Teaching the basic principles of electrocardiography experimentally.

Electrocardiography (ECG) is one of the most widely used methods in clinical diagnosis. Here we describe an experimental approach that offers hands-on learning of its basic principles. An experimental model that consists of a rubber foil with a low electrical conductivity and a DC power unit is used to simulate the body and the electric dipole of the heart. It enables students to learn about the main features of the electric dipole and to visualize the induced electric potential in the body. The determination of the characteristic equipotential lines around the dipole and the measurement of simple electrocardiograms, comprising bipolar and unipolar leads, are made with a low-cost voltmeter. To make the exercise more relevant to clinical ECG, as well as making it more interesting, the students are additionally tasked to measure their own electrocardiogram with a simple, personal handheld ECG device.

Membrane Curvature, Trans-Membrane Area Asymmetry, Budding, Fission and Organelle Geometry.

In biology, the modern scientific fashion is to mostly study proteins. Much less attention is paid to lipids. However, lipids themselves are extremely important for the formation and functioning of cellular membrane organelles. Here, the role of the geometry of the lipid bilayer in regulation of organelle shape is analyzed. It is proposed that during rapid shape transition, the number of lipid heads and their size (i.e., due to the change in lipid head charge) inside lipid leaflets modulates the geometrical properties of organelles, in particular their membrane curvature. Insertion of proteins into a lipid bilayer and the shape of protein trans-membrane domains also affect the trans-membrane asymmetry between surface areas of luminal and cytosol leaflets of the membrane. In the cases where lipid molecules with a specific shape are not predominant, the shape of lipids (cylindrical, conical, or wedge-like) is less important for the regulation of membrane curvature, due to the flexibility of their acyl chains and their high ability to diffuse.

Keratin Dynamics and Spatial Distribution in Wild-Type and K14 R125P Mutant Cells-A Computational Model.

Keratins are one of the most abundant proteins in epithelial cells. They form a cytoskeletal filament network whose structural organization seriously conditions its function. Dynamic keratin particles and aggregates are often observed at the periphery of mutant keratinocytes related to the hereditary skin disorder epidermolysis bullosa simplex, which is due to mutations in keratins 5 and 14. To account for their emergence in mutant cells, we extended an existing mathematical model of keratin turnover in wild-type cells and developed a novel 2D phase-field model to predict the keratin distribution inside the cell. This model includes the turnover between soluble, particulate and filamentous keratin forms. We assumed that the mutation causes a slowdown in the assembly of an intermediate keratin phase into filaments, and demonstrated that this change is enough to account for the loss of keratin filaments in the cell's interior and the emergence of keratin particles at its periphery. The developed mathematical model is also particularly tailored to model the spatial distribution of keratins as the cell changes its shape.

Crowd-Sourcing of Membrane Fission: How crowding of non-specialized membrane-bound proteins contributes to cellular membrane fission.

Fission of cellular membranes is ubiquitous and essential for life. Complex protein machineries, such as the dynamin and ESCRT spirals, have evolved to mediate membrane fission during diverse cellular processes, for example, vesicle budding. A new study suggests that non-specialized membrane-bound proteins can induce membrane fission through mass action due to protein crowding. Because up to 2/3 of the mass of cellular membranes is contributed by proteins, membrane protein crowding is an important physiological parameter. Considering the complexity of membrane shape transitions during a fission reaction, spatial and temporal variability in protein distribution, and the abundance of intrinsically disordered regions in proteins on an invaginating membrane, protein crowding can have diverse consequences for fission in the cell. The question is, how and to what extent this mechanism combines with the action of dedicated fission machineries.

Membrane bending: the power of protein imbalance.

Many cellular processes require membrane deformation, which is driven by specialized protein machinery and can often be recapitulated using pure lipid bilayers. However, biological membranes contain a large amount of embedded proteins. Recent research suggests that membrane-bound proteins with asymmetric distribution of mass across the bilayer can influence membrane bending in a nonspecific manner due to molecular crowding. This mechanism is physical in nature and arises from collisions between such 'mushroom-shaped' proteins. It can either facilitate or impede the action of protein coats, for example COPII, during vesicle budding. We describe the physics of how molecular crowding can influence membrane bending and discuss the implications for other cellular processes, such as sorting of glycosylphosphatidylinositol-anchored proteins (GPI-APs) and production of intraluminal vesicles.

Membrane bending by protein crowding is affected by protein lateral confinement.

Crowding of asymmetrically-distributed membrane proteins has been recently recognized as an important factor in remodeling of biological membranes, for example during transport vesicle formation. In this paper, we theoretically analyze the effect of protein crowding on membrane bending and examine its dependence on protein size, shape, transmembrane asymmetry and lateral confinement. We consider three scenarios of protein lateral organization, which are highly relevant for cellular membranes in general: freely diffusing membrane proteins without lateral confinement, the presence of a diffusion barrier and interactions with a vesicular coat. We show that protein crowding affects vesicle formation even if the proteins are distributed symmetrically across the membrane and that this effect depends significantly on lateral confinement. The largest crowding effect is predicted for the proteins that are confined to the forming vesicle by a diffusion barrier. We calculate the bending properties of a crowded membrane and find that its spontaneous curvature depends primarily on the degree of transmembrane asymmetry, and its effective bending modulus on the type of lateral confinement. Using the example of COPII vesicle formation from the endoplasmic reticulum, we analyze the energetic cost of vesicle formation. The results provide a novel insight into the effects of lateral and transmembrane organization of membrane proteins, and can guide data interpretation and future experimental approaches.

Trans-membