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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., The arrival of migratory cells at their targets relies on following precise routes within tissues. Here the authors demonstrate that the cell adhesion molecule E-cadherin can control the path of cell migration by confining the site where bleb-type protrusions form within the cell front.

Significance Bleb-driven cell migration plays important roles in diverse biological processes. Here, we present the mechanism for polarity establishment and maintenance in blebbing cells in vivo. We show that actin polymerization defines the leading edge, the position where blebs form. We show that the cell front can direct the formation of the rear by facilitating retrograde flow of proteins that limit the generation of blebs at the opposite aspect of the cell. Conversely, localization of bleb-inhibiting proteins at one aspect of the cell results in the establishment of the cell front at the opposite side. These antagonistic interactions result in robust polarity that can be initiated in a random direction, or oriented by a chemokine gradient., To study the mechanisms controlling front-rear polarity in migrating cells, we used zebrafish primordial germ cells (PGCs) as an in vivo model. We find that polarity of bleb-driven migrating cells can be initiated at the cell front, as manifested by actin accumulation at the future leading edge and myosin-dependent retrograde actin flow toward the other side of the cell. In such cases, the definition of the cell front, from which bleb-inhibiting proteins such as Ezrin are depleted, precedes the establishment of the cell rear, where those proteins accumulate. Conversely, following cell division, the accumulation of Ezrin at the cleavage plane is the first sign for cell polarity and this aspect of the cell becomes the cell back. Together, the antagonistic interactions between the cell front and back lead to a robust polarization of the cell. Furthermore, we show that chemokine signaling can bias the establishment of the front-rear axis of the cell, thereby guiding the migrating cells toward sites of higher levels of the attractant. We compare these results to a theoretical model according to which a critical value of actin treadmilling flow can initiate a positive feedback loop that leads to the generation of the front-rear axis and to stable cell polarization. Together, our in vivo findings and the mathematical model, provide an explanation for the observed nonoriented migration of primordial germ cells in the absence of the guidance cue, as well as for the directed migration toward the region where the gonad develops.

Tension and mechanical properties of muscle tissue are tightly related to proper skeletal muscle function, which makes experimental access to the biomechanics of muscle tissue formation a key requirement to advance our understanding of muscle function and development. Recently developed elastic in vitro culture chambers allow for raising 3D muscle tissue under controlled conditions and to measure global tissue force generation. However, these chambers are inherently incompatible with high-resolution microscopy limiting their usability to global force measurements, and preventing the exploitation of modern fluorescence based investigation methods for live and dynamic measurements. Here, we present a new chamber design pairing global force measurements, quantified from post-deflection, with local tension measurements obtained from elastic hydrogel beads embedded in muscle tissue. High-resolution 3D video microscopy of engineered muscle formation, enabled by the new chamber, shows an early mechanical tissue homeostasis that remains stable in spite of continued myotube maturation.

Nucleus centering in mouse oocytes depends on a gradient of actin-positive vesicle persistence. Modeling coupled to 3D simulations and experimental testing of predictions coming from the simulations demonstrate that this gradient nonspecifically centers large objects during prophase I and meiosis I in oocytes., Nucleus centering in mouse oocytes results from a gradient of actin-positive vesicle activity and is essential for developmental success. Here, we analyze 3D model simulations to demonstrate how a gradient in the persistence of actin-positive vesicles can center objects of different sizes. We test model predictions by tracking the transport of exogenous passive tracers. The gradient of activity induces a centering force, akin to an effective pressure gradient, leading to the centering of oil droplets with velocities comparable to nuclear ones. Simulations and experimental measurements show that passive particles subjected to the gradient exhibit biased diffusion toward the center. Strikingly, we observe that the centering mechanism is maintained in meiosis I despite chromosome movement in the opposite direction; thus, it can counteract a process that specifically off-centers the spindle. In conclusion, our findings reconcile how common molecular players can participate in the two opposing functions of chromosome centering versus off-centering.

