Abstract The investigation of morphogenesis during organism development has massively benefited from the interaction between developmental biology and biophysics, which gave new quantitative insights into nature’s physical working principles. The process of zebrafish epiboly provides a beautiful model system as cells exhibit collective migration, meanwhile requiring symmetry breaking for gastrulation and the subsequent tissue differentiation. These elegantly robust processes are facilitated by both biochemical and mechanical cues. Here we test the hypothesis that the future body axis is robust against mechanical perturbations of tissue flow and tissue stress fields. For this, we trigger tissue flow by photoablation, which leads to an externally enforced symmetry breaking before natural symmetry breaking of gastrulation occurs. Using 3D light sheet microscopy, we obtained nuclei trajectories as a proxy for the migration of individual cells. Analyzing the derived tissue velocity field, we observed that epiboly is highly robust and mostly unaffected by ablation damage. However, the photoablation induced a convergent motion of cells towards an azimuthal angle resembling shield formation. We extensively characterized this convergent motion and checked if this tissue flow is able to reorient natural symmetry breaking during shield formation. While ablations can transiently disrupt or reorient the convergent motion, we cannot confirm any redirection of the location where the future body axis of the animal develops. The observations were also highly dependent on the developmental stage of the embryo, which cast a greater and more permanent impact, suggesting strong control exerted by factors other than mechanical signals. Significance statement Morphogenesis emerges from the interplay between biochemical patterning and mechanical forces, yet the extent to which mechanical cues influence embryo developmental remains unclear. Using zebrafish epiboly as model system, we imposed controlled mechanical perturbations through targeted photoablation and tracking resulting 3D cell movements. We show that although ablations generate transient convergent motions, epiboly is remarkably robust to externally induced tissue flows. Our findings indicate that the future body axis is strongly buffered against mechanical disturbances, highlighting the dominant role of developmental stage and non-mechanical cues in guiding development.

The mechanical properties of the cytoplasm and nucleoplasm are crucial for the correct and robust functioning of a cell and play a key role in understanding how mechanical signals are transferred to the nucleus. Here, we demonstrate remarkable shape mimicry between the cellular and nuclear shape of oocytes, following the externally applied deformation without direct contact between the cell cortex and the nucleus. This effect arises from a surprisingly soft and fluid-like nucleoplasm that barely resists external strain, while the viscoelastic cytoplasm drives shape transmission. Comparative studies in jellyfish, starfish, and mouse oocytes reveal that lower cytoplasmic elasticity in jellyfish leads to reduced nuclear shape mimicry, highlighting the role of cytoplasmic mechanics in nuclear deformation.

Intracellular organization is a highly regulated homeostatic state maintained to ensure eukaryotic cells’ correct and efficient functioning. Thanks to decades of research, vast knowledge of the proteins involved in intracellular transport and organization has been acquired. However, how these influence and potentially regulate the intracellular mechanical properties of the cell is largely unknown. There is a deep knowledge gap between the understanding of cortical mechanics, which is accessible by a series of experimental tools, and the intracellular situation that has been largely neglected due to the difficulty of performing intracellular mechanics measurements. Recently, tools required for such quantitative and localized analysis of intracellular mechanics have been introduced. Here, we review how these approaches and the resulting viscoelastic models lead the way to a full mechanical description of the cytoplasm, which is instrumental for a quantitative characterization of the intracellular life of cells.

Disruptions of the eukaryotic plasma membrane due to chemical and mechanical challenges are frequent and detrimental and thus need to be repaired to maintain proper cell function and avoid cell death. However, the cellular mechanisms involved in wound resealing and restoration of homeostasis are diverse and contended. Here, it is shown that clathrin-mediated endocytosis is induced at later stages of plasma membrane wound repair following the actual resealing of the wound. This compensatory endocytosis occurs near the wound, predominantly at sites of previous early endosome exocytosis which is required in the initial stage of membrane resealing, suggesting a spatio-temporal co-ordination of exo- and endocytosis during wound repair. Using cytoskeletal alterations and modulations of membrane tension and membrane area, membrane tension is identified as a major regulator of the wounding-associated exo- and endocytic events that mediate efficient wound repair. Thus, membrane tension changes are a universal trigger for plasma membrane wound repair modulating the exocytosis of early endosomes required for resealing and subsequent clathrin-mediated endocytosis acting at later stages to restore cell homeostasis and function.

