Micropipette Aspiration Experiments Research Articles

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 if both should be included. To clarify this, we performed microindentation experiments on cells with microindenters of various tip radii, including micron-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 while 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.

The liquid-to-solid transition of FUS is promoted by the condensate surface

A wide range of macromolecules can undergo phase separation, forming biomolecular condensates in living cells. These membraneless organelles are typically highly dynamic, formed reversibly, and carry out essential functions in biological systems. Crucially, however, a further liquid-to-solid transition of the condensates can lead to irreversible pathological aggregation and cellular dysfunction associated with the onset and development of neurodegenerative diseases. Despite the importance of this liquid-to-solid transition of proteins, the mechanism by which it is initiated in normally functional condensates is unknown. Here we show, by measuring the changes in structure, dynamics, and mechanics in time and space, that single-component FUS condensates do not uniformly convert to a solid gel, but rather that liquid and gel phases coexist simultaneously within the same condensate, resulting in highly inhomogeneous structures. Furthermore, our results show that this transition originates at the interface between the condensate and the dilute continuous phase, and once initiated, the gelation process propagates toward the center of the condensate. To probe such spatially inhomogeneous rheology during condensate aging, we use a combination of established micropipette aspiration experiments together with two optical techniques, spatial dynamic mapping and reflective confocal dynamic speckle microscopy. These results reveal the importance of the spatiotemporal dimension of the liquid-to-solid transition and highlight the interface of biomolecular condensates as a critical element in driving pathological protein aggregation.

Micromechanics of Biomembranes.

Micromechanics techniques are playing an increasing role in characterization of biomembranes. The mechanical properties of membranes play an important role for a whole range of cellular processes. Lipid-protein biomembranes display lateral heterogeneity, domain formation, and morphological changes at mesoscopic and nanoscopic length scales. An attempt is made to introduce how membrane's material properties can be measured. Both fluctuation analysis and micro-pipette aspiration experiments have been used to quantify the micromechanics of membranes. The relationship between the structure and function of biomembranes is a critical concern in modern biology. This overview calls for a deeper understanding of how the cell complexity might be related to the mechanical properties of the lipid-protein membrane. Mechanical properties can influence cellular response to processes like adhesion, transport, differentiation, proliferation and migration.

Mechanical Characterization and Modelling of Subcellular Components of Oocytes

The early steps of embryogenesis are controlled exclusively by the quality of oocyte that linked closely to its mechanical properties. The mechanical properties of an oocyte were commonly characterized by assuming it was homogeneous such that the result deviated significantly from the true fact that it was composed of subcellular components. In this work, we accessed and characterized the subcellular components of the oocytes and developed a layered high-fidelity finite element model for describing the viscoelastic responses of an oocyte under loading. The zona pellucida (ZP) and cytoplasm were isolated from an oocyte using an in-house robotic micromanipulation platform and placed on AFM to separately characterizing their mechanical profiling by analyzing the creep behavior with the force clamping technique. The spring and damping parameters of a Kelvin–Voigt model were derived by fitting the creeping curve to the model, which were used to define the shear relaxation modulus and relaxation time of ZP or cytoplasm in the ZP and cytoplasm model. In the micropipette aspiration experiment, the model was accurate sufficiently to deliver the time-varying aspiration depth of the oocytes under the step negative pressure of a micropipette. In the micropipette microinjection experiment, the model accurately described the intracellular strain introduced by the penetration. The developed oocyte FEM model has implications for further investigating the viscoelastic responses of the oocytes under different loading settings.

Quantifying Membrane Curvature Sensing of Peripheral Proteins by Simulated Buckling and Umbrella Sampling

Membrane curvature plays an essential role in the organization and trafficking of membrane associated proteins. Comparison or prediction of the experimentally resolved protein concentrations adopted at different membrane curvatures requires direct quantification of the relative partitioning free energy. Here, we present a highly efficient and simple to implement a free-energy calculation method which is able to directly resolve the relative partitioning free energy of proteins as a direct function of membrane curvature, i.e., a curvature sensing profile, within (coarse-grained) molecular dynamics simulations. We demonstrate its utility by resolving these profiles for two known curvature sensing peptides, namely ALPS and α-synuclein, for a membrane curvature ranging from -1/6.5 to +1/6.5 nm-1. We illustrate that the difference in relative partitioning (binding) free energy between these two extrema is only about 13 kBT for both peptides, illustrating that the driving force of curvature sensing is subtle. Furthermore, we illustrate that ALPS and α-synuclein sense curvature via a contrasting mechanism, which is differentially affected by membrane composition. In addition, we demonstrate that the intrinsic spontaneous curvature of both of these peptides lies beyond the range of membrane curvature accessible in micropipette aspiration experiments, being about 1/7 nm -1. Our approach offers an efficient and simple to implement in silico tool for exploring and screening the membrane curvature sensing mechanisms of proteins.

