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Nucleus deformation activates a mechanosensitive lipase signaling pathway regulating myosin II activity. (A) Relative cortical myosin II intensity for progenitor cells cultured in suspension versus 7 µm confinement conditions for control cells (DMEM), with cPLA 2 inhibitor, or injected with cPLA 2 MO and cPLA2 morpholino+cPLA 2 mRNA. (B) Exemplary confocal
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The physical microenvironment regulates cell behavior during tissue development and homeostasis. How single cells decode information about their geometrical shape under mechanical stress and physical space constraints within their local environment remains largely unknown. Here we show that the nucleus, the biggest cellular organelle, functions as...
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... of cortical myosin II enrichment were evident, with myosin II re-localization increasing for larger cell deformations (Fig. 1C). A cell confinement height below 35 7 µm caused a pronounced increase in cell lysis during compression, defining a maximal threshold deformation of ~30% of the initial cell diameter, given a blastula cell size of d~25 µm (Fig. S3G). Overall, these data support that the physical microenvironment defines a specific set point level of cortical contractility as a function of physical cell deformation in confined microenvironments. 40 We have previously shown that an increase in myosin II-mediated cortical contractility induced a stochastic motility switch into a ...
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... next sought to identify potential mechanisms that control cellular shape deformation sensing and adaptive morphodynamic behavior. Cortical myosin re-localization occurred on passivated confinement surfaces independently of adhesive substrate coating (Fig. S2B, 3A) and cell-cell contact formation (Fig. S3B). These observations support that the activation of cortical 35 contractility in confinement occurs independently of adhesion-dependent mechano-transduction pathways (27). The temporal characteristics of myosin re-localization in confined cells (fast, stable and reversible accumulation of cortical myosin), suggest that shape ...
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... copyright holder for this preprint (which . http://dx.doi.org/10.1101/865949 doi: bioRxiv preprint first posted online Dec. 5, 2019; cells (29)) showed no significant reduction in cortical myosin accumulation under cell deformation (Fig. S3C), despite the presence of functional Piezo1 channels in these cells (Fig. S3D). Interestingly, we observed that cortical myosin II enrichment only started to occur below a threshold confinement height (~13 µm) that correlated with the spatial dimension of the nucleus ( Fig. 2A and Fig. S3G). Analyzing nuclear shape change versus ...
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... copyright holder for this preprint (which . http://dx.doi.org/10.1101/865949 doi: bioRxiv preprint first posted online Dec. 5, 2019; cells (29)) showed no significant reduction in cortical myosin accumulation under cell deformation (Fig. S3C), despite the presence of functional Piezo1 channels in these cells (Fig. S3D). Interestingly, we observed that cortical myosin II enrichment only started to occur below a threshold confinement height (~13 µm) that correlated with the spatial dimension of the nucleus ( Fig. 2A and Fig. S3G). Analyzing nuclear shape change versus cortical myosin accumulation 5 revealed a bi-phasic behavior, with a first phase in ...
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... a threshold deformation of ~13 µm ( Fig. 2C-E, Fig. S3E, Movie 6). In addition, analysis of nucleus membrane curvature for confined versus control cells in suspension indicated INM surface unfolding that remained stable over the measurement time of 60 min (Fig. 2F,G, Movie 6), with no significant 15 difference in total nuclear volume and surface (Fig. S3F). Nucleus deformation further correlated with cortical myosin II accumulation in the endogenous in vivo context during the blastula to gastrula transition, when a gradient of cellular packing density appears from the animal pole towards the lateral margin (30) (Fig. ...
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... further probe the dependence of cortical myosin II accumulation on nucleus size, we dissociated 20 primary embryonic stem cells from early and late blastula stages as cells reduce their size in consecutive rounds of early cleavage divisions (Fig. S3G). Deforming cells of different sizes under similar confinement heights revealed that myosin II accumulation is correlated with relative changes in nucleus deformation but not cell deformation (Fig. 2H). To test a functional role of the nucleus in regulating cortical contractility levels during cellular shape deformation, we analyzed 25 ...