In both physiological processes and disease contexts, migrating cells have the ability to adapt to conditions in their environment. As an in vivo model for this process, we use zebrafish primordial germ cells that migrate throughout the developing embryo. When migrating within an ectodermal environment, the germ cells form fewer and smaller blebs when compared with their behavior within mesodermal environment. We find that cortical tension of neighboring cells is a parameter that affects blebbing frequency. Interestingly, the change in blebbing activity is accompanied by the formation of more actin-rich protrusions. These alterations in cell behavior that correlate with changes in RhoA activity could allow the cells to maintain dynamic motility parameters, such as migration speed and track straightness, in different settings. In addition, we find that the polarity of the cells can be affected by stiff structures positioned in their migration path This article has an associated 'The people behind the papers' interview.

Optical tweezers are tools made of light that enable contactless pushing, trapping, and manipulation of objects, ranging from atoms to space light sails. Since the pioneering work by Arthur Ashkin in the 1970s, optical tweezers have evolved into sophisticated instruments and have been employed in a broad range of applications in the life sciences, physics, and engineering. These include accurate force and torque measurement at the femtonewton level, microrheology of complex fluids, single micro- and nano-particle spectroscopy, single-cell analysis, and statistical-physics experiments. This roadmap provides insights into current investigations involving optical forces and optical tweezers from their theoretical foundations to designs and setups. It also offers perspectives for applications to a wide range of research fields, from biophysics to space exploration.

Living cells are complex entities that perform many different complex tasks with astonishing robustness. While the direct dependence of biological processes on controlled protein expression is well established, we only begin to understand how intracellular mechanical characteristics guide and support biological function. This is in stark contrast to the expected functional role that intracellular mechanical properties should have for many core cellular functions such as organization, homeostasis and transport. From a mechanical point of view, cells are complex viscoelastic materials that are continuously driven out of thermodynamic equilibrium, which makes both a physical measurement and mathematical modeling of its properties difficult. Here, we define a “mechanical fingerprint” that can not only characterize the intracellular mechanical state, but also carve out the mechanical differences between cell types with the potential to relate these to proper cell function. By analyzing the frequency-dependent viscoelastic properties and intracellular activity of cells using microrheology, we distilled the complex active mechanical state into just 6 parameters that comprise the mechanical fingerprint. The systematic investigation of the fingerprint illustrates a parameter tuning that can be explained by the functional cellular requirements. However, the full potential of the mechanical fingerprint is given by a statistical analysis of its parameters across all investigated cell types, which suggests that cells adjust mechanical parameters in a correlated way to position their intracellular mechanical properties within a well defined phase-space that is spanned between activity, mechanical resistance and fluidity. This paves the way for a systematic study of the interdependence of biological function and intracellular active mechanics.

Cellular membrane area is a key parameter for any living cell that is tightly regulated to avoid membrane damage. Changes in area-to-volume ratio are known to be critical for cell shape, but are mostly investigated by changing the cell volume via osmotic shocks. In turn, many important questions relating to cellular shape, membrane tension homeostasis and local membrane area cannot be easily addressed because experimental tools for controlled modulation of cell membrane area are lacking. Here we show that photoswitching an amphiphilic azobenzene can trigger its intercalation into the plasma membrane of various mammalian cells ranging from erythrocytes to myoblasts and cancer cells. The photoisomerization leads to a rapid (250-500 ms) and highly reversible membrane area change (ca 2 % for erythrocytes) that triggers a dramatic shape modulation of living cells.

While matrix stiffness regulates cell behavior on 2D substrates, recent studies using synthetic hydrogels have suggested that in 3D environments, cell behavior is primarily impacted by matrix degradability, independent of stiffness. However, these studies did not consider the potential impact of other confounding matrix parameters that typically covary with changes in stiffness, particularly, hydrogel swelling and hydrolytic stability, which may explain the previously observed distinctions in cell response in 2D versus 3D settings. To investigate how cells sense matrix stiffness in 3D environments, a nonswelling, hydrolytically stable, linearly elastic synthetic hydrogel model is developed in which matrix stiffness and degradability can be tuned independently. It is found that matrix degradability regulates cell spreading kinetics, while matrix stiffness dictates the final spread area once cells achieve equilibrium spreading. Importantly, the differentiation of human mesenchymal stromal cells toward adipocytes or osteoblasts is regulated by the spread state of progenitor cells upon initiating differentiation. These studies uncover matrix stiffness as a major regulator of cell function not just in 2D, but also in 3D environments, and identify matrix degradability as a critical microenvironmental feature in 3D that in conjunction with matrix stiffness dictates cell spreading, cytoskeletal state, and stem cell differentiation outcomes.

