Transport across cell membranes is modulated by lipid order

This study measures the uptake of various dyes into HeLa cells and determines simultaneously the degree of membrane lipid chain order on a single cell level by spectral analysis of the membrane-embedded dye Laurdan. First, this study finds that the mean generalized polarization (GP) value of single cells varies within a population in a range that is equivalent to a temperature variation of 9 K. This study exploits this natural variety of membrane order to examine the uptake as a function of GP at constant temperature. It is shown that transport across the cell membrane correlates with the membrane phase state. Specifically, higher membrane transport with increasing lipid chain order is observed. As a result, hypothermal-adapted cells with reduced lipid membrane order show less transport. Environmental factors influence transport as well. While increasing temperature reduces lipid order, it is found that locally high cell densities increase lipid order and in turn lead to increased dye uptake. To demonstrate the physiological relevance, membrane state and transport during an in vitro wound healing process are analyzed. While the uptake within a confluent cell layer is high, it decreases toward the center where the membrane lipid chain order is lowest

ARAM: an automated image analysis software to determine rosetting parameters and parasitaemia in Plasmodium samples

Additional file 3: Figure S3. Bland-Altman diagrams. Left Comparison of the cell detection by ARAM and an operator. Right Comparison of the determined rosette size by ARAM and an operator

Acetylcholinesterase activity influenced by lipid membrane area and surface acoustic waves

According to the current model of nerve propagation, the function of acetylcholinesterase (AChE) is to terminate synaptic transmission of nerve signals by hydrolyzing the neurotransmitter acetylcholine (ACh) in the synaptic cleft to acetic acid (acetate) and choline. However, extra-synaptic roles, which are known as ‘non-classical’ roles, have not been fully elucidated. Here, we measured AChE activity with the enzyme bound to lipid membranes of varying area per enzyme in vitro using the Ellman assay. We found that the activity was not affected by density fluctuations in a supported lipid bilayer (SLB) induced by standing surface acoustic waves. Nevertheless, we found twice as high activity in the presence of small unilamellar vesicles (SUV) compared to lipid-free samples. We also showed that the increase in activity scaled with the available membrane area per enzyme

Directed invasion of cancer cell spheroids inside 3D collagen matrices oriented by microfluidic flow in experiment and simulation

Invasion is strongly influenced by the mechanical properties of the extracellular matrix. Here, we use microfluidics to align fibers of a collagen matrix and study the influence of fiber orientation on invasion from a cancer cell spheroid. The microfluidic setup allows for highly oriented collagen fibers of tangential and radial orientation with respect to the spheroid, which can be described by finite element simulations. In invasion experiments, we observe a strong bias of invasion towards radial as compared to tangential fiber orientation. Simulations of the invasive behavior with a Brownian diffusion model suggest complete blockage of migration perpendicularly to fibers allowing for migration exclusively along fibers. This slows invasion toward areas with tangentially oriented fibers down, but does not prevent it

Transient permeabilization of living cells: combining shear flow and acoustofluidic trapping for the facilitated uptake of molecules

Here, we present a novel approach for the transient permeabilization of cells. We combined laminar shear flow in a microchannel with chaotic advection employing surface acoustic waves. First, as a fundamental result on the one hand, and as a kind of reference measurement for the more complex acoustofluidic approach on the other hand, we studied the permeabilization of cells in pure shear flow in a microchannel with Y-geometry. As a proof of principle, we used fluorescent dyes as model drugs and investigated their internalization into HeLa cells. We found that drug uptake scaled non-linearly with flow rate and thus shear stress. For calcein, we obtained a maximal enhancement factor of about 12 at an optimum flow rate of Q = 500 µL/h in the geometry used here compared to static incubation. This result is discussed in the light of structural phase transitions of lipid membranes accompanied by non-linear effects, as the plasma membrane is the main barrier to overcome. Second, we demonstrated the enhanced permeabilization of acoustically trapped cells in surface acoustic wave induced vortices in a microchannel, with an enhancement factor of about 18 compared to quasi-static incubation. Moreover, we optimized the trapping conditions regarding flow rate, the power level of the surface acoustic wave, and trapping time. Finally, we showed that our method is not limited to small molecules but can also be applied to compounds with higher molecular weight

