Straining the root on and off triggers local calcium signalling.

A fundamental function of an organ is the ability to perceive mechanical cues. Yet, how this is accomplished is not fully understood, particularly in plant roots. In plants, the majority of studies dealing with the effects of mechanical stress have investigated the aerial parts. However, in natural conditions roots are also subjected to mechanical cues, for example when the root encounters a hard obstacle during its growth or when the soil settles. To investigate root cellular responses to root compression, we developed a microfluidic system associated with a microvalve allowing the delivery of controlled and reproducible mechanical stimulations to the root. In this study, examining plants expressing the R-GECO1-mTurquoise calcium reporter, we addressed the root cell deformation and calcium increase induced by the mechanical stimulation. Lateral pressure applied on the root induced a moderate elastic deformation of root cortical cells and elicited a multicomponent calcium signal at the onset of the pressure pulse, followed by a second one at the release of the pressure. This indicates that straining rather than stressing of tissues is relevant to trigger the calcium signal. Although the intensity of the calcium response increases with the pressure applied, successive pressure stimuli led to a remarkable attenuation of the calcium signal. The calcium elevation was restricted to the tissue under pressure and did not propagate. Strain sensing, spatial restriction and habituation to repetitive stimulation represent the fundamental properties of root signalling in response to local mechanical stimulation. These data linking mechanical properties of root cells to calcium elevation contribute to elucidating the pathway allowing the root to adapt to the mechanical cues generated by the soil.

PPP2R1A regulates migration persistence through the NHSL1-containing WAVE Shell Complex.

The RAC1-WAVE-Arp2/3 signaling pathway generates branched actin networks that power lamellipodium protrusion of migrating cells. Feedback is thought to control protrusion lifetime and migration persistence, but its molecular circuitry remains elusive. Here, we identify PPP2R1A by proteomics as a protein differentially associated with the WAVE complex subunit ABI1 when RAC1 is activated and downstream generation of branched actin is blocked. PPP2R1A is found to associate at the lamellipodial edge with an alternative form of WAVE complex, the WAVE Shell Complex, that contains NHSL1 instead of the Arp2/3 activating subunit WAVE, as in the canonical WAVE Regulatory Complex. PPP2R1A is required for persistence in random and directed migration assays and for RAC1-dependent actin polymerization in cell extracts. PPP2R1A requirement is abolished by NHSL1 depletion. PPP2R1A mutations found in tumors impair WAVE Shell Complex binding and migration regulation, suggesting that the coupling of PPP2R1A to the WAVE Shell Complex is essential to its function.

Distinct timing of neutrophil spreading and stiffening during phagocytosis.

Phagocytic cells form the first line of defense in an organism, engulfing microbial pathogens. Phagocytosis involves cell mechanical changes that are not yet well understood. Understanding these mechanical modifications promises to shed light on the immune processes that trigger pathological complications. Previous studies showed that phagocytes undergo a sequence of spreading events around their target followed by an increase in cell tension. Seemingly in contradiction, other studies observed an increase in cell tension concomitant with membrane expansion. Even though phagocytes are viscoelastic, few studies have quantified viscous changes during phagocytosis. It is also unclear whether cell lines behave mechanically similarly to primary neutrophils. We addressed the question of simultaneous versus sequential spreading and mechanical changes during phagocytosis by using immunoglobulin-G-coated 8- and 20-µm-diameter beads as targets. We used a micropipette-based single-cell rheometer to monitor viscoelastic properties during phagocytosis by both neutrophil-like PLB cells and primary human neutrophils. We show that the faster expansion of PLB cells on larger beads is a geometrical effect reflecting a constant advancing speed of the phagocytic cup. Cells become stiffer on 20- than on 8-µm beads, and the relative timing of spreading and stiffening of PLB cells depends on target size: on larger beads, stiffening starts before maximal spreading area is reached but ends after reaching maximal area. On smaller beads, the stiffness begins to increase after cells have engulfed the bead. Similar to PLB cells, primary cells become stiffer on larger beads but start spreading and stiffen faster, and the stiffening begins before the end of spreading on both bead sizes. Our results show that mechanical changes in phagocytes are not a direct consequence of cell spreading and that models of phagocytosis should be amended to account for causes of cell stiffening other than membrane expansion.

Luminal flow actuation generates coupled shear and strain in a microvessel-on-chip.