A key behavior observed during morphogenesis, wound healing, and cancer invasion is that of collective and coordinated cellular motion. Hence, understanding the different aspects of such coordinated migration is fundamental for describing and treating cancer and other pathological defects. In general, individual cells exert forces on their environment in order to move, and collective motion is coordinated by cell–cell adhesion-based forces. However, this notion ignores other mechanisms that encourage cellular movement, such as pressure differences. Here, using model tumors, it is found that increased pressure drove coordinated cellular motion independent of cell–cell adhesion by triggering cell swelling in a soft extracellular matrix (ECM). In the resulting phenotype, a rapid burst-like stream of cervical cancer cells emerged from 3D aggregates embedded in soft collagen matrices (0.5 mg mL −1 ). This fluid-like pushing mechanism, recorded within 8 h after embedding, shows high cell velocities and super-diffusive motion. Because the swelling in this model system critically depends on integrin-mediated cell–ECM adhesions and cellular contractility, the swelling is likely triggered by unsustained mechanotransduction, providing new evidence that pressure-driven effects must be considered to more completely understand the mechanical forces involved in cell and tissue movement as well as invasion.

Coordinated Cell Motion In article number 2104808 by Swetha Raghuraman, Timo Betz, and co-workers, a novel migration phenomenon is presented wherein cancer cells burst out from tumor aggregates as an act of pressure release into the surrounding in-homogeneous soft extra-cellular matrix (ECM) made up of collagen. The pressure within tumor aggregates manifests due to cellular swelling, leading to a super-diffusive coordinated expulsion of cells within 12 hours, directed towards regions providing least mechanical resistance

Collective migration of cells is a key behaviour observed during morphogenesis, wound healing and cancer cell invasion. Hence, understanding the different aspects of collective migration is at the core of further progress in describing and treating cancer and other pathological defects. The standard dogma in cell migration is that cells exert forces on the environment to move and cell-cell adhesion-based forces provide the coordination for collective migration. Here, we report a new collective migration mechanism that is independent of pulling forces on the extra-cellular matrix (ECM), as it is driven by the pressure difference generated inside model tumours. We observe a striking collective migration phenotype, where a rapid burst-like stream of HeLa cervical cancer cells emerges from the 3D aggregate embedded in matrices with low collagen concentration (0.5 mg ml − 1 ). This invasion-like behaviour is recorded within 8 hours post embedding (hpe), and is characterised by high cell velocity and super-diffusive collective motion. We show that cellular swelling, triggered by the soft matrix, leads to a rise in intrinsic pressure, which eventually drives an invasion-like phenotype of HeLa cancer aggregates. These dynamic observations provide new evidence that pressure-driven effects need to be considered for a complete description of the mechanical forces involved in collective migration and invasion.

Here we present the design of a high-resolution, high-magnification microscope using LEGO® bricks and easily-available lenses. With the provided workflow and suggested experiments, we show that 9-to-13-year old students significantly increased their understanding of microscopy.

To investigate the relation between structural and local mechanical properties in biological tissues, we develop a light sheet microscope combined with optical tweezers for in vivo imaging and microrheology.

The life and death of an organism depends largely on correct cell division. While the overall biochemical signaling and morphological processes during mitosis are well understood, the importance of mechanical forces and material properties is only just starting to be discovered. Recent studies of global cell stiffening during cell division may imply an understanding of the cytosol mechanics that is mistaken. Here we show that in contrast to the stiffening process in the cell cortex, the interior of the cell undergoes a softening and fluidification that is accompanied by a decrease of active forces driving particle mobility. Using optical tweezers-based microrheology we capture the complex active and passive material state of the cytoplasm using only six relevant parameters. We demonstrate that the softening occurs because of a surprising role switch between microtubules and actin, where the intracellular, actin-based mechanics is largely controlled by a formin-mediated network. ### Competing Interest Statement The authors have declared no competing interest.