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... processes at the INM interface are involved in the regulation of myosin II activity and cortical contractility. Among a set of molecules tested under confinement 10 conditions, we identified cytosolic phospholipase A2 (cPLA2) as a key molecular target mediating the activation of cortical myosin II enrichment under cell compression (Fig. 3A,B). Inhibition of cPLA2 by pharmacological or genetic interference robustly blocked cortical myosin II relocalization under varying confinement heights (Fig. S5A). Overexpression of cPLA2 mRNA rescued the morphant phenotype and led to a comparable myosin II accumulation as in control 15 cells (Fig. 3A,B). To exclude that other mechanisms ...
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... myosin II enrichment under cell compression (Fig. 3A,B). Inhibition of cPLA2 by pharmacological or genetic interference robustly blocked cortical myosin II relocalization under varying confinement heights (Fig. S5A). Overexpression of cPLA2 mRNA rescued the morphant phenotype and led to a comparable myosin II accumulation as in control 15 cells (Fig. 3A,B). To exclude that other mechanisms such as structural changes in the actin network prevent cortical myosin II re-localization under cPLA2 inhibition, we added LPA as an exogenous myosin II activator to cPLA2 inhibited cells. Under this condition, myosin II was strongly accumulated at the cell cortex (Fig. S5B,C) and induced cell ...
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... other mechanisms such as structural changes in the actin network prevent cortical myosin II re-localization under cPLA2 inhibition, we added LPA as an exogenous myosin II activator to cPLA2 inhibited cells. Under this condition, myosin II was strongly accumulated at the cell cortex (Fig. S5B,C) and induced cell polarization and amoeboid motility (Fig. 3C), suggesting that myosin II can be activated by alternative pathways when cPLA2 20 signaling is inhibited and is competent to bind to the cell ...
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... to the INM (32). We thus tested a role of cPLA2 in the nucleus by generating a modified cPLA2 construct 25 containing a nuclear export sequence (NES). Using Leptomycin B as a blocker of nuclear export, an accumulation of cPLA2-NES-GFP within the nucleus was observed, showing a concomitant increase of cortical myosin II levels in confined cells (Fig. 3D,E). These data support, that cPLA2 localization in the nucleus is required for myosin II enrichment at the ...
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... further validated that cortical myosin II enrichment in cells of different sizes (early versus late 30 blastula cells) and different embryonic cell lineages (mesendoderm/ectoderm) depends on the activation of cPLA2 signaling. Pharmacological inhibition of cPLA2 activity blocked cortical myosin re-localization under cell deformation in confinement (Fig. 3F), supporting a consistent role of cPLA2 activation under physical cell deformation across early to late developmental stages. These data support that activation of cPLA2 signaling in the nucleus mediates adaptive 35 cytoskeletal and morphodynamic behavior under cell ...
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... acid (AA) is the primary cleavage product generated by cPLA2 activity (33). To directly validate whether nucleus deformation in confinement triggers cPLA2 activity, we measured the release of AA by Raman spectroscopy. The analysis of Raman spectra confirmed the specific production of AA in confined cells (Fig. 3G and Fig. S5D), with the increase in AA 40 production in confined versus control cells being specifically blocked in the presence of cPLA2 inhibitor (Fig. 3H). We further observed that AA was exclusively detected in the cytoplasm of confined cells, arguing that AA is directly released from nuclear membranes into the ...
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... in confinement triggers cPLA2 activity, we measured the release of AA by Raman spectroscopy. The analysis of Raman spectra confirmed the specific production of AA in confined cells (Fig. 3G and Fig. S5D), with the increase in AA 40 production in confined versus control cells being specifically blocked in the presence of cPLA2 inhibitor (Fig. 3H). We further observed that AA was exclusively detected in the cytoplasm of confined cells, arguing that AA is directly released from nuclear membranes into the ...
