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Phagocytosis is the process of engulfment and internalization of comparatively large particles by cells, and plays a central role in the functioning of our immune system. We study the process of phagocytosis by considering a simplified coarse grained model of a three-dimensional vesicle, having a uniform adhesion interaction with a rigid particle, and containing curved membrane-bound protein complexes or curved membrane nano-domains, which in turn recruit active cytoskeletal forces. Complete engulfment is achieved when the bending energy cost of the vesicle is balanced by the gain in the adhesion energy. The presence of curved (convex) proteins reduces the bending energy cost by self-organizing with a higher density at the highly curved leading edge of the engulfing membrane, which forms the circular rim of the phagocytic cup that wraps around the particle. This allows the engulfment to occur at much smaller adhesion strength. When the curved membrane-bound protein complexes locally recruit actin polymerization machinery, which leads to outward forces being exerted on the membrane, we found that engulfment is achieved more quickly and at a lower protein density. We consider spherical and non-spherical particles and found that non-spherical particles are more difficult to engulf in comparison to the spherical particles of the same surface area. For non-spherical particles, the engulfment time crucially depends on the initial orientation of the particles with respect to the vesicle. Our model offers a mechanism for the spontaneous self-organization of the actin cytoskeleton at the phagocytic cup, in good agreement with recent high-resolution experimental observations.

Modern electronics can be utilized to provide automated means of monitoring sponta-neous blood sedimentation and by implementing wireless power and data transfer can be used in centrifugation systems as well. Here, we present the method of building the optical linear charge coupled device (CCD) sensor with microcontroller circuit for re-cording and monitoring the sedimentation process. Brief overview of the engineering be-hind the communication protocol is given together with key microcontroller techniques that are required for fast analogue voltage signal sampling. Proposed system has adapt-able light integration protocol and shows exceptional durability under centrifugal force loads that are commonly used in platelet-rich plasma. The parts for building the system are sourced at common electronics stores and provides easy means for observing optical changes in blood samples with emphasis on sedimentation and forming layers with dif-ferent optical properties. Keywords: Blood sedimentation; Plasma; Platelet-rich plasma; Microcontroller; Optical sensor

Cells often migrate on curved surfaces inside the body, such as curved tissues, blood vessels or highly curved protrusions of other cells. Recentin-vitroexperiments provide clear evidence that motile cells are affected by the curvature of the substrate on which they migrate, preferring certain curvatures to others, termed “curvotaxis”. The origin and underlying mechanism that gives rise to this curvature sensitivity are not well understood. Here, we employ a “minimal cell” model which is composed of a vesicle that contains curved membrane protein complexes, that exert protrusive forces on the membrane (representing the pressure due to actin polymerization). This minimal-cell model gives rise to spontaneous emergence of a motile phenotype, driven by a lamellipodia-like leading edge. By systematically screening the behaviour of this model on different types of curved substrates (sinusoidal, cylinder and tube), we show that minimal ingredients and energy terms capture the experimental data. The model recovers the observed migration on the sinusoidal substrate, where cells move along the grooves (minima), while avoiding motion along the ridges. In addition, the model predicts the tendency of cells to migrate circumferentially on convex substrates and axially on concave ones. Both of these predictions are verified experimentally, on several cell types. Altogether, our results identify the minimization of membrane-substrate adhesion energy and binding energy between the membrane protein complexes as key players of curvotaxis in cell migration.

It is known that giant vesicles undergo dynamic morphological changes when exposed to a detergent. The solubilization process may take multiple pathways. In this work, we identify lipid vesicle shape dynamics before the solubilization of 1,2-dioleoyl-sn-glycero-3-phosphocholine giant vesicles with Triton X-100 (TR) detergent. The violent lipid vesicle dynamics was observed with laser confocal scanning microscopy and was qualitatively explained via a numerical simulation. A three-dimensional Monte Carlo scheme was constructed that emulated the nonequilibrium conditions at the beginning stages of solubilization, accounting for a gradual addition of TR detergent molecules into the lipid bilayers. We suggest that the main driving factor for morphology change in lipid vesicles is the associative tendency of the TR molecules, which induces spontaneous curvature of the detergent inclusions, an intrinsic consequence of their molecular shape. The majority of the observed lipid vesicle shapes in the experiments were found to correspond very well to the numerically calculated shapes in the phase space of possible solutions. The results give an insight into the early stages of lipid vesicle solubilization by amphiphilic molecules, which is nonequilibrium in nature and very difficult to study.