Cancer-associated fibroblasts (CAFs) promote cancer cell invasion and dissemination by remodeling the extracellular matrix; however, the mechanism by which CAFs remodel the matrix is still unknown. Attieh et al. show that CAFs induce cancer cell invasion through fibronectin matrix assembly that is mainly mediated by integrin-αvβ3., Cancer-associated fibroblasts (CAFs) are the most abundant cells of the tumor stroma. Their capacity to contract the matrix and induce invasion of cancer cells has been well documented. However, it is not clear whether CAFs remodel the matrix by other means, such as degradation, matrix deposition, or stiffening. We now show that CAFs assemble fibronectin (FN) and trigger invasion mainly via integrin-αvβ3. In the absence of FN, contractility of the matrix by CAFs is preserved, but their ability to induce invasion is abrogated. When degradation is impaired, CAFs retain the capacity to induce invasion in an FN-dependent manner. The level of expression of integrins αv and β3 and the amount of assembled FN are directly proportional to the invasion induced by fibroblast populations. Our results highlight FN assembly and integrin-αvβ3 expression as new hallmarks of CAFs that promote tumor invasion.

Tissue homeostasis is dependent on the controlled localization of specific cell types and the correct composition of the extracellular stroma. While the role of the cancer stroma in tumour progression has been well characterized, the specific contribution of the matrix itself is unknown. Furthermore, the mechanisms enabling normal—not cancer—stroma to provide tumour-suppressive signals and act as an antitumorigenic barrier are poorly understood. Here we show that extracellular matrix (ECM) generated by normal fibroblasts (NFs) is softer than the CAF matrix, and its physical and structural features regulate cancer cell proliferation. We find that normal ECM triggers downregulation and nuclear exit of the histone demethylase JMJD1a resulting in the epigenetic growth restriction of carcinoma cells. Interestingly, JMJD1a positively regulates transcription of many target genes, including YAP/TAZ (WWTR1), and therefore gene expression in a stiffness-dependent manner. Thus, normal stromal restricts cancer cell proliferation through JMJD1a-dependent modulation of gene expression., The tumour stroma has altered stiffness and matrix architecture compared to normal tissue, which favours proliferation, and invasion. Here, the authors find that the extracellular matrix produced by normal fibroblasts inhibits cancer cell proliferation through mechanosensitive downregulation of JMJD1a.

Living cells are active mechanical systems that are able to generate forces. Their structure and shape are primarily determined by biopolymer filaments and molecular motors that form the cytoskeleton. Active force generation requires constant consumption of energy to maintain the nonequilibrium activity to drive organization and transport processes necessary for their function. To understand this activity it is necessary to develop new approaches to probe the underlying physical processes. Active cell mechanics incorporates active molecular-scale force generation into the traditional framework of mechanics of materials. This review highlights recent experimental and theoretical developments towards understanding active cell mechanics. We focus primarily on intracellular mechanical measurements and theoretical advances utilizing the Langevin framework. These developing approaches allow a quantitative understanding of nonequilibrium mechanical activity in living cells. This article is part of a Special Issue entitled: Mechanobiology.

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial–stromal interactions. Mice lacking SHARPIN expression in all cells ( Sharpin cpdm ), and mice with a stromal ( S100a4‐Cre ) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpin cpdm mammary epithelial cells transplanted in vivo into wild‐type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpin cpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpin cpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro . Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.

Understanding embryonic development requires the characterization of the forces and the mechanical features that shape cells and tissues within the organism. In addition, experimental application of forces on cells and altering cell and organelle shape allows determining the role such forces play in morphogenesis. Here, we present a holographic optical tweezers-based new microscopic platform for in vivo applications in the context of a developing vertebrate embryo that unlike currently used setups allows simultaneous trapping of multiple objects and rapid comparisons of viscoelastic properties in different locations. This non-invasive technique facilitates a dynamic analysis of mechanical properties of cells and tissues without intervening with embryonic development. We demonstrate the application of this platform for manipulating organelle shape and for characterizing the mechanobiological properties of cells in live zebrafish embryos. The method of holographic optical tweezers as described here is of general interest and can be easily transferred to studying a range of developmental processes in zebrafish, thereby establishing a versatile platform for similar investigations in other organisms. Fluorescent beads injected into zebrafish embryos at 1-cell stage are maintained within the embryos and do not affect their development as observed in the presented 1-day old embryo.