The life and death of an organism rely on correct cell division, which occurs through the process of mitosis. Although the biochemical signalling and morphogenetic processes during mitosis are well understood, the importance of mechanical forces and material properties is only just starting to be discovered. Recent studies have revealed that the layer of proteins beneath the cell membrane—the so-called cell cortex—stiffens during mitosis, but it is as yet unclear whether mechanical changes occur in the rest of the material in the cell, contained in the cytoplasm. Here we show that, in contrast to the cortical stiffening, the interior of the cell undergoes a softening and an increase in dissipative timescale, similar to viscoelastic relaxation. These mechanical changes are accompanied by a decrease in the active forces that drive particle mobility. Using optical tweezers to perform microrheology measurements, we capture the complex active and passive material states of the cytoplasm using six relevant parameters, of which only two vary considerably during mitosis. We demonstrate a role switch between microtubules and actin that could contribute to the observed softening.

Microscopy is an essential tool in many fields of science. However, due to their costs and fragility, the usage of microscopes is limited in classroom settings and nearly absent at home. In this article we present the construction of a microscope using LEGO ® bricks and low-cost, easily available lenses. We demonstrate that the obtained magnification and resolution are sufficient to resolve micrometer-sized objects and propose a series of experiments that explore various biophysical principles. Finally, a study with students in the age range of 9 to 13 shows that the understanding of microscopy increases significantly after working with the LEGO microscope.

The biophysical and biochemical properties of live tissues are important in the context of development and disease. Methods for evaluating these properties typically involve destroying the tissue or require specialized technology and complicated analyses. Here, we present a novel, noninvasive methodology for determining the spatial distribution of tissue features within embryos, making use of nondirectionally migrating cells and software we termed "Landscape," which performs automatized high-throughput three-dimensional image registration. Using the live migrating cells as bioprobes, we identified structures within the zebrafish embryo that affect the distribution of the cells and studied one such structure constituting a physical barrier, which, in turn, influences amoeboid cell polarity. Overall, this work provides a unique approach for detecting tissue properties without interfering with animal's development. In addition, Landscape allows for integrating data from multiple samples, providing detailed and reliable quantitative evaluation of variable biological phenotypes in different organisms.

The migration of many cell types relies on the formation of actomyosin-dependent protrusions called blebs, but the mechanisms responsible for focusing this kind of protrusive activity to the cell front are largely unknown. Here, we employ zebrafish primordial germ cells (PGCs) as a model to study the role of cell-cell adhesion in bleb-driven single-cell migration in vivo. Utilizing a range of genetic, reverse genetic and mathematical tools, we define a previously unknown role for E-cadherin in confining bleb-type protrusions to the leading edge of the cell. We show that E-cadherin-mediated frictional forces impede the backwards flow of actomyosin-rich structures that define the domain where protrusions are preferentially generated. In this way, E-cadherin confines the bleb-forming region to a restricted area at the cell front and reinforces the front-rear axis of migrating cells. Accordingly, when E-cadherin activity is reduced, the bleb-forming area expands, thus compromising the directional persistence of the cells.

Fibrin is an elastomeric protein forming highly extensible fiber networks that provide the scaffold of blood clots. Here we reveal the molecular mechanisms that explain the large extensibility of fibrin networks by performing in situ small angle X-ray scattering measurements while applying a shear deformation. We simultaneously measure shear-induced alignment of the fibers and changes in their axially ordered molecular packing structure. We show that fibrin networks exhibit distinct structural responses that set in consecutively as the shear strain is increased. They exhibit an entropic response at small strains (<5%), followed by progressive fiber alignment (>25% strain) and finally changes in the fiber packing structure at high strain (>100%). Stretching reduces the fiber packing order and slightly increases the axial periodicity, indicative of molecular unfolding. However, the axial periodicity changes only by 0.7%, much less than the 80% length increase of the fibers, indicating that fiber elongation mainly stems from uncoiling of the natively disordered αC-peptide linkers that laterally bond the molecules. Upon removal of the load, the network structure returns to the original isotropic state, but the fiber structure becomes more ordered and adopts a smaller packing periodicity compared to the original state. We conclude that the hierarchical packing structure of fibrin fibers, with built-in disorder, makes the fibers extensible and allows for mechanical annealing. Our results provide a basis for interpreting the molecular basis of haemostatic and thrombotic disorders associated with clotting and provide inspiration to design resilient bio-mimicking materials.