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... phosphorylation (35). We tested the involvement of Rho/ROCK and Calcium-MLCK signaling that act as key regulatory pathways of myosin II activity (4). MLCK inhibition showed no 5 significant effect on myosin II enrichment in confined cells, while a pronounced reduction of cortical myosin recruitment was observed under inhibition of Rho activity (Fig. 3I). Using a RhoAFret sensor further indicated an increased RhoA activity in confined cells versus control cells in suspension (Fig. S5E,F). These data support that AA production by cPLA2 activity engages upon nuclear envelope unfolding, regulating phosphorylation-dependent myosin II activity at the 10 cell ...
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... of cortical myosin II enrichment were evident, with myosin II re-localization increasing for larger cell deformations (Fig. 1C). A cell confinement height below 35 7 µm caused a pronounced increase in cell lysis during compression, defining a maximal threshold deformation of ~30% of the initial cell diameter, given a blastula cell size of d~25 µm (Fig. S3G). Overall, these data support that the physical microenvironment defines a specific set point level of cortical contractility as a function of physical cell deformation in confined microenvironments. 40 We have previously shown that an increase in myosin II-mediated cortical contractility induced a stochastic motility switch into a ...
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... next sought to identify potential mechanisms that control cellular shape deformation sensing and adaptive morphodynamic behavior. Cortical myosin re-localization occurred on passivated confinement surfaces independently of adhesive substrate coating (Fig. S2B, 3A) and cell-cell contact formation (Fig. S3B). These observations support that the activation of cortical 35 contractility in confinement occurs independently of adhesion-dependent mechano-transduction pathways (27). The temporal characteristics of myosin re-localization in confined cells (fast, stable and reversible accumulation of cortical myosin), suggest that shape ...
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... deformation sensing due to rapid turnover of the cell cortex (28). Testing for the activation of mechanosensitive ion channels using GsMTx4 (an inhibitor of the tension-dependent Piezo1 channel that is activated following confinement of human cancer cells (29)) showed no significant reduction in cortical myosin accumulation under cell deformation (Fig. S3C), despite the presence of functional Piezo1 channels in these cells (Fig. S3D). Interestingly, we observed that cortical myosin II enrichment only started to occur below a threshold confinement height (~13 µm) that correlated with the spatial dimension of the nucleus ( Fig. 2A and Fig. S3G). Analyzing nuclear shape change versus ...
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... the activation of mechanosensitive ion channels using GsMTx4 (an inhibitor of the tension-dependent Piezo1 channel that is activated following confinement of human cancer cells (29)) showed no significant reduction in cortical myosin accumulation under cell deformation (Fig. S3C), despite the presence of functional Piezo1 channels in these cells (Fig. S3D). Interestingly, we observed that cortical myosin II enrichment only started to occur below a threshold confinement height (~13 µm) that correlated with the spatial dimension of the nucleus ( Fig. 2A and Fig. S3G). Analyzing nuclear shape change versus cortical myosin accumulation 5 revealed a bi-phasic behavior, with a first phase in ...
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... continuously reduced when nucleus deformation started to occur at a threshold deformation of ~13 µm ( To further probe the dependence of cortical myosin II accumulation on nucleus size, we dissociated 20 primary embryonic stem cells from early and late blastula stages as cells reduce their size in consecutive rounds of early cleavage divisions (Fig. S3G). Deforming cells of different sizes under similar confinement heights revealed that myosin II accumulation is correlated with relative changes in nucleus deformation but not cell deformation (Fig. 2H). To test a functional role of the nucleus in regulating cortical contractility levels during cellular shape deformation, we analyzed 25 ...