Preparation of Giant Polymersomes via Inkjet Printing and Confined Geometry Hydration Applied to Micropipette Aspiration Experiments

AbstractA novel facile approach to creating many localized giant polymersomes (GPs) (vesicles) is developed. This method allows for the easy capture and testing of the bulk properties of the GPs. Inkjet printing is shown to produce vesicles of a non‐ionic block copolymer, poly(ethylene oxide)16‐poly(butylene oxide)22 (E16B22). The printer deposits a polymer solution (i.e., water/glucose/polymer) on a hydrophobic glass slide. Upon drying, glucose is left between polymer lamellar. Subsequent hydration of the sample dissolves glucose, and as a result, a concentration gradient is generated, which provides the energy necessary for separating the neutral polymer layers. After about 2 h in water, GPs are observed and are free of organic solvents. This novel methodology provides for the efficient formation of localized GPs. The distribution of the solution allows for a metered approach to releasing the GPs. This combination facilitates the collection of vesicles for micropipette aspiration experiments.

Active probing of the mechanical properties of biological and synthetic vesicles

BackgroundThe interest in mechanics of synthetic and biological vesicles has been continuously growing during the last decades. Liposomes serve as model systems for investigating fundamental membrane processes and properties. More recently, extracellular vesicles (EVs) have been investigated mechanically as well. EVs are widely studied in fundamental and applied sciences, but their material properties remained elusive until recently. Elucidating the mechanical properties of vesicles is essential to unveil the mechanisms behind a variety of biological processes, e.g. budding, vesiculation and cellular uptake mechanisms. Scope of reviewThe importance of mechanobiology for studies of vesicles and membranes is discussed, as well as the different available techniques to probe their mechanical properties. In particular, the mechanics of vesicles and membranes as obtained by nanoindentation, micropipette aspiration, optical tweezers, electrodeformation and electroporation experiments is addressed. Major conclusionsEVs and liposomes possess an astonishing rich, diverse behavior. To better understand their properties, and for optimization of their applications in nanotechnology, an improved understanding of their mechanical properties is needed. Depending on the size of the vesicles and the specific scientific question, different techniques can be chosen for their mechanical characterization. General significanceUnderstanding the mechanical properties of vesicles is necessary to gain deeper insight in the fundamental biological mechanisms involved in vesicle generation and cellular uptake. This furthermore facilitates technological applications such as using vesicles as targeted drug delivery vehicles. Liposome studies provide insight into fundamental membrane processes and properties, whereas the role and functioning of EVs in biology and medicine are increasingly elucidated.

The roles of contact time and contact pressure on the coalescence of water droplets suspended in concentrated bitumen solutions

The coalescence of water drops in diluted bitumen solutions has been previously studied via micropipette experiments in which water drops are brought to contact, and the onset of drop coalescence is evaluated as a function of bitumen dilution, type of solvent, etc. Due to the opacity of concentrated bitumen solutions, most of these studies have been conducted in highly diluted systems. Under those conditions (typically solvent/bitumen or S/B mass ratios greater than 4), asphaltenes tend to form skins at the oil/water interface that prevent coalescence altogether. In ‘naphthenic’ froth treatment (NFT) process, however, S/B ratios of 0.5–2 are often used. To study S/B ratios relevant to NFT operations, we developed a Hele-Shaw version of the micropipette aspiration experiment, in which drops held on micropipettes are sandwiched to disks by two hydrophobically-treated plates spaced less than a few hundred microns apart. The resulting thin layer of bitumen is optically transparent in brightfield microscopy, allowing us to execute micropipette contacting experiments at industrially relevant bitumen dilutions. Another departure from almost all the literature in the past is that we have measured the time, tc, required to observe coalescence from the moment two disk-shaped drops are contacted under an imposed contact pressure, P. tc was observed to decrease when P, pH or bitumen dilution was increased. Deasphalting bitumen also led to a significant decrease in tc, while increasing the aging time of the interface from 15 to 60 min dramatically increased tc. The coalescence time was found to obey an exponential relationship with P, and was explained to be a result of an activated process related to the formation of a bridge of water across the thin film of bitumen in the contact zone between the drops. The tc-P correlations measured in this work provide the foundation for designing coalescers that coarsen the drop size distribution in water-in-bitumen emulsions under given conditions of bitumen concentration and water pH.