In the microvasculature, blood flow-derived forces are key regulators of vascular structure and function. Consequently, the development of hydrogel-based microvessel-on-chip systems that strive to mimic thein vivocellular organization and mechanical environment has received great attention in recent years. However, despite intensive efforts, current microvessel-on-chip systems suffer from several limitations, most notably failure to produce physiologically relevant wall strain levels. In this study, a novel microvessel-on-chip based on the templating technique and using luminal flow actuation to generate physiologically relevant levels of wall shear stress and circumferential stretch is presented. Normal forces induced by the luminal pressure compress the surrounding soft collagen hydrogel, dilate the channel, and create large circumferential strain. The fluid pressure gradient in the system drives flow forward and generates realistic pulsatile wall shear stresses. Rigorous characterization of the system reveals the crucial role played by the poroelastic behavior of the hydrogel in determining the magnitudes of the wall shear stress and strain. The experimental measurements are combined with an analytical model of flow in both the lumen and the porous hydrogel to provide an exceptionally versatile user manual for an application-based choice of parameters in microvessels-on-chip. This unique strategy of flow actuation adds a dimension to the capabilities of microvessel-on-chip systems and provides a more general framework for improving hydrogel-basedin vitroengineered platforms.

Pericyte mechanics and mechanobiology.

Pericytes are mural cells of the microvasculature, recognized by their thin processes and protruding cell body. Pericytes wrap around endothelial cells and play a central role in regulating various endothelial functions, including angiogenesis and inflammation. They also serve as a vascular support and regulate blood flow by contraction. Prior reviews have examined pericyte biological functions and biochemical signaling pathways. In this Review, we focus on the role of mechanics and mechanobiology in regulating pericyte function. After an overview of the morphology and structure of pericytes, we describe their interactions with both the basement membrane and endothelial cells. We then turn our attention to biophysical considerations, and describe contractile forces generated by pericytes, mechanical forces exerted on pericytes, and pericyte responses to these forces. Finally, we discuss 2D and 3D engineered in vitro models for studying pericyte mechano-responsiveness and underscore the need for more evolved models that provide improved understanding of pericyte function and dysfunction.

Rapid viscoelastic changes are a hallmark of early leukocyte activation.

To accomplish their critical task of removing infected cells and fighting pathogens, leukocytes activate by forming specialized interfaces with other cells. The physics of this key immunological process are poorly understood, but it is important to understand them because leukocytes have been shown to react to their mechanical environment. Using an innovative micropipette rheometer, we show in three different types of leukocytes that, when stimulated by microbeads mimicking target cells, leukocytes become up to 10 times stiffer and more viscous. These mechanical changes start within seconds after contact and evolve rapidly over minutes. Remarkably, leukocyte elastic and viscous properties evolve in parallel, preserving a well-defined ratio that constitutes a mechanical signature specific to each cell type. Our results indicate that simultaneously tracking both elastic and viscous properties during an active cell process provides a new, to our knowledge, way to investigate cell mechanical processes. Our findings also suggest that dynamic immunomechanical measurements can help discriminate between leukocyte subtypes during activation.

Micropipette force probe to quantify single-cell force generation: application to T-cell activation.

In response to engagement of surface molecules, cells generate active forces that regulate many cellular processes. Developing tools that permit gathering mechanical and morphological information on these forces is of the utmost importance. Here we describe a new technique, the micropipette force probe, that uses a micropipette as a flexible cantilever that can aspirate at its tip a bead that is coated with molecules of interest and is brought in contact with the cell. This technique simultaneously allows tracking the resulting changes in cell morphology and mechanics as well as measuring the forces generated by the cell. To illustrate the power of this technique, we applied it to the study of human primary T lymphocytes (T-cells). It allowed the fine monitoring of pushing and pulling forces generated by T-cells in response to various activating antibodies and bending stiffness of the micropipette. We further dissected the sequence of mechanical and morphological events occurring during T-cell activation to model force generation and to reveal heterogeneity in the cell population studied. We also report the first measurement of the changes in Young's modulus of T-cells during their activation, showing that T-cells stiffen within the first minutes of the activation process.

Mechanical Criterion for the Rupture of a Cell Membrane under Compression.

We investigate the mechanical conditions leading to the rupture of the plasma membrane of an endothelial cell subjected to a local, compressive force. Membrane rupture is induced by tilted microindentation, a technique used to perform mechanical measurements on adherent cells. In this technique, the applied force can be deduced from the measured horizontal displacement of a microindenter's tip, as imaged with an inverted microscope and without the need for optical sensors to measure the microindenter's deflection. We show that plasma membrane rupture of endothelial cells occurs at a well-defined value of the applied compressive stress. As a point of reference, we use numerical simulations to estimate the magnitude of the compressive stresses exerted on endothelial cells during the deployment of a stent.

T-lymphocyte passive deformation is controlled by unfolding of membrane surface reservoirs.