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... processes at the INM interface are involved in the regulation of myosin II activity and cortical contractility. Among a set of molecules tested under confinement 10 conditions, we identified cytosolic phospholipase A2 (cPLA2) as a key molecular target mediating the activation of cortical myosin II enrichment under cell compression (Fig. 3A,B). Inhibition of cPLA2 by pharmacological or genetic interference robustly blocked cortical myosin II relocalization under varying confinement heights (Fig. S5A). Overexpression of cPLA2 mRNA rescued the morphant phenotype and led to a comparable myosin II accumulation as in control 15 cells (Fig. 3A,B). To exclude that other mechanisms ...
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... myosin II enrichment under cell compression (Fig. 3A,B). Inhibition of cPLA2 by pharmacological or genetic interference robustly blocked cortical myosin II relocalization under varying confinement heights (Fig. S5A). Overexpression of cPLA2 mRNA rescued the morphant phenotype and led to a comparable myosin II accumulation as in control 15 cells (Fig. 3A,B). To exclude that other mechanisms such as structural changes in the actin network prevent cortical myosin II re-localization under cPLA2 inhibition, we added LPA as an exogenous myosin II activator to cPLA2 inhibited cells. Under this condition, myosin II was strongly accumulated at the cell cortex (Fig. S5B,C) and induced cell ...
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... other mechanisms such as structural changes in the actin network prevent cortical myosin II re-localization under cPLA2 inhibition, we added LPA as an exogenous myosin II activator to cPLA2 inhibited cells. Under this condition, myosin II was strongly accumulated at the cell cortex (Fig. S5B,C) and induced cell polarization and amoeboid motility (Fig. 3C), suggesting that myosin II can be activated by alternative pathways when cPLA2 20 signaling is inhibited and is competent to bind to the cell ...
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... to the INM (32). We thus tested a role of cPLA2 in the nucleus by generating a modified cPLA2 construct 25 containing a nuclear export sequence (NES). Using Leptomycin B as a blocker of nuclear export, an accumulation of cPLA2-NES-GFP within the nucleus was observed, showing a concomitant increase of cortical myosin II levels in confined cells (Fig. 3D,E). These data support, that cPLA2 localization in the nucleus is required for myosin II enrichment at the ...
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... further validated that cortical myosin II enrichment in cells of different sizes (early versus late 30 blastula cells) and different embryonic cell lineages (mesendoderm/ectoderm) depends on the activation of cPLA2 signaling. Pharmacological inhibition of cPLA2 activity blocked cortical myosin re-localization under cell deformation in confinement (Fig. 3F), supporting a consistent role of cPLA2 activation under physical cell deformation across early to late developmental stages. These data support that activation of cPLA2 signaling in the nucleus mediates adaptive 35 cytoskeletal and morphodynamic behavior under cell ...
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... acid (AA) is the primary cleavage product generated by cPLA2 activity (33). To directly validate whether nucleus deformation in confinement triggers cPLA2 activity, we measured the release of AA by Raman spectroscopy. The analysis of Raman spectra confirmed the specific production of AA in confined cells (Fig. 3G and Fig. S5D), with the increase in AA 40 production in confined versus control cells being specifically blocked in the presence of cPLA2 inhibitor (Fig. 3H). We further observed that AA was exclusively detected in the cytoplasm of confined cells, arguing that AA is directly released from nuclear membranes into the ...
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... in confinement triggers cPLA2 activity, we measured the release of AA by Raman spectroscopy. The analysis of Raman spectra confirmed the specific production of AA in confined cells (Fig. 3G and Fig. S5D), with the increase in AA 40 production in confined versus control cells being specifically blocked in the presence of cPLA2 inhibitor (Fig. 3H). We further observed that AA was exclusively detected in the cytoplasm of confined cells, arguing that AA is directly released from nuclear membranes into the ...
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... phosphorylation (35). We tested the involvement of Rho/ROCK and Calcium-MLCK signaling that act as key regulatory pathways of myosin II activity (4). MLCK inhibition showed no 5 significant effect on myosin II enrichment in confined cells, while a pronounced reduction of cortical myosin recruitment was observed under inhibition of Rho activity (Fig. 3I). Using a RhoAFret sensor further indicated an increased RhoA activity in confined cells versus control cells in suspension (Fig. S5E,F). These data support that AA production by cPLA2 activity engages upon nuclear envelope unfolding, regulating phosphorylation-dependent myosin II activity at the 10 cell ...