International audience; The mechanical properties of tumors and the tumor environment provide important information for the progression and characterization of cancer. Tumors are surrounded by an extracellular matrix (ECM) dominated by collagen I. The geometrical and mechanical properties of the ECM play an important role for the initial step in the formation of metastasis, presented by the migration of malignant cells towards new settlements as well as the vascular and lymphatic system. The extent of this cell invasion into the ECM is a key medical marker for cancer prognosis. In vivo studies reveal an increased stiffness and different architecture of tumor tissue when compared to its healthy counterparts. The observed parallel collagen organization on the tumor border and radial arrangement at the invasion zone has raised the question about the mechanisms organizing these structures. Here we study the effect of contractile forces originated from model tumor spheroids embedded in a biomimetic collagen I matrix. We show that contractile forces act immediately after seeding and deform the ECM, thus leading to tensile radial forces within the matrix. Relaxation of this tension via cutting the collagen does reduce invasion, showing a mechanical relation between the tensile state of the ECM and invasion. In turn, these results suggest that tensile forces in the ECM facilitate invasion. Furthermore, simultaneous contraction of the ECM and tumor growth leads to the condensation and reorientation of the collagen at the spheroid’s surface. We propose a tension-based model to explain the collagen organization and the onset of invasion by forces originating from the tumor.

Many biochemical processes in the growth cone finally target its biomechanical properties, such as stiffness and force generation, and thus permit and control growth cone movement. Despite the immense progress in our understanding of biochemical processes regulating neuronal growth, growth cone biomechanics remains poorly understood. Here, we combine different experimental approaches to measure the structural and mechanical properties of a growth cone and to simultaneously determine its actin dynamics and traction force generation. Using fundamental physical relations, we exploited these measurements to determine the internal forces generated by the actin cytoskeleton in the lamellipodium. We found that, at timescales longer than the viscoelastic relaxation time of τ = 8.5 ± 0.5 sec, growth cones show liquid-like characteristics, whereas at shorter time scales they behaved elastically with a surprisingly low elastic modulus of E = 106 ± 21 Pa. Considering the growth cone’s mechanical properties and retrograde actin flow, we determined the internal stress to be on the order of 30 pN per μm 2 . Traction force measurements confirmed these values. Hence, our results indicate that growth cones are particularly soft and weak structures that may be very sensitive to the mechanical properties of their environment.

Neuronal growth is an extremely complex yet reliable process that is directed by a dynamic lamellipodial structure at the tip of every growing neurite, called the growth cone. Lamellipodial edge fluctuations are controlled by the interplay between actin polymerization pushing the edge forward and molecular motor driven retrograde actin flow retracting the actin network. The leading edge switches randomly between extension and retraction processes. We identify switching of "on/off" states in actin polymerization as the main determinant of lamellipodial advancement. Our analysis of motility statistics allows for a prediction of growth direction. This was used in simulations explaining the amazing signal detection capabilities of neuronal growth by the experimentally found biased stochastic processes. Our measurements show that the intensity of stochastic fluctuations depend on changes in the underlying active intracellular processes and we find a power law eta = a*x(alpha) with exponent alpha = 2.63 +/- 0.12 between noise intensity eta and growth cone activity x, defined as the sum of protrusion and retraction velocity. Differences in the lamellipodial dynamics between primary neurons and a neuronal cell line further suggests that active processes tune the observed stochastic fluctuations. This hints at a possible role of noise intensity in determining signal detection sensitivity.

In somatic cells, the position of the cell centroid is dictated by the centrosome. The centrosome is instrumental in nucleus positioning, the two structures being physically connected. Mouse oocytes have no centrosomes, yet harbour centrally located nuclei. We demonstrate how oocytes define their geometric centre in the absence of centrosomes. Using live imaging of oocytes, knockout for the formin 2 actin nucleator, with off-centred nuclei, together with optical trapping and modelling, we discover an unprecedented mode of nucleus positioning. We document how active diffusion of actin-coated vesicles, driven by myosin Vb, generates a pressure gradient and a propulsion force sufficient to move the oocyte nucleus. It promotes fluidization of the cytoplasm, contributing to nucleus directional movement towards the centre. Our results highlight the potential of active diffusion, a prominent source of intracellular transport, able to move large organelles such as nuclei, providing in vivo evidence of its biological function.

