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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.

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.

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.

The mechanical properties and tension of muscle tissue are tightly related to proper skeletal muscle function, which makes experimental access to the biomechanics of muscle tissue development 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 measurements of 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 the muscle tissue. High resolution 3D video microscopy of engineered muscle development, enabled by the new chamber, shows an early mechanical tissue homeostasis that remains stable in spite of continued myotube maturation.

Funding: We acknowledge funding from the German Science Foundation (DFG grant numbers RH 147/1-1 to J.R., EXC 1003 CiM to T.B.), the Herchel Smith Foundation (postdoctoral fellowship to A.D.), the Royal Society (University Research Fellowship to K.J.C.), the UK EPSRC (programme grant number EP/P030017/1 to M.C.G.), the Human Frontier Science Program (HFSP grant number RGP0018/2017 to T.B.), the European Research Council (consolidator grant numbers 772798 to K.J.C., 771201 to T.B., 647186 to G.C. and 772426 to K.F.), and the UK BBSRC (equipment grant number BB/R000042/1 to G.C. and research project grant number BB/N006402/1 to K.F.). Cortical stiffness is an important cellular property that changes during migration, adhesion and growth. Previous atomic force microscopy (AFM) indentation measurements of cells cultured on deformable substrates have suggested that cells adapt their stiffness to that of their surroundings. Here we show that the force applied by AFM to a cell results in a significant deformation of the underlying substrate if this substrate is softer than the cell. This ‘soft substrate effect’ leads to an underestimation of a cell’s elastic modulus when analysing data using a standard Hertz model, as confirmed by finite element modelling and AFM measurements of calibrated polyacrylamide beads, microglial cells and fibroblasts. To account for this substrate deformation, we developed a ‘composite cell–substrate model’. Correcting for the substrate indentation revealed that cortical cell stiffness is largely independent of substrate mechanics, which has major implications for our interpretation of many physiological and pathological processes. Postprint

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.

The mechanisms by which cells exert forces on their nuclei to migrate through openings smaller than the nuclear diameter remain unclear. In microfluidic devices, the hourglass shape of the nucleus and its strain patterns as it translocates through narrow constrictions suggest pulling forces. We use CRISPR/Cas9 to label nesprin-2 giant, a protein that links the cytoskeleton to the interior of the nucleus. We demonstrate that nesprin-2 giant accumulates at the front of the nucleus during nuclear deformation through narrow constrictions, independently of the nuclear lamina. We find that nesprins are more mobile than lamin A/C, and hypothesize that nesprin accumulation at the front is a consequence of forward pulling by the cytoskeleton. Using artificial constructs, we show indeed that the actin-binding domain of nesprin-2 is necessary and sufficient to generate this accumulation, and that microtubules are not involved. Actin filaments are organized in a barrel structure around the moving nucleus in the direction of movement. This and estimates of nesprin elongation suggest that actin pulling forces on nesprins are distributed around the deformed nucleus towards the front.

SUMMARYThe mechanisms by which cells exert forces on their nuclei to migrate through openings smaller than the nuclear diameter remain unclear. In microfluidic devices, the hourglass shape of the nucleus and its strain patterns as it translocates through narrow constrictions suggest pulling forces. We use CRISPR/Cas9 to fluorescently label nesprin-2 giant, a protein that links the cytoskeleton to the interior of the nucleus. We demonstrate that nesprin-2 giant accumulates at the front of the nucleus during nuclear deformation through narrow constrictions, independently of the nuclear lamina. We find that nesprins are more mobile than lamin A/C, at time scales similar to that of the accumulation. Using artificial constructs, we show that the actin-binding domain of nesprin-2 is necessary and sufficient to generate this accumulation, and that microtubules are not necessary. Actin filaments are organized in a barrel structure around the moving nucleus in the direction of movement, suggesting that this structure is responsible for redistribution of nesprins towards the front of the nucleus. Two-photon ablation and the use of drugs inhibiting the cytoskeleton demonstrate a pulling force on the nucleus from the front of the cell that is dependent on formin and actomyosin contractility. This elastic recoil is significantly reduced when nesprins are reduced at the nuclear envelope. We thus show that actin redistributes nesprin-2 giant towards the front of the nucleus and contributes to pulling the nucleus through narrow constrictions, in concert with myosin.

The mechanism for nucleus centering in mouse oocytes results from a gradient of actin-positive vesicles. By microinjecting oil droplets and fluorescent beads, we analyze the consequences of the gradient of activity on transport of exogenous tracer particles of different sizes. We also use optical tweezers to probe rheological properties of the cytoplasm. We find that the gradient activity induces a general centering force, akin to an effective pressure gradient, leading to centering of oil droplets with velocities comparable to nuclear ones. High temporal resolution measurements reveal that passive particles, larger than 1µm, experience the activity gradient by a biased diffusion towards the cell center. Unexpectedly, this general and size dependent mechanism is maintained in Meiosis I but contrasted by a further process that specifically off-centers the spindle. These antagonizing processes depend on myosin activity, thus we reconcile how the same molecular actors can have two opposite functions (centering versus off-centering).