Extracellular vesicles (EVs) are membrane-enclosed structures of nanometer dimensions. They are formed by cells and can be found in isolates from body fluids. It is indicated that they play important role in intercellular communication in health and disease. In this work we observed the morpholoical parameters of EVs isolated by differential centrifugation from washed erythrocytes, in which vesiculation was accelerated by addition of detergent (sodium dodecyl sulfate). Aliquots of the isolate were suspended medium of two different osmolarities. Isolates were imaged with a scan-ning electron microscope (SEM) and the images were analyzed by using the contours of EVs from which the volume V, surface area S, and relative volume v = (36S3/V2)1/2 were assessed by a geomet-rical model. EVs were considered as spheres or spheroids. The isolates were rich in erythrocyte EVs so that representative sets (86 vesicles at osmolarity of 50 mOsmol/L and 109 vesicles at osmolarity of 300 mOsmol/L) could be outlined. The EVs at osmolarity of 50 mOsmol/L had shapes close to a sphere, while at osmolarity of 300 mOsmol/L they had elongated shapes. The shapes were approxi-mated by prolate spheroids. The average volume/surface area of EVs at osmolarity 50 mOsmol/L were 3.18×105 nm3/2.20×104 nm2, and at osmolarity 300 mOsmol/L they were 4.3×105 nm3/2.79×104 nm2. The respective differences in favor of the values at 300 mOsmol/L were statistically significant and of sufficient power. The relative volume of EVs at 50 mOsmol/L and 300 mOsmol/L were 1 and 0.96, respectively. While the difference in v suggests that, similarly to erythrocytes, water enters EVs in order to attain the Donnan equilibrium, the differences in V and A suggest that selective popping of (larger) EVs in the hypoosmolar sample took place. Keywords: Extracellular vesicles; Erythrocytes; Osmolarity; Differential centrifugation; Scanning electron microscopy (SEM); Morphology; Interdisciplinary connection; Medicine

Biological membranes are composed of isotropic and anisotropic curved nanodomains. Anisotropic membrane components, such as Bin/Amphiphysin/Rvs (BAR) superfamily protein domains, could trigger/facilitate the growth of membrane tubular protrusions, while isotropic curved nanodomains may induce undulated (necklace-like) membrane protrusions. We review the role of isotropic and anisotropic membrane nanodomains in stability of tubular and undulated membrane structures generated or stabilized by cyto- or membrane-skeleton. We also describe the theory of spontaneous self-assembly of isotropic curved membrane nanodomains and derive the critical concentration above which the spontaneous necklace-like membrane protrusion growth is favorable. We show that the actin cytoskeleton growth inside the vesicle or cell can change its equilibrium shape, induce higher degree of segregation of membrane nanodomains or even alter the average orientation angle of anisotropic nanodomains such as BAR domains. These effects may indicate whether the actin cytoskeleton role is only to stabilize membrane protrusions or to generate them by stretching the vesicle membrane. Furthermore, we demonstrate that by taking into account the in-plane orientational ordering of anisotropic membrane nanodomains, direct interactions between them and the extrinsic (deviatoric) curvature elasticity, it is possible to explain the experimentally observed stability of oblate (discocyte) shapes of red blood cells in a broad interval of cell reduced volume. Finally, we present results of numerical calculations and Monte-Carlo simulations which indicate that the active forces of membrane skeleton and cytoskeleton applied to plasma membrane may considerably influence cell shape and membrane budding.

