competition for space. A mechanical crosstalk can arise from this competition. In this article, we dissect the biomechanical sequence caused by a controlled compressive stress on multicellular spheroids (MCSs) used as a tumor model system. On timescales of minutes, we show that a compressive stress causes a reduction of the MCS volume, linked to a reduction of the cell volume in the core of the MCS. On timescales of hours, we observe a reversible induction of the proliferation inhibitor, p27Kip1, from the center to the periphery of the spheroid. On timescales of days, we observe that cells are blocked in the cell cycle at the late G1 checkpoint, the restriction point. We show that the effect of pressure on the proliferation can be antagonized by silencing p27Kip1. Finally, we quantify a clear correlation between the pressure-induced volume change and the growth rate of the spheroid. The compression-induced proliferation arrest that we studied is conserved for five cell lines, and is completely reversible. It demonstrates a generic crosstalk between mechanical stresses and the key players of cell cycle regulation. Our results suggest a role of volume change in the sensitivity to pressure, and that p27Kip1 is strongly influenced by this change. (view pdf)
2013Delarue, Morgan, Fabien Montel, Ouriel Caen, Jens Elgeti, Jean-Michel Siaugue, Danijela Vignjevic, Jacques Prost, Jean-François Joanny, and Giovanni Cappello. Mechanical control of cell flow in multicellular spheroids. Physical review letters 110, no. 13 (2013): 138103.
Collective cell motion is observed in a wide range of biological processes. In tumors, physiological gradients of nutrients, growth factors, or even oxygen give rise to gradients of proliferation. We show using fluorescently labeled particles that these gradients drive a velocity field resulting in a cellular flow in multicellular spheroids. Under mechanical stress, the cellular flow is drastically reduced. We describe the results with a hydrodynamic model that considers only convection of the particles by the cellular flow. (view pdf)
2012Montel, Fabien, Morgan Delarue, Jens Elgeti, Danijela Vignjevic, Giovanni Cappello, and Jacques Prost. Isotropic stress reduces cell proliferation in tumor spheroids. New Journal of Physics 14, no. 5 (2012): 055008.
In most instances, tumors have to push their surroundings in order to grow. Thus, during their development, tumors must be able to both exert and sustain mechanical stresses. Using a novel experimental procedure, we study quantitatively the effect of an applied mechanical stress on the long-term growth of a spherical cell aggregate. Our results indicate the possibility to modulate tumor growth depending on the applied pressure. Moreover, we demonstrate quantitatively that the cells located in the core of the spheroid display a different response to stress than those in the periphery. We compare the results to a simple numerical model developed for describing the role of mechanics in cancer progression. (view pdf)
2011Montel, Fabien, Morgan Delarue, Jens Elgeti, Laurent Malaquin, Markus Basan, Thomas Risler, Bernard Cabane et al. Stress clamp experiments on multicellular tumor spheroids. Physical review letters 107, no. 18 (2011): 188102.
The precise role of the microenvironment on tumor growth is poorly understood. Whereas the tumor is in constant competition with the surrounding tissue, little is known about the mechanics of this interaction. Using a novel experimental procedure, we study quantitatively the effect of an applied mechanical stress on the long-term growth of a spheroid cell aggregate. We observe that a stress of 10 kPa is sufficient to drastically reduce growth by inhibition of cell proliferation mainly in the core of the spheroid. We compare the results to a simple numerical model developed to describe the role of mechanics in cancer progression. (view pdf)
2010Roquelet, Cyrielle, Jean‐Sébastien Lauret, Valérie Alain‐Rizzo, Christophe Voisin, Romain Fleurier, Morgan Delarue, Damien Garrot et al. Π‐Stacking Functionalization of Carbon Nanotubes through Micelle Swelling. ChemPhysChem 11, no. 8 (2010): 1667-1672.
We report on a new, original and efficient method for Π-stacking functionalization of single-wall carbon nanotubes. This method is applied to the synthesis of a high-yield light-harvesting system combining single-wall carbon nanotubes and porphyrin molecules. We developed a micelle-swelling technique that leads to controlled and stable complexes presenting an efficient energy transfer. We demonstrate the key role of the organic solvent in the functionalization mechanism. By swelling the micelles, the solvent helps the non-water-soluble porphyrins to reach the micelle core and allows a strong enhancement of the interaction between porphyrins and nano- tubes. This technique opens new avenues for the functionalization of carbon nanostructures. (view pdf)