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.

Mechanics is at the heart of many cellular processes and its importance has received considerable attention during the last two decades. In particular, the tension of cell membranes, and more specifically of the cell cortex, is a key parameter that determines the mechanical behavior of the cell periphery. However, the measurement of tension remains challenging due to its dynamic nature. Here we show that a noninvasive interferometric technique can reveal time-resolved effective tension measurements by a high-accuracy determination of edge fluctuations in expanding cell blebs of filamin-deficient melanoma cells. The introduced technique shows that the bleb tension is ∼10–100 pN/μm and increases during bleb growth. Our results directly confirm that the subsequent stop of bleb growth is induced by an increase of measured tension, possibly mediated by the repolymerized actin cytoskeleton.

Red blood cells, or erythrocytes, are seen to flicker under optical microscopy, a phenomenon initially described as thermal fluctuations of the cell membrane. But recent studies have suggested the involvement of non-equilibrium processes, without definitively ruling out equilibrium interpretations. Using active and passive microrheology to directly compare the membrane response and fluctuations on single erythrocytes, we report here a violation of the fluctuation–dissipation relation, which is a direct demonstration of the non-equilibrium nature of flickering. With an analytical model of the composite erythrocyte membrane and realistic stochastic simulations, we show that several molecular mechanisms may explain the active fluctuations, and we predict their kinetics. We demonstrate that tangential metabolic activity in the network formed by spectrin, a cytoskeletal protein, can generate curvature-mediated active membrane motions. We also show that other active membrane processes represented by direct normal force dipoles may explain the observed membrane activity. Our findings provide solid experimental and theoretical frameworks for future investigations of the origin and function of active motion in cells. The membranes of red blood cells exhibit a flickering motion that has long been ascribed a thermal origin. Microrheology experiments provide direct evidence that flickering is an active process characterized by non-equilibrium dynamics.

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.

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.

KrF excimer laser irradiation was used to remove organic moieties from UV-transparent films of organosilanes on borosilicate glass. High-resolution patterns with different functional groups on glass were obtained by a combination of laser modification and silanisation steps. The local material modification near the ablation threshold of glass was investigated by white light interference microscopy. Change in chemical properties of irradiated surface areas were studied by fluorescence microscopy after an appropriate dying of exposed samples. From the results, the domination of thermo-chemical effects induced by the laser irradiation is derived. Finally, an example is given how the patterned organosilane films can be applied to influence cell growth on glass.

Cell motility and division are complex mechanisms that involve drastic cell shape changes. A crucial role is played by the actin cortex, which, together with the molecular motor myosin, regulates the production of cellular tension (1). A better understanding of the interplay between actin network dynamics and myosin-driven contractility is necessary to elucidate the detailed mechanism of contraction in a dynamic, out of equilibrium, constantly polymerizing and depolymerizing actin structure. In order to dissect this dual actin-myosin contribution we make advantage of biomimetic systems: cell-sized liposomes indeed offer the possibility to retain only the essential players involved in contraction mechanisms and to finely control the physical and biochemical conditions to mimic cell-like shape changes (2).Particularly we study acto-myosin contraction mechanisms on cell-sized doublets, which enable overcoming the spherical symmetry of single liposomes: with this geometry, shape changes, and hence tension build-up, can be fully characterised by recording contact angle variations. A polymerizing actin network is built at the outer surface of doublets. A fluorescently labelled actin is used and simultaneous detection of phase contrast and fluorescence images allow following actin cortex dynamics and further shape changes induced by the addition of myosin motors (3). A micropipette aspiration technique is also adopted to further characterize these cell-like shape changes (4).This reconstituted doublet system allows us to decouple biochemical and physical aspects of actin network dynamics and fully dissect the dual role of actin and myosin contractility, thus significantly improving our understanding of acto-myosin driven cell shape changes.1.Blanchoin L et al. Physiol Rev 2014, 94:235-263.2.Murrell M et al. Nat Rev Mol Cell Biol. 2015, 16(8):486-98.3.Carvalho K et al. Proc Natl Acad Sci U S A. 2013, 110(41):16456-16461.4.Campillo C et al. Biophys J 2013, 104:1248-1256.

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.

The blood stage malaria parasite, the merozoite, has a small window of opportunity during which it must successfully target and invade a human erythrocyte. The process of invasion is nonetheless remarkably rapid. To date, mechanistic models of invasion have focused predominantly on the parasite actomyosin motor contribution to the energetics of entry. Here, we have conducted a numerical analysis using dimensions for an archetypal merozoite to predict the respective contributions of the host-parasite interactions to invasion, in particular the role of membrane wrapping. Our theoretical modeling demonstrates that erythrocyte membrane wrapping alone, as a function of merozoite adhesive and shape properties, is sufficient to entirely account for the first key step of the invasion process, that of merozoite reorientation to its apex and tight adhesive linkage between the two cells. Next, parasite-induced reorganization of the erythrocyte cytoskeleton and release of parasite-derived membrane can also account for a considerable energetic portion of actual invasion itself, through membrane wrapping. Thus, contrary to the prevailing dogma, wrapping by the erythrocyte combined with parasite-derived membrane release can markedly reduce the expected contributions of the merozoite actomyosin motor to invasion. We therefore propose that invasion is a balance between parasite and host cell contributions, evolved toward maximal efficient use of biophysical forces between the two cells.