Phagocytosis is the process of engulfment and internalization of comparatively large particles by the cell, that plays a central role in the functioning of our immune system. We study the process of phagocytosis by considering a simplified coarse grained model of a three-dimensional vesicle, having uniform adhesion interaction with a rigid particle, in the presence of curved membrane proteins and active cytoskeletal forces. Complete engulfment is achieved when the bending energy cost of the vesicle is balanced by the gain in the adhesion energy. The presence of curved (convex) proteins reduces the bending energy cost by self-organizing with higher density at the highly curved leading edge of the engulfing membrane, which forms the circular rim of the phagocytic cup that wraps around the particle. This allows the engulfment to occur at much smaller adhesion strength. When the curved proteins exert outwards protrusive forces, representing actin polymerization, at the leading edge, we find that engulfment is achieved more quickly and at lower protein density. We consider spherical as well as non-spherical particles, and find that non-spherical particles are more difficult to engulf in comparison to the spherical particles of the same surface area. For non-spherical particles, the engulfment time crucially depends upon the initial orientation of the particles with respect to the vesicle. Our model offers a mechanism for the spontaneous self-organization of the actin cytoskeleton at the phagocytic cup, in good agreement with recent high-resolution experimental observations., 27 pages, 23 figures

Eukaryotic cells adhere to extracellular matrix during the normal development of the organism, forming static adhesion as well as during cell motility. We study this process by considering a simplified coarse-grained model of a vesicle that has uniform adhesion energy with a flat substrate, mobile curved membrane proteins and active forces. We find that a high concentration of curved proteins alone increases the spreading of the vesicle, by the self-organization of the curved proteins at the high curvature vesicle-substrate contact line, thereby reducing the bending energy penalty at the vesicle rim. This is most significant in the regime of low bare vesicle-substrate adhesion. When these curved proteins induce protrusive forces, representing the actin cytoskeleton, we find efficient spreading, in the form of sheet-like lamellipodia. Finally, the same mechanism of spreading is found to include a minimal set of ingredients needed to give rise to motile phenotypes., 30 pages, 27 figures

Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments., Cells display a high degree of plasticity in migration, but how polarity is organized in different microenvironments has remained unclear. This study uses mechanical perturbations to reveal that migration using actin-rich or bleb-based protrusions are both organized around Rac GTPase.

Eukaryote cells have a flexible shape, which dynamically changes according to the function performed by the cell. One mechanism for deforming the cell membrane into the desired shape is through the expression of curved membrane proteins. Furthermore, these curved membrane proteins are often associated with the recruitment of the cytoskeleton, which then applies active forces that deform the membrane. This coupling between curvature and activity was previously explored theoretically in the linear limit of small deformations, and low dimensionality. Here we explore the unrestricted shapes of vesicles that contain active curved membrane proteins, in three-dimensions, using Monte-Carlo numerical simulations. The activity of the proteins is in the form of protrusive forces that push the membrane outwards, as may arise from the cytoskeleton of the cell due to actin or microtubule polymerization occurring near the membrane. For proteins that have an isotropic convex shape, the additional protrusive force enhances their tendency to aggregate and form membrane protrusions (buds). In addition, we find another transition from deformed spheres with necklace type aggregates, to flat pancake-shaped vesicles, where the curved proteins line the outer rim. This second transition is driven by the active forces, coupled to the spontaneous curvature, and the resulting configurations may shed light on the formation of sheet-like protrusions and lamellipodia of adhered and motile cells.

Two different uses of Monte Carlo methods in soft matter physics are presented in the following work. Firstly, the Monte Carlo simulated annealing is used to minimize the elastistic energy of the inverted hexagonal phase (HII) optimal geometry. We will do a brief overview on the mechanics of the HII lipid phase. In our model the expression for the lipid monolayer free energy consists of two energy contributions: the bending energy which involves also a deviatoric term, and the interstitial energy which describes the deformation energy due to stretching of the phospholipid molecule chains. On the basis of the derived expression for the lipid monolayer free energy, we will theoretically predict optimal geometry and physical conditions for the stability of the inverted hexagonal phase. Using the Monte Carlo simulated annealing method, we will theoretically describe first steps in the LαHII phase transition. Another interesting subject investigated by means of Monte Carlo simulations are the thermal fluctuations of nearly spherical vesicles. The theoretical basis of this analysis was done by Milner and Safran (Phys Rev A 36(9):4371–4379, 1987. doi: 10.1103/PhysRevA.36.4371 ) that uses the mean field approximation. In this work we will show the application of the Monte Carlo simulations and show the correlation between the time mean simulated thermal fluctuations decomposed into spherical harmonics and the bending stiffness in 2-dimensional and 3-dimensional space.

Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted Arp2/3 and WAVE complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments.

We studied biological membranes of spherical topology within the framework of the spontaneous curvature model. Both Monte Carlo simulations and the numerical minimization of the curvature energy were used to obtain the shapes of the vesicles. The shapes of the vesicles and their energy were calculated for different values of the reduced volume. The vesicles which exhibit in-plane ordering were also studied. Minimal models have been developed in order to study the orientational ordering in colloids coated with a thin sheet of nematic liquid crystal (nematic shells). The topological defects are always present on the surfaces with the topology of a sphere. The location of the topological defects depends strongly on the curvature of the surface. We studied the nematic ordering and the formation of topological defects on vesicles obtained by the minimization of the spontaneous curvature energy.

Biological membranes are composed of different components and there is no a priori reason to assume that all components are isotropic. It was previously shown that the anisotropic properties of membrane components may explain the stability of membrane tubular protrusions even without the application of external force. Our theoretical study focuses on the role of anisotropic membrane components in the stability of membrane tubular structures generated or stabilized by actin filaments. We show that the growth of the actin cytoskeleton inside the vesicle can induce the partial lateral segregation of different membrane components. The entropy of mixing of membrane components hinders the total lateral segregation of the anisotropic and isotropic membrane components. Self-assembled aggregates formed by anisotropic membrane components facilitate the growth of long membrane tubular protrusions. Protrusive force generated by actin filaments favors strong segregation of membrane components by diminishing the opposing effect of mixing entropy.

Due to insufficient adhesion of sputtered Ti/NiV/Ag metallization scheme on n^+Si substrate when annealed below 550^oC, investigation was focused on the influence of process parameters on adhesion properties. Adhesion between metallic stack and Si substrate was found to be a strong function of annealing temperature. Additional investigations showed that the cause for poor adhesion was also rather high residual stress in metal thin layers, particularly stress in sputtered NiV layer. High residual stress was measured for NiV (1883+/-252MPa) and lower for Ti (334+/-60MPa) and Ag (310+/-10MPa) layer, respectively. The influence of sputtering parameters on the stress behavior was experimentally verified. By reducing the cathode DC power and Ar working pressure during NiV sputtering we were able to reduce the stress within the structure. A clear correlation was found between the residual stress magnitude and adhesion properties within the temperature range from room temperature to 550^oC. By residual stress reduction within a metallic stack, the necessary annealing temperature to obtain optimal adhesion was reduced from 550 to 500^oC.

Design and realization of a digitally controlled closed loop calibration system for smart sensors, capable of self-learning, is reported. Closed loop design enables analysis of sensor properties and optimization of calibration procedure. Dedicated software was developed, enabling control of acquired data and calibration procedure. Resulting statistical data provide closed loop feedback variable for failure analysis. Calibration system enables digital temperature compensation by acquisition of calibration points, calculation of sensor polynomial coefficients and storing the calculated data in the sensor memory. Due to the proposed digital temperature compensation method, temperature output error of pressure sensors was significantly reduced (typically from 0.15% FSO/°C to 0.05% FSO/°C, based on calibration of 29482 manifold absolute pressure sensors). Modular approach results in reduction of calibration time (typically down to 42 s/sensor), thus enabling mass calibration.

The membrane bending stiffness of nearly spherical lipid vesicles can be deduced from the analysis of their thermal shape fluctuations. The theoretical basis of this analysis [Milner and Safran, Phys. Rev. A: At., Mol., Opt. Phys., 1987, 36, 4371–4379] uses the mean field approximation. In this work we apply Monte Carlo simulations and estimate the error in the determination of the bending stiffness due to the approximations applied in the theory. It is less than 10%. The method presented in this work can also be used to determine the changes of the bending stiffness of biological membranes due to their chemical and/or structural modifications.