Cell Surface Deformation during an Action Potential

The excitation of many cells and tissues is associated with cell mechanical changes. The evidence presented herein corroborates that single cells deform during an action potential. It is demonstrated that excitation of plant cells (Chara braunii internodes) is accompanied by out-of-plane displacements of the cell surface in the micrometer range (∼1–10 μm). The onset of cellular deformation coincides with the depolarization phase of the action potential. The mechanical pulse: 1) propagates with the same velocity as the electrical pulse (within experimental accuracy, ∼10 mm s−1), 2) is reversible, 3) in most cases is of biphasic nature (109 out of 152 experiments), and 4) is presumably independent of actin-myosin-motility. The existence of transient mechanical changes in the cell cortex is confirmed by micropipette aspiration experiments. A theoretical analysis demonstrates that this observation can be explained by a reversible change in the mechanical properties of the cell surface (transmembrane pressure, surface tension, and bending rigidity). Taken together, these findings contribute to the ongoing debate about the physical nature of cellular excitability.

Mathematical study of a single leukocyte in microchannel flow

The recruitment of leukocytes and subsequent rolling, activation, adhesion and transmigration are essential stages of an inflammatory response. Chronic inflammation may entail atherosclerosis, one of the most devastating cardiovascular diseases. Understanding this mechanism is of crucial importance in immunology and in the development of anti-inflammatory drugs. Micropipette aspiration experiments show that leukocytes behave as viscoelastic drops during suction. The flow of non-Newtonian viscoelastic fluids can be described by differential, integral and rate-type constitutive equations. In this study, the rate-type Oldroyd-B model is used to capture the viscoelasticity of the leukocyte which is considered as a drop. Our main goal is to analyze a mathematical model describing the deformation and flow of an individual leukocyte in a microchannel flow. In this model we consider a coupled problem between a simplified Oldroyd-B system and a transport equation which describes the density considered as non constant in the Navier–Stokes equations. First we present the mathematical model and we prove the existence of solution, then we describe its numerical approximation using the level set method. Through the numerical simulations we analyze the hemodynamic effects of three inlet velocity values. We note that the hydrodynamic forces pushing the cell become higher with increasing inlet velocities.

Probing eukaryotic cell mechanics via mesoscopic simulations.

Cell mechanics has proven to be important in many biological processes. Although there is a number of experimental techniques which allow us to study mechanical properties of cell, there is still a lack of understanding of the role each sub-cellular component plays during cell deformations. We present a new mesoscopic particle-based eukaryotic cell model which explicitly describes cell membrane, nucleus and cytoskeleton. We employ Dissipative Particle Dynamics (DPD) method that provides us with the unified framework for modeling of a cell and its interactions in the flow. Data from micropipette aspiration experiments were used to define model parameters. The model was validated using data from microfluidic experiments. The validated model was then applied to study the impact of the sub-cellular components on the cell viscoelastic response in micropipette aspiration and microfluidic experiments.

Improved Measurement of Elastic Properties of Cells by Micropipette Aspiration and Its Application to Lymphocytes.

Mechanical deformability of cells is an important property for their function and development, as well as a useful marker of cell state. The classical technique of micropipette aspiration allows single-cell studies and we provide here a method to measure the two basic mechanical parameters, elastic modulus and Poisson's ratio. The proposed method, developed from finite-element analysis of micropipette aspiration experiments, may be implemented in future technologies for the automated measurement of mechanical properties of cells, based on the micropipette aspiration technique or on the cell transit through flow constrictions. We applied this method to measure the elastic parameters of lymphocytes, in which the mechanical properties depend on their activation state. Additionally, we discuss in this work the accuracy of previous models to estimate the elastic modulus of cells, in particular the analytical model by Theret et al., widely used in the field. We show the necessity of using an improved model, taking into account the finite size of the cells, to obtain new insights that may remain hidden otherwise.