Red blood cells (RBCs) are soft and flexible biconcave discs, which are able to pass through capillaries with diameters several times smaller than the RBC size. The RBC deformability also results in noticeable membrane fluctuations, which must be correlated with RBC membrane properties. However, it is still under debate whether RBC membrane fluctuations are simply passive thermal undulations or whether a red cell also experiences active fluctuations which are driven by a metabolic activity or other cell processes. We will present direct evidence that the RBC undulations are not solely passive thermal fluctuations, which has been obtained from a set of different experiments and simulations using a high spatiotemporal resolution: from 10 microseconds to several seconds in time and up to 20 nanometers in space. Experimental results show a violation of the fluctuation-dissipation theorem (FDT) for freshly prepared RBCs indicating the existence of active processes. However, the FDT is satisfied for starved cells demonstrating that the membrane fluctuations are passive when the energy supply is absent. Experiments also show a considerable change in the fluctuation amplitudes for fresh and starved cells. Subsequently, we perform simulations which fully mimic and quantify the experiments. We are able to quantitatively extract RBC membrane properties including shear elasticity, bending rigidity, and membrane viscosity. Furthermore, we test several models for active fluctuations, which mimic different possible mechanisms including spectrin network remodeling, ion pumps, and change in the spontaneous membrane curvature. Simulation results agree well with experimental data and suggest that several processes mentioned above may contribute to active RBC fluctuations. We will discuss which processes are more likely to take place.

Red blood cells are amazingly deformable structures able to recover their initial shape even after large deformations as when passing through tight blood capillaries. The reason for this exceptional property is found in the composition of the membrane and the membrane-cytoskeleton interaction. We investigate the mechanics and the dynamics of RBCs by a unique noninvasive technique, using weak optical tweezers to measure membrane fluctuation amplitudes with μs temporal and sub nm spatial resolution. This enhanced edge detection method allows to span over >4 orders of magnitude in frequency. Hence, we can simultaneously measure red blood cell membrane mechanical properties such as bending modulus κ = 2.8 ± 0.3 × 10 −19 J = 67.6 ± 7.2 k B T , tension σ = 6.5 ± 2.1 × 10 −7 N/m, and an effective viscosity η eff = 81 ± 3.7 × 10 −3 Pa s that suggests unknown dissipative processes. We furthermore show that cell mechanics highly depends on the membrane-spectrin interaction mediated by the phosphorylation of the interconnection protein 4.1R. Inhibition and activation of this phosphorylation significantly affects tension and effective viscosity. Our results show that on short time scales (slower than 100 ms) the membrane fluctuates as in thermodynamic equilibrium. At time scales longer than 100 ms, the equilibrium description breaks down and fluctuation amplitudes are higher by 40% than predicted by the membrane equilibrium theory. Possible explanations for this discrepancy are influences of the spectrin that is not included in the membrane theory or nonequilibrium fluctuations that can be accounted for by defining a nonthermal effective energy of up to E eff = 1.4 ± 0.1 k B T , that corresponds to an actively increased effective temperature.

Isolation of individual cells from a heterogeneous cell population is an invaluable step in the analysis of single cell properties. The demands in molecular and cellular biology as well as molecular medicine are the selection, isolation, and monitoring of single cells and cell clusters of biopsy material. Of particular interest are methods which complement a passive optical or spectroscopic selection with a variety of active single cell processing techniques such as mechanical, biochemical, or genetic manipulation prior to isolation. Sophisticated laser-based cell processing systems are available which can perform single cell processing in a contact-free and sterile manner. Until now, however, these multipurpose turnkey systems offer only basic micromanipulation and are not easily modified or upgraded, whereas laboratory situations often demand simple but versatile and adaptable solutions. We built a flexible laser micromanipulation platform combining contact-free microdissection and catapulting capabilities using a pulsed ultraviolet (337nm) laser with simultaneous generation of optical tweezing forces using a continuous wave infrared (1064nm) laser. The potential of our platform is exemplified with techniques such as local laser-induced injection of biomolecules into individual living cells, laser surgery, isolation of single cells by laser catapulting, and control of neuronal growth using optical gradient forces. Arbitrary dynamic optical force patterns can be created by fast laser scanning with acousto-optical deflectors and galvanometer mirrors, allowing multibeam contact-free micromanipulation, a prerequisite for reliable handling of material in laboratory-on-a-chip applications. All common microscopy techniques can be used simultaneously with the offered palette of micromanipulation methods. Taken together, we show that advanced optical micromanipulation systems can be designed which combine quality, cost efficiency, and adaptability.

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.