Quantifying both viscoelasticity and surface tension: Why sharp tips overestimate cell stiffness.

Quantifying the mechanical properties of cells is important to better understand how mechanics constrain cellular processes. Furthermore, because pathologies are usually paralleled by altered cell mechanical properties, mechanical parameters can be used as a novel way to characterize the pathological state of cells. Key features used in models are cell tension, cell viscoelasticity (representing the average of the cell bulk), or a combination of both. It is unclear which of these features is the most relevant or whether both should be included. To clarify this, we performed microindentation experiments on cells with microindenters of various tip radii, including micrometer-sized microneedles. We obtained different cell-indenter contact radii and measured the corresponding contact stiffness. We derived a model predicting that this contact stiffness should be an affine function of the contact radius and that, at vanishing contact radius, the cell stiffness should be equal to the cell tension multiplied by a constant. When microindenting leukocytes and both adherent and trypsinized adherent cells, the contact stiffness was indeed an affine function of the contact radius. For leukocytes, the deduced surface tension was consistent with that measured using micropipette aspiration. For detached endothelial cells, agreement between microindentation and micropipette aspiration was better when considering these as only viscoelastic when analyzing micropipette aspiration experiments. This work suggests that indenting cells with sharp tips but neglecting the presence of surface tension leads to an effective elastic modulus whose origin is in fact surface tension. Accordingly, using sharp tips when microindenting a cell is a good way to directly measure its surface tension without the need to let the viscoelastic modulus relax.

Clogging of a Rectangular Slit by a Spherical Soft Particle.

The capture of a soft spherical particle in a rectangular slit leads to a nonmonotonic pressure-flow rate relation at low Reynolds number. Simulations reveal that the flow induced deformations of the trapped particle focus the streamlines and pressure drop to a small region. This increases the resistance to flow by several orders of magnitude as the driving pressure is increased. As a result, two regimes are observed in experiments and simulations: a flow-dominated regime for small particle deformations, where flow rate increases with pressure, and an elastic-dominated regime in which solid deformations block the flow.

Big data analytics capability in healthcare operations and supply chain management: the role of green process innovation.

Green approaches remain little disseminated in the healthcare sector despite growing interest in recent years from practitioners and researchers. Big Data Analytics Capability (BDAC) can play a critical role in the integration of environmental concerns into operations and supply chain management (OSCM) and further strengthen the environmental performance of healthcare facilities. According to the literature, the integration of the environment into operations process remains insufficient to achieve high levels of performance and requires efforts in green process innovation. However, this relationship between BDAC and green process innovation remains poorly justified empirically. To address this theoretical gap, we investigated the relationship between BDAC, environmental process integration, green process innovation in OSCM and environmental performance. The main contribution of this study is the valuable knowledge on how BDAC influences environmental process integration and green process innovation to enhance environmental performance. Moreover, the study highlights the mediating role of green process innovation on environmental performance, a finding that has not been mentioned in the extant literature. The paper provides valuable insight for managers and stakeholders that can assist them in supporting the application of BDAC in healthcare OSCM to create sustainable value.

Measuring Cell Mechanical Properties Using Microindentation.

Quantifying cell mechanical properties is of interest to better understand both physiological and pathological cellular processes. Cell mechanical properties are quantified by a finite set of parameters such as the effective Young's modulus or the effective viscosity. These parameters can be extracted by applying controlled forces to a cell and by quantifying the resulting deformation of the cell.Microindentation consists in pressing a cell with a calibrated spring terminated by a rigid tip and by measuring the resulting indentation of the cell. We have developed a microindentation technique that uses a flexible micropipette as a spring. The micropipette has a microbead at its tip, and this spherical geometry allows using analytical models to extract cell mechanical properties from microindentation experiments. We use another micropipette to hold the cell to be indented, which makes this technique well suited to study nonadherent cells, but we also describe how to use this technique on adherent cells.

A Multi-Omics Common Data Model for Primary Immunodeficiencies.