An Active Contraction Model of Valvular Interstitial Cells

Valvular interstitial cells (VICs) play a critical role in the pathophysiology of heart valves. The objective of this study was to investigate the roles of different subcellular structures of the VICs, especially α-SMA stress fibers, to the VIC mechanical responses under different mechanical loading conditions and activation states. We modeled the VIC as a continuum with two distinct domains: the cell nucleus and cytoplasm. The nucleus was modeled as an incompressible neo-Hookean material while the cytoplasm was modeled as a mixture of two solid phases: the basal, isotropic cytoskeleton phase and α-SMA stress fiber phase, which exhibit some orientations at each point described by an orientation density function. The α-SMA stress fibers also exhibit passive elastic and active contractile responses in the direction of the orientations. We developed VIC mechanical model, which integrated the data from two experiments: micropipette aspiration (MA) and atomic force microscopy (AFM) of the aortic VIC (AVIC) and pulmonary VIC (PVIC) that each exhibits different expression levels of the α-SMA and contraction strength. In the MA experiment, VICs are in inactivated states while in AFM experiment, VICs are in activated states, exhibiting higher level of α-SMA expression and contraction. Thus, using our model in conjunction with the experimental data, we investigated how the expression level and active contraction of the α-SMA fibers affect the mechanical responses of the VICs. We implemented our model on the finite element method. We determined a ∼10-fold difference in the contractions strength between the AVICs and PVICs, implying that not only the expression level of the α-SMA fibers but also the contraction level increases from PVICs compared to AVICs. The higher expression of the α-SMA fibers may facilitate more efficient contraction and increased ability to synthesize collagen and other ECM components.

An active contraction model of the valvular interstitial cell

An active contraction model of the valvular interstitial cell

On intrinsic stress fiber contractile forces in semilunar heart valve interstitial cells using a continuum mixture model

Heart valve interstitial cells (VICs) play a critical role in the maintenance and pathophysiology of heart valve tissues. Normally quiescent in the adult, VICs can become activated in periods of growth and disease. When activated, VICs exhibit increased levels of cytokines and extracellular matrix (ECM) synthesis, and upregulated expression and strong contraction of α-smooth muscle actin (α-SMA) fibers. However, it remains unknown how expression and contraction of the α-SMA fibers, which vary among different VIC types, contribute to the overall VIC mechanical responses, including the nucleus and cytoskeleton contributions. In the present study, we developed a novel solid-mixture model for VIC biomechanical behavior that incorporated 1) the underlying cytoskeletal network, 2) the oriented α-SMA stress fibers with passive elastic and active contractile responses, 3) a finite deformable elastic nucleus. We implemented the model in a full 3D finite element simulation of a VIC based on known geometry. Moreover, we examined the respective mechanical responses of aortic and pulmonary VICs (AVICs and PVICs, respectively), which are known to have different levels of α-SMA expression levels and contractile behaviors. To calibrate the model, we simulated the combined mechanical responses of VICs in both micropipette aspiration (MA) and atomic force microscopy (AFM) experiments. These two states were chosen as the VICs were under significantly different mechanical loading conditions and activation states, with the α-SMA fibers inactivated in the MA studies while fully activated in the AFM studies. We also used the AFM to study the mechanical property of the nucleus. Our model predicted that the substantial differences found in stiffening of the AVIC compared to the PVICs was due to a 9 to 16 times stronger intrinsic AVIC α-SMA stress fiber contractile force. Model validation was done by simulating a traction force microscopy experiment to estimate the forces the VICs exert on the underlying substrate, and found good agreement with reported traction force microscopy results. Further, estimated nuclear stiffness for both AVICs and PVICs were similar and comparable to the literature, and were both unaffected by VIC activation level. These results suggest substantial functional differences between AVICs and PVICs at the subcellular level. Moreover, this first VIC computational biomechanical model is but a first step in developing a comprehensive, integrated view of the VIC pathophysiology and interactions with the valve ECM micro-environment based on simulation technologies.

Cell shape regulation through mechanosensory feedback control.

Cells undergo controlled changes in morphology in response to intracellular and extracellular signals. These changes require a means for sensing and interpreting the signalling cues, for generating the forces that act on the cell's physical material, and a control system to regulate this process. Experiments on Dictyostelium amoebae have shown that force-generating proteins can localize in response to external mechanical perturbations. This mechanosensing, and the ensuing mechanical feedback, plays an important role in minimizing the effect of mechanical disturbances in the course of changes in cell shape, especially during cell division, and likely in other contexts, such as during three-dimensional migration. Owing to the complexity of the feedback system, which couples mechanical and biochemical signals involved in shape regulation, theoretical approaches can guide further investigation by providing insights that are difficult to decipher experimentally. Here, we present a computational model that explains the different mechanosensory and mechanoresponsive behaviours observed in Dictyostelium cells. The model features a multiscale description of myosin II bipolar thick filament assembly that includes cooperative and force-dependent myosin-actin binding, and identifies the feedback mechanisms hidden in the observed mechanoresponsive behaviours of Dictyostelium cells during micropipette aspiration experiments. These feedbacks provide a mechanistic explanation of cellular retraction and hence cell shape regulation.

Small membranes under negative surface tension.