Dynamic effective elasticity of melanoma cells under shear and elongational flow confirms estimation from force spectroscopy

The detection and enrichment of circulating melanoma cells is a challenge, as the cells are very heterogeneous in terms of their biomechanical properties and surface markers. In addition, there is a lack of valid and reliable biomarkers predicting progress and therapeutic response. In this study, we analyze the elasticity of A375 melanoma cells by applying force spectroscopy and a microfluidic method. To identify and eventually separate freely circulating tumor cells, it is crucial to know their physical properties precisely. First, we use standard AFM force spectroscopy, where the elasticity of the cells is calculated from indentation with a pyramidal tip. To extend the limits of the measurements with a tip, we then use cantilevers without a tip to apply force over a larger area of the cells. The resulting Young’s moduli are slightly lower and vary less without the tip, presumably because of the spatial inhomogeneity of the cells. Finally, we implement our microfluidic method: we measure single cell elasticity by analyzing their deformation in high-speed micrographs while passing a stenosis. Combining the force field and the change in shape provides the basis for a stress–strain diagram. The results from the microfluidic deformation analysis were well in accordance with the results from force spectroscopy. The microfluidic method, however, provides advantages over conventional methods, as it is less invasive and less likely to harm the cell during the measurement. The whole cell is measured as one entity without having contact to a stiff substrate, while force spectroscopy is limited to the contact area of the tip, and in some cases dependent of the cell substrate interaction. Consequently, microfluidic deformation analysis allows us to predict the overall elastic behavior of the whole, inhomogeneous cell in three-dimensional force fields. This method may contribute to improve the detection of circulating melanoma cells in the clinical practice

Intake of silica nanoparticles by giant lipid vesicles: influence of particle size and thermodynamic membrane state

The uptake of nanoparticles into cells often involves their engulfment by the plasma membrane and a fission of the latter. Understanding the physical mechanisms underlying these uptake processes may be achieved by the investigation of simple model systems that can be compared to theoretical models. Here, we present experiments on a massive uptake of silica nanoparticles by giant unilamellar lipid vesicles (GUVs). We find that this uptake process depends on the size of the particles as well as on the thermodynamic state of the lipid membrane. Our findings are discussed in the light of several theoretical models and indicate that these models have to be extended in order to capture the interaction between nanomaterials and biological membranes correctly

Intake of silica nanoparticles by giant lipid vesicles: influence of particle size and thermodynamic membrane state

The uptake of nanoparticles into cells often involves their engulfment by the plasma membrane and a fission of the latter. Understanding the physical mechanisms underlying these uptake processes may be achieved by the investigation of simple model systems that can be compared to theoretical models. Here, we present experiments on a massive uptake of silica nanoparticles by giant unilamellar lipid vesicles (GUVs). We find that this uptake process depends on the size of the particles as well as on the thermodynamic state of the lipid membrane. Our findings are discussed in the light of several theoretical models and indicate that these models have to be extended in order to capture the interaction between nanomaterials and biological membranes correctly

Survival of Plasmodium falciparum infected Red Blood Cell Aggregates in Elongational Shear Flow

Rosetting, the formation of red blood cell aggregates, is a life-threatening condition in malaria tropica and not yet fully understood. We study rosette stability using a set of microfluidic stenotic channels, with varied narrowing angle and erythrocytes of blood groups O and A. We find reduced ability of a rosette to pass a stenosis without disruption, the longer the tapered part of the constriction and the narrower the stenosis is. In general, this ability increases with rosette size and is 5-15% higher in blood group A. The experimental results are substantiated by equivalent experiments using lectin-induced red blood cell aggregates and a simulation of the underlying protein binding kinetics.</p