T-lymphocytes in the human body routinely undergo large deformations, both passively, when going through narrow capillaries, and actively, when transmigrating across endothelial cells or squeezing through tissue. We investigate physical factors that enable and limit such deformations and explore how passive and active deformations may differ. Employing micropipette aspiration to mimic squeezing through narrow capillaries, we find that T-lymphocytes maintain a constant volume while they increase their apparent membrane surface area upon aspiration. Human resting T-lymphocytes, T-lymphoblasts, and the leukemic Jurkat T-cells all exhibit membrane rupture above a critical membrane area expansion that is independent of either micropipette size or aspiration pressure. The unfolded membrane matches the excess membrane contained in microvilli and membrane folds, as determined using scanning electron microscopy. In contrast, during transendothelial migration, a form of active deformation, we find that the membrane surface exceeds by a factor of two the amount of membrane stored in microvilli and folds. These results suggest that internal membrane reservoirs need to be recruited, possibly through exocytosis, for large active deformations to occur.

A simple microfluidic device to study cell-scale endothelial mechanotransduction.

Atherosclerosis is triggered by chronic inflammation of arterial endothelial cells (ECs). Because atherosclerosis develops preferentially in regions where blood flow is disturbed and where ECs have a cuboidal morphology, the interplay between EC shape and mechanotransduction events is of primary interest. In this work we present a simple microfluidic device to study relationships between cell shape and EC response to fluid shear stress. Adhesive micropatterns are used to non-invasively control EC elongation and orientation at both the monolayer and single cell levels. The micropatterned substrate is coupled to a microfluidic chamber that allows precise control of the flow field, high-resolution live-cell imaging during flow experiments, and in situ immunostaining. Using micro particle image velocimetry, we show that cells within the chamber alter the local flow field so that the shear stress on the cell surface is significantly higher than the wall shear stress in regions containing no cells. In response to flow, we observe the formation of lamellipodia in the downstream portion of the EC and cell retraction in the upstream portion. We quantify flow-induced calcium mobilization at the single cell level for cells cultured on unpatterned surfaces or on adhesive lines oriented either parallel or orthogonal to the flow. Finally, we demonstrate flow-induced intracellular calcium waves and show that the direction of propagation of these waves is determined by cell polarization rather than by the flow direction. The combined versatility and simplicity of this microfluidic device renders it very useful for studying relationships between EC shape and mechanosensitivity.

Elastocapillary Instability in Mitochondrial Fission.

Mitochondria are dynamic cell organelles that constantly undergo fission and fusion events. These dynamical processes, which tightly regulate mitochondrial morphology, are essential for cell physiology. Here we propose an elastocapillary mechanical instability as a mechanism for mitochondrial fission. We experimentally induce mitochondrial fission by rupturing the cell's plasma membrane. We present a stability analysis that successfully explains the observed fission wavelength and the role of mitochondrial morphology in the occurrence of fission events. Our results show that the laws of fluid mechanics can describe mitochondrial morphology and dynamics.

Characterizing cell adhesion by using micropipette aspiration.

We have developed a technique to directly quantify cell-substrate adhesion force using micropipette aspiration. The micropipette is positioned perpendicular to the surface of an adherent cell and a constant-rate aspiration pressure is applied. Since the micropipette diameter and the aspiration pressure are our control parameters, we have direct knowledge of the aspiration force, whereas the cell behavior is monitored either in brightfield or interference reflection microscopy. This setup thus allows us to explore a range of geometric parameters, such as projected cell area, adhesion area, or pipette size, as well as dynamical parameters such as the loading rate. We find that cell detachment is a well-defined event occurring at a critical aspiration pressure, and that the detachment force scales with the cell adhesion area (for a given micropipette diameter and loading rate), which defines a critical stress. Taking into account the cell adhesion area, intrinsic parameters of the adhesion bonds, and the loading rate, a minimal model provides an expression for the critical stress that helps rationalize our experimental results.

Ultrasound internal tattooing.

PURPOSE: The ability of remotely tagging tissues in a controlled and three-dimensional manner during preoperative imaging could greatly help surgeons to identify targets for resection. The authors' objective is to selectively and noninvasively deposit markers under image guidance for such internal tattooing. METHODS: This study describes the production of new ultrasound-inducible droplets carrying large payloads of fluorescent markers and the in vivo proof of concept of their remote and controlled deposition via focused ultrasound. The droplets are monodispersed multiple emulsions produced in a microfluidic system, consisting of aqueous fluorescein in perfluorocarbon in water. Their conversion (either by vaporization or cavitation) is performed remotely using a clinical ultrasonic imaging probe. RESULTS: When submitted to 5 MHz imaging pulses, the droplets vaporize in vitro at 1.4 MPa peak-negative pressure and eject their content. After several seconds, a brightly fluorescent spot (0.5 mm diameter) is observed at the focus of the transducer. Experiments in the chorioallantoique membrane of chicken eggs and chicken embryo demonstrate that the spot is stable and is easily seen by naked eye. CONCLUSIONS: These ultrasound-inducible multiple emulsions could be used to deliver large amounts of contrast agents, chemotherapy, and genetic materials in vivo using a conventional ultrasound scanner.