Combined phase-sensitive acoustic microscopy (PSAM) at 1.2 GHz and confocal laser scanning microscopy (CLSM) in reflection and fluorescence has been implemented and applied to polymer blend films and fluorescently labeled fibroblasts and neuronal cells in order to explore the prospects and the various contrast mechanisms of this powerful technique. Topographic contrast is available for appropriate samples from CLSM in reflection and, with significantly higher precision, from the acoustic phase images. Material contrast can be gained from acoustic amplitude V(z) graphs. In the case of the biological cells investigated, the optical and acoustic images are very different and exhibit different features of the samples.

Cell migration is essential for morphogenesis, for organ formation and homeostasis, with relevance for clinical conditions. The migration of primordial germ cells (PGCs) is a useful model to study this process in the context of the developing embryo. Zebrafish PGC migration depends on the formation of cellular protrusions in form of blebs, a type of protrusion found in various cell types. Here we report on the mechanisms allowing the inflation of the membrane during bleb formation. We show that the rapid expansion of the protrusion depends on membrane invaginations that are localized preferentially at the cell front. The formation of these invaginations requires the function of Cdc42, and their unfolding allows bleb inflation and dynamic cell-shape changes performed by migrating cells. Inhibiting the formation and release of the invaginations strongly interfered with bleb formation, cell motility and with the ability of the cells to reach their target.

Summary Contact inhibition of locomotion (CIL) is a multifaceted process that causes many cell types to repel each other upon collision. During development, this seemingly uncoordinated reaction is a critical driver of cellular dispersion within embryonic tissues. Here, we show that Drosophila hemocytes require a precisely orchestrated CIL response for their developmental dispersal. Hemocyte collision and subsequent repulsion involves a stereotyped sequence of kinematic stages that are modulated by global changes in cytoskeletal dynamics. Tracking actin retrograde flow within hemocytes in vivo reveals synchronous reorganization of colliding actin networks through engagement of an inter-cellular adhesion. This inter-cellular actin-clutch leads to a subsequent build-up in lamellar tension, triggering the development of a transient stress fiber, which orchestrates cellular repulsion. Our findings reveal that the physical coupling of the flowing actin networks during CIL acts as a mechanotransducer, allowing cells to haptically sense each other and coordinate their behaviors., Graphical Abstract, Highlights • CIL in embryonic dispersing hemocytes requires choreographed changes in motility • Tracking actin flow in vivo reveals synchronous changes in colliding actin networks • An inter-cellular actin-clutch between colliding cells induces lamellar tension • Tension buildup and release orchestrate the CIL response, Contact inhibition of locomotion in Drosophila macrophages involves mechanical coupling of colliding actin networks. The resultant buildup in lamellar tension orchestrates the response and choreographs cell repulsion.

Recent results indicate that, in addition to chemical cues, mechanical stimuli may also impact neuronal growth. For instance, unlike most other cell types, neurons prefer soft substrates. However, the mechanisms responsible for the neuronal affinity for soft substrates have not yet been identified. In this study, we show that, in vitro, neurons continuously probe their mechanical environment. Growth cones visibly deform substrates with a compliance commensurate with their own. To understand the sensing of stiff substrates by growth cones, we investigated their precise temporal response to well-defined mechanical stress. When the applied stress exceeded a threshold of 274 +/- 41 pN/microm(2), neurons retracted and re-extended their processes, thereby enabling exploration of alternative directions. A calcium influx through stretch-activated ion channels and the detachment of adhesion sites were prerequisites for this retraction. Our data illustrate how growing neurons may detect and avoid stiff substrates--as a mechanism involved in axonal branch pruning--and provide what we believe is novel support of the idea that mechanics may act as guidance cue for neuronal growth.