We use computer simulations and a simple free energy model to study the response of a bilayer membrane to the application of a negative (compressive) mechanical tension. Such a tension destabilizes the long wavelength undulation modes of giant vesicles, but it can be sustained when small membranes and vesicles are considered. Our negative tension simulation results reveal two regimes-(i) a weak negative tension regime characterized by stretching-dominated elasticity and (ii) a strong negative tension regime featuring bending-dominated elastic behavior. This resembles the findings of the classic Evans and Rawicz micropipette aspiration experiment in giant unilamellar vesicles (GUVs) [E. Evans and W. Rawicz, Phys, Rev. Lett. 64, 2094 (1990)]. However, in GUVs the crossover between the two elasticity regimes occurs at a small positive surface tension, while in smaller membranes it takes place at a moderate negative tension. Another interesting observation concerning the response of a small membrane to negative surface tension is related to the relationship between the mechanical and fluctuation tensions, which are equal to each other for non-negative values. When the tension decreases to negative values, the fluctuation tension γ drops somewhat faster than the mechanical tension τ in the small negative tension regime, before it saturates (and becomes larger than τ) for large negative tensions. The bending modulus exhibits an "opposite" trend. It remains almost unchanged in the stretching-dominated elastic regime, and decreases in the bending-dominated regime. Both the amplitudes of the thermal height undulations and the projected area variations diverge at the onset of mechanical instability.

A poroviscohyperelastic model for numerical analysis of mechanical behavior of single chondrocyte

The aim of this paper is to use a poroviscohyperelastic (PVHE) model, which is developed based on the porohyperelastic (PHE) model to explore the mechanical deformation properties of single chondrocytes. Both creep and relaxation responses are investigated by using finite element analysis models of micropipette aspiration and atomic force microscopy experiments, respectively. The newly developed PVHE model is compared thoroughly with the standard neo-Hookean solid and PHE models. It has been found that the PVHE can accurately capture both creep and stress relaxation behaviors of chondrocytes better than other two models. Hence, the PVHE is a promising model to investigate mechanical properties of single chondrocytes.

Effects of an antimitotic drug on mechanical behaviours of the cytoskeleton in distinct grades of colon cancer cells

Biomechanical behaviours of cells change during cancer progression due to alterations in the main cytoskeletal proteins. Microtubules play a vital role in mitosis and in supporting the integrity of the cell due to their ability to withstand high compressive loads. Accordingly, microtubule-targeting agents (MTAs) have become one of the most promising classes of drugs in cancer therapy. This study evaluated changes in visco-elastic parameters induced by an appropriate concentration of an antimitotic drug in two different grades of colon cancer cells. Actin microfilaments and microtubules contents in the cells were evaluated by Western blot analysis and fluorescence intensity calculation. Micropipette aspiration experiments showed that the MTA had distinct mechanical effects on different cell lines. The more aggressive the cells, the greater the reduction in elasticity and viscosity. Invasive cells had a higher initial instantaneous Young’s modulus than primary cells, but this reduced to approximately one half of the values for primary cells after 48h of drug treatment. A considerable association was seen between the changes in mechanical properties and the microtubule to F-actin microfilament content ratio, which decreased with MTA treatment.

Finite element models and molecular dynamic simulations for studying the response of mast cell under mechanical activation

In micropipette aspiration experiment, increasing mechanical stress applied to cell membrane induced degranulation of mast cell as well as a current that could be inhibited by an inhibitor, which is specific for the transient receptor potential vanilloid (TRPVs) channels. To determine the sensitivity of TRPVs to membrane strain and tension, and to gain new insights into the activation mechanism of TRPVs, finite element models of mast cell and molecular dynamic simulations of human aquaporin-1 are presented. During the finite element simulations, the cell membrane sustained to micropipette aspiration was simulated, and the strain distribution along membrane thickness direction was obtained. Besides, combining the finite element models of osteoblast aspirated into micropipette and other compared models, we examined the relationship between cell mechanical attributes and mechanical stimulations and presented a new perspective to determine the cell equivalent elastic modulus. Considering the indetermination of TRPV crystal structure, human aquaporin-1, one kind of the channel membrane proteins, substituting for TRPV, has been studied with molecular dynamic (MD) simulations, under different external lateral tensions which have been obtained in mast cell finite element simulations, to investigate the mechanical stimulation effects on the membrane channels. The simulations show that human aquaporin-1 undergoes significant conformational change and expands in accordance with lateral tension, which not only confirms the tendency of the previous electrophysiological experiments but also leads us to a better understanding of TRPVs. The multi-scale study combining finite element simulation and MD simulation is a significant breakthrough in the field of mechanical mechanism in cell system.