Primary Immunodeficiencies (PIDs) are associated with more than 400 rare monogenic diseases affecting various biological functions (e.g., development, regulation of the immune response) with a heterogeneous clinical expression (from no symptom to severe manifestations). To better understand PIDs, the ATRACTion project aims to perform a multi-omics analysis of PIDs cases versus a control group patients, including single-cell transcriptomics, epigenetics, proteomics, metabolomics, metagenomics and lipidomics. In this study, our goal is to develop a common data model integrating clinical and omics data, which can be used to obtain standardized information necessary for characterization of PIDs patients and for further systematic analysis. For that purpose, we extend the OMOP Common Data Model (CDM) and propose a multi-omics ATRACTion OMOP-CDM to integrate multi-omics data. This model, available for the community, is customizable for other types of rare diseases (https://framagit.org/imagine-plateforme-bdd/pub-rhu4-atraction).

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.

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.

Influence of external forces on actin-dependent T cell protrusions during immune synapse formation.

BACKGROUND INFORMATION: We have previously observed that in response to antigenic activation, T cells produce actin-rich protrusions that generate forces involved in T cell activation. These forces are influenced by the mechanical properties of antigen-presenting cells (APCs). However, how external forces, which can be produced by APCs, influence the dynamic of the actin protrusion remains unknown. In this study, we quantitatively characterised the effects of external forces in the dynamic of the protrusion grown by activated T cells. RESULTS: Using a micropipette force probe, we applied controlled compressive or pulling forces on primary T lymphocytes activated by an antibody-covered microbead, and measured the effects of these forces on the protrusion generated by T lymphocytes. We found that the application of compressive forces slightly decreased the length, the time at which the protrusion stops growing and retracts and the velocity of the protrusion formation, whereas pulling forces strongly increased these parameters. In both cases, the applied forces did not alter the time required for the T cells to start growing the protrusion (delay). Exploring the molecular events controlling the dynamic of the protrusion, we showed that inhibition of the Arp2/3 complex impaired the dynamic of the protrusion by reducing both its maximum length and its growth speed and increasing the delay to start growing. Finally, T cells developed similar protrusions in more physiological conditions, that is, when activated by an APC instead of an activating microbead. CONCLUSIONS: Our results suggest that the formation of the force-generating protrusion by T cells is set by an intracellular constant time and that its dynamic is sensitive to external forces. They also show that actin assembly mediated by actin-related protein Arp2/3 complex is involved in the formation and dynamic of the protrusion. SIGNIFICANCE: Actin-rich protrusions developed by T cells are sensory organelles that serve as actuators of immune surveillance. Our study shows that forces experienced by this organelle modify their dynamic suggesting that they might modify immune responses. Moreover, the quantitative aspects of our analysis should help to get insight into the molecular mechanisms involved in the formation of the protrusion.

Diacylglycerol kinase ζ promotes actin cytoskeleton remodeling and mechanical forces at the B cell immune synapse.

Diacylglycerol kinases (DGKs) limit antigen receptor signaling in immune cells by consuming the second messenger diacylglycerol (DAG) to generate phosphatidic acid (PA). Here, we showed that DGKζ promotes lymphocyte function-associated antigen 1 (LFA-1)-mediated adhesion and F-actin generation at the immune synapse of B cells with antigen-presenting cells (APCs), mostly in a PA-dependent manner. Measurement of single-cell mechanical force generation indicated that DGKζ-deficient B cells exerted lower forces at the immune synapse than did wild-type B cells. Nonmuscle myosin activation and translocation of the microtubule-organizing center (MTOC) to the immune synapse were also impaired in DGKζ-deficient B cells. These functional defects correlated with the decreased ability of B cells to present antigen and activate T cells in vitro. The in vivo germinal center response of DGKζ-deficient B cells was also reduced compared with that of wild-type B cells, indicating that loss of DGKζ in B cells impaired T cell help. Together, our data suggest that DGKζ shapes B cell responses by regulating actin remodeling, force generation, and antigen uptake-related events at the immune synapse. Hence, an appropriate balance in the amounts of DAG and PA is required for optimal B cell function.

Shigella impairs human T lymphocyte responsiveness by hijacking actin cytoskeleton dynamics and T cell receptor vesicular trafficking.