We present a novel technique to noninvasively control the growth and turning behavior of an extending neurite. A highly focused infrared laser, positioned at the leading edge of a neurite, has been found to induce extension/turning toward the beam's center. This technique has been used successfully to guide NG108-15 and PC12 cell lines [Ehrlicher, A., Betz, T., Stuhrmann, B., Koch, D. Milner, V. Raizen, M. G., and Kas, J. (2002). Guiding neuronal growth with light. Proc. Natl. Acad. Sci. USA 99, 16024-16028], as well as primary rat and mouse cortical neurons [Stuhrmann, B., Goegler, M., Betz, T., Ehrlicher, A., Koch, D., and Kas, J. (2005). Automated tracking and laser micromanipulation of cells. Rev. Sci. Instr. 76, 035105]. Optical guidance may eventually be used alone or with other methods for controlling neurite extension in both research and clinical applications.

In the absence of simple noninvasive measurements, the knowledge of temporal and spatial variations of axons mechanics remains scarce. By extending thermal fluctuation spectroscopy (TFS) to long protrusions, we determine the transverse amplitude thermal fluctuation spectra that allow direct and simultaneous access to three key mechanics parameters: axial tension, bending flexural rigidity and plasma membrane tension. To test our model, we use PC12 cell protrusions-a well-know biophysical model of axons-in order to simplify the biological system under scope. For instance, axial and plasma membrane tension are found in the range of nano Newton and tens of pico Newtons per micron respectively. Furthermore, our results shows that the TFS technique is capable to distinguish quasi-identical protrusions. Another advantage of our approach is the time resolved nature of the measurements. Indeed, in the case of long term experiments on PC12 protrusions, TFS has revealed large temporal, correlated variations of the protrusion mechanics, displaying extraordinary feedback control over the axial tension in order to maintain a constant tension value.

Confocal and multiphoton microscopy are essential tools in modern life sciences. They allow fast and highly resolved imaging of a steadily growing number of fluorescent markers, ranging from fluorescent proteins to quantum dots and other fluorophores, used for the localization of molecules and the quantitative detection of molecular properties within living cells and organisms. Up to now, only one physical limitation seemed to be unavoidable. Both confocal and multiphoton microscopy rely on lasers as excitation sources, and their monochromatic radiation allows only a limited number of simultaneously usable dyes, which depends on the specific number of laser lines available in the used microscope. We have overcome this limitation by successfully replacing all excitation lasers in a standard confocal microscope with pulsed white light ranging from 430 to 1300 nm generated in a tapered silica fiber. With this easily reproducible method, simultaneous confocal and multiphoton microscopy was demonstrated. By developing a coherent and intense laser source with spectral width comparable to a mercury lamp, we provide the flexibility to excite any desired fluorophore combination.

Control over neuronal growth is a fundamental objective in neuroscience, cell biology, developmental biology, biophysics, and biomedicine and is particularly important for the formation of neural circuits in vitro , as well as nerve regeneration in vivo [Zeck, G. & Fromherz, P. (2001) Proc. Natl. Acad. Sci. USA 98, 10457–10462]. We have shown experimentally that we can use weak optical forces to guide the direction taken by the leading edge, or growth cone, of a nerve cell. In actively extending growth cones, a laser spot is placed in front of a specific area of the nerve's leading edge, enhancing growth into the beam focus and resulting in guided neuronal turns as well as enhanced growth. The power of our laser is chosen so that the resulting gradient forces are sufficiently powerful to bias the actin polymerization-driven lamellipodia extension, but too weak to hold and move the growth cone. We are therefore using light to control a natural biological process, in sharp contrast to the established technique of optical tweezers [Ashkin, A. (1970) Phys. Rev. Lett. 24, 156–159; Ashkin, A. & Dziedzic, J. M. (1987) Science 235, 1517–1520], which uses large optical forces to manipulate entire structures. Our results therefore open an avenue to controlling neuronal growth in vitro and in vivo with a simple, noncontact technique.

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved., Cells sense mechanical forces from their environment, but the precise mechanical variable sensed by cells is unclear. Here, the authors show that cells can sense the rate of force application, known as the loading rate, with effects on YAP nuclear localization and cytoskeletal stiffness remodelling.