This work presents the theoretical basis for Monte Carlo simulations of thermally fluctuating lipid bilayers. The purpose of such simulations is to obtain additional insight into the mechanical processes involved in thermal fluctuations of lipid bilayers and to test and limit some of the assumptions used in established theoretical models employed in measurements of the elastic properties of lipid membranes. The two-dimensional and three-dimensional models are explained and discussed. A dynamically triangulated surface model of the membrane is adapted to the thermally fluctuating vesicle with constant volume and local membrane stretching. The microstates of the vesicle are sampled according to the Metropolis algorithm, where fluctuations that conserve the volume of the vesicle are produced, while the energy of any given microstate of the vesicle consists of the bending and stretching energies of the vesicle. The vesicle shape is then approximated by a set of spherical harmonics. The results of the spectral analysis of thermally fluctuating vesicles in our simulations open questions that are discussed in the concluding section.

Aim To investigate morphological alterations of malignant cancer cells (T24) of urothelial origin seeded on flat titanium (Ti) and nanotubular titanium dioxide (TiO2) nanostructures. Methods Using anodization method, TiO2 surfaces composed of vertically aligned nanotubes of 50-100 nm diameters were produced. The flat Ti surface was used as a reference. The alteration in the morphology of cancer cells was evaluated using scanning electron microscopy (SEM). A computational model, based on the theory of membrane elasticity, was constructed to shed light on the biophysical mechanisms responsible for the observed changes in the contact area of adhesion. Results Large diameter TiO2 nanotubes exhibited a significantly smaller contact area of adhesion (P

In this paper the design, fabrication methods and characterization of thin film meandered Pt resistive heater (size20×20mm2) with integrated Pt sensors on Si platform is presented. Pt heaters and temperature sensors were fabricated simultaneously by DC sputtering method. It was found that the fabrication process has significant influence on the electrical properties of the realized thin film resistive layers, which also explains the discrepancies between the calculated and measured values that were obtained during this work. Annealing temperature of the Pt layers was found to influence significantly the final resistance of the deposited layer and was performed at 500 °C. To reduce the heat loss, the heater and temperature sensors were covered by Pyrex glass with prefabricated cavity. By this new approach, the power consumption was reduced due to improved thermal insulation. A comparative study was performed and showed that we can decrease the power consumption by more than 25% only by this approach. Measured temperature coefficient of resistance (TCR) for temperature sensors and the heater was between 1400−1700 ppm and showed that also the heater can be used as temperature sensing element.

SCREAM process for releasing micromechanical MEMS structures using plasma polymerized fluorocarbon thin film for trench sidewall passivation is reported. The developed process is designed as a one mask process, and all etchings and depositions are fabricated in the same etching system (standard capacitive coupled RIE), as one-step-one-run process.

Closed biological membranes were considered within the spontaneous curvature model. Ground state membrane shapes were compared with Monte Carlo simulations in the thermal equilibrium, where membranes are subject to thermal uctuations. The results of the two approaches correspond well with each other. The oblate discocyte membrane shapes are obtained in the ground state but can become metastable when thermal uctuations are taken into account. The nematic ordering in oblate and stomatocyte vesicle membranes was also studied. It was conrmed

In recent years, liposomes encapsulated with nanoparticles have found enormous scopes in various biomedical fields such as drug design, transport, imaging, targeted delivery and therapy. These applications require a clear understanding about the interaction of nanoparticles with cell membranes. The present work aims to investigate the effect of encapsulation of uncharged and positively charged nanoparticles in three different types of lipids such as1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC),1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine and1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine(SOPC-POPS) mixture and archaeal lipids. Through the temperature dependent fluorescence anisotropy measurements, we have found that the entrapment of nanoparticles in the bilayer has decreased the lipid transition temperature and increased the membrane fluidity of all three types of lipid vesicles. The results were more predominant in SOPC-POPS mixture because of high density encapsulation of nanoparticles in the vesicles due to electrostatic interaction between negatively charged membrane and positively charged iron oxide nanoparticles.

In this mini-review, a brief historical survey of the mechanisms which determine the shapes of liposomes and cells and the budding and fission of their membrane is presented. Special attention is given to the role of orientational ordering of membrane components in thin membrane necks which connect the membrane buds (daughter vesicles) to the parent membrane. It is indicated that topological anti-defects in membrane necks may induce the rupture of the neck and the fission of the membrane daughter vesicles.