Strategies employed by pathogenic enteric bacteria, such as Shigella, to subvert the host adaptive immunity are not well defined. Impairment of T lymphocyte chemotaxis by blockage of polarised edge formation has been reported upon Shigella infection. However, the functional impact of Shigella on T lymphocytes remains to be determined. Here, we show that Shigella modulates CD4+ T cell F-actin dynamics and increases cell cortical stiffness. The scanning ability of T lymphocytes when encountering antigen-presenting cells (APC) is subsequently impaired resulting in decreased cell-cell contacts (or conjugates) between the two cell types, as compared with non-infected T cells. In addition, the few conjugates established between the invaded T cells and APCs display no polarised delivery and accumulation of the T cell receptor to the contact zone characterising canonical immunological synapses. This is most likely due to the targeting of intracellular vesicular trafficking by the bacterial type III secretion system (T3SS) effectors IpaJ and VirA. The collective impact of these cellular reshapings by Shigella eventually results in T cell activation dampening. Altogether, these results highlight the combined action of T3SS effectors leading to T cell defects upon Shigella infection.

Intermittent Pili-Mediated Forces Fluidize Neisseria meningitidis Aggregates Promoting Vascular Colonization.

Neisseria meningitidis, a bacterium responsible for meningitis and septicemia, proliferates and eventually fills the lumen of blood capillaries with multicellular aggregates. The impact of this aggregation process and its specific properties are unknown. We first show that aggregative properties are necessary for efficient infection and study their underlying physical mechanisms. Micropipette aspiration and single-cell tracking unravel unique features of an atypical fluidized phase, with single-cell diffusion exceeding that of isolated cells. A quantitative description of the bacterial pair interactions combined with active matter physics-based modeling show that this behavior relies on type IV pili active dynamics that mediate alternating phases of bacteria fast mutual approach, contact, and release. These peculiar fluid properties proved necessary to adjust to the geometry of capillaries upon bacterial proliferation. Intermittent attractive forces thus generate a fluidized phase that allows for efficient colonization of the blood capillary network during infection.

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.

Optical Tweezers-Based Measurements of Forces and Dynamics at Microtubule Ends.

Microtubules are dynamic cytoskeletal polymers that polymerize and depolymerize while interacting with different proteins and structures within the cell. The highly regulated dynamic properties as well as the pushing and pulling forces generated by dynamic microtubule ends play important roles in processes such as in cell division. For instance, microtubule end-binding proteins are known to affect dramatically the dynamic properties of microtubules, and cortical dyneins are known to mediate pulling forces on microtubule ends. We discuss in this chapter our efforts to reconstitute these systems in vitro and mimic their interactions with structures within the cell using micro-fabricated barriers. Using an optical tweezers setup, we investigate the dynamics and forces of microtubules growing against functionalized barriers in the absence and presence of end-binding proteins and barrier-attached motor proteins. This setup allows high-speed as well as nanometer and piconewton resolution measurements on dynamic microtubules.

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.

Measuring Cell Viscoelastic Properties Using a Microfluidic Extensional Flow Device.

The quantification of cellular mechanical properties is of tremendous interest in biology and medicine. Recent microfluidic technologies that infer cellular mechanical properties based on analysis of cellular deformations during microchannel traversal have dramatically improved throughput over traditional single-cell rheological tools, yet the extraction of material parameters from these measurements remains quite complex due to challenges such as confinement by channel walls and the domination of complex inertial forces. Here, we describe a simple microfluidic platform that uses hydrodynamic forces at low Reynolds number and low confinement to elongate single cells near the stagnation point of a planar extensional flow. In tandem, we present, to our knowledge, a novel analytical framework that enables determination of cellular viscoelastic properties (stiffness and fluidity) from these measurements. We validated our system and analysis by measuring the stiffness of cross-linked dextran microparticles, which yielded reasonable agreement with previously reported values and our micropipette aspiration measurements. We then measured viscoelastic properties of 3T3 fibroblasts and glioblastoma tumor initiating cells. Our system captures the expected changes in elastic modulus induced in 3T3 fibroblasts and tumor initiating cells in response to agents that soften (cytochalasin D) or stiffen (paraformaldehyde) the cytoskeleton. The simplicity of the device coupled with our analytical model allows straightforward measurement of the viscoelastic properties of cells and soft, spherical objects.

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.

Cytotoxic T Cells Use Mechanical Force to Potentiate Target Cell Killing.

The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals.

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.