Citations
... More globally, recent work by our lab and others (Lomakin et al., 2019;Venturini et al., 2019) showed that contractility is activated upon confinement by a mechanoresponse pathway mediated by the release of calcium and the activation of the enzyme cPLA2 (Enyedi et al., 2016). These works proposed that the contractility activation happens under 5µm compression due to nuclear stretching and that the stretch depended on the cell cycle stage or the state of the nuclear lamina. ...
Previously associated with apoptosis, blebs have arisen in the past decade as important structures for amoeboid cell migration, particularly for cancer cells. Blebs are formed when the plasma membrane detaches from the actomyosin cortex. They retract exerting friction forces and allowing cells to migrate. In recent years, a few independent studies have reported large and stable blebs in cells under non-adhesive confinement. This universal switch to bleb-based migration has been found in amoeba, choanoflagellates, immortalized cell lines and primary cultures. Unlike previous blebs described, they are able to overcome retraction and stabilize a constant flow. Stable blebs are a new type of cellular structures that amoeboid cells use to migrate, analogous to filopodia or lamellipodia for mesenchymal cells. In a single cell, multiple blebs form and compete against each other, so that eventually a single bleb drives the migration. Thus, it is important to know how single blebs are stabilized to understand how single-bleb amoeboid cells polarize. More generally, stable actomyosin flows constitute the basis of fast migration in numerous cell types, including also immune cells. During my Ph.D. I studied bleb morphogenesis and bleb stabilization in confined cancer cells, using advanced microfluidic techniques to control the confinement of cells. The first part of my project describes the blebs forming as an immediate response of cells to confinement and what differentiates it from a classical retracting bleb. The second part of my project focuses on the mechanism leading to the establishment of a retrograde flow. Based on the results I obtained with my experiments, we propose that bleb stabilization depends on 1) the depletion of actin by myosin contractility and 2) the particular actin filament arrangement at the bleb tip caused by the membrane topology of a confined cell. I completed this work with advanced imaging which allowed observation of single actin filaments and tagged cytoskeleton-associated molecules at the bleb tip, under different perturbations. This unique set of observations allowed to complete a model for the stabilization of motile blebs, with conclusions that can be generally applied to any flowing actomyosin cortex. My results show three cortex regimes in blebs: 1) Assembling loose cortex: localized at the tip, composed of single filaments poorly attached to the membrane. If this region is lost, the bleb retracts. 2) Crosslinked cortex: actin filaments and fibers bind together to form a network which gradually gets denser and reticulated but do not contract (this region is devoid of Myosin II motors). 3) Contractile cortex: towards the base of the bleb. Myosin-II starts to get enriched contracting the dense actin network, driving the entire retrograde actin flow up to the tip of the bleb, generating new actin free regions at the tip and pressurizing the bleb, leading to membrane protrusion at the very front.
... Relying on this ruler, cells can measure the degree of their environmental confinement and rapidly tailor specific behaviors to adapt to the confinement at time scales shorter than those associated with changes in gene expression. In the context of cell migration, such tailored cellular behaviors might help cells avoid environmental entrapment, which is relevant to cancer cell invasion, immune cell patrolling of peripheral tissues, and progenitor cell motility within a highly crowded cell mass of a developing embryo (33). The nuclear ruler mechanism defines an active function for the nucleus in cell migration, potentially explaining why enucleated cells show a poor motile capacity in dense collagen gels (34). ...
The nucleus makes the rules
Single cells continuously experience and react to mechanical challenges in three-dimensional tissues. Spatial constraints in dense tissues, physical activity, and injury all impose changes in cell shape. How cells can measure shape deformations to ensure correct tissue development and homeostasis remains largely unknown (see the Perspective by Shen and Niethammer). Working independently, Venturini et al. and Lomakin et al. now show that the nucleus can act as an intracellular ruler to measure cellular shape variations. The nuclear envelope provides a gauge of cell deformation and activates a mechanotransduction pathway that controls actomyosin contractility and migration plasticity. The cell nucleus thereby allows cells to adapt their behavior to the local tissue microenvironment.
Science , this issue p. eaba2644 , p. eaba2894 ; see also p. 295
... Nuclear probing depends on the biophysical properties of the nucleus, such as stiffness and size [52], but the detailed molecular mechanisms remain to be discovered. Recent data from HeLa, fibrosarcoma, and zebrafish primary progenitor stem cells show that spatial confinement stretches the nuclear envelope, thereby upregulating cellular contractility via myosin [80,81]. Thus, one may speculate that nuclear protrusions into small pores could lead to local nuclear envelope stretching and myosin activation, thereby inducing cellular and nuclear retraction from small pores. ...
Migration of leukocytes is essential for the induction, maintenance, and regulation of immune responses. On their trafficking routes, leukocytes encounter microenvironments of diverse mechanochemical composition, such as epithelial sheets, fibrillar networks, and cell-dense lymphatic organs. These microenvironments impose fundamental challenges on leukocytes, which include adhesive crawling under high shear stress, extreme cellular deformation while crossing physical barriers, and pathfinding in maze-like 3D environments. Crossing these microenvironments in a fast and efficient manner is a hallmark of leukocyte biology. We review the underlying cell biological principles and molecular mechanisms. By integrating knowledge from physiological in vivo and reductionistic in vitro approaches, we developed a holistic view of leukocyte migration strategies, including misregulation in disease and mechanistic hijacking by tumor cells.
Exogenous mechanical cues are transmitted from the extracellular matrix to the nuclear envelope (NE), where mechanical stress on the NE mediates shuttling of transcription factors and other signaling cascades that dictate downstream cellular behavior and fate decisions. To systematically study how nuclear morphology can change across various physiologic microenvironmental contexts, we cultured mesenchymal progenitor cells (MSCs) in engineered 2D and 3D hyaluronic acid hydrogel systems. Across multiple contexts we observed highly ‘wrinkled’ nuclear envelopes, and subsequently developed a quantitative single-cell imaging metric to better evaluate how wrinkles in the nuclear envelope relate to progenitor cell mechanotransduction. We determined that in soft 2D environments the NE is predominately wrinkled, and that increases in cellular mechanosensing (indicated by cellular spreading, adhesion complex growth, and nuclear localization of YAP/TAZ) occurred only in absence of nuclear envelope wrinkling. Conversely, in 3D hydrogel and tissue contexts, we found NE wrinkling occurred along with increased YAP/TAZ nuclear localization. We further determined that these NE wrinkles in 3D were largely generated by actin impingement, and compared to other nuclear morphometrics, the degree of nuclear wrinkling showed the greatest correlation with nuclear YAP/TAZ localization. These findings suggest that the degree of nuclear envelope wrinkling can predict mechanotransduction state in mesenchymal progenitor cells and highlights the differential mechanisms of NE stress generation operative in 2D and 3D microenvironmental contexts.
The EMBO/EMBL Symposium 'Mechanical Forces in Development' was held in Heidelberg, Germany, on 3-6 July 2019. This interdisciplinary symposium brought together an impressive and diverse line-up of speakers seeking to address the origin and role of mechanical forces in development. Emphasising the importance of integrative approaches and theoretical simulations to obtain comprehensive mechanistic insights into complex morphogenetic processes, the meeting provided an ideal platform to discuss the concepts and methods of developmental mechanobiology in an era of fast technical and conceptual progress. Here, we summarise the concepts and findings discussed during the meeting, as well as the agenda it sets for the future of developmental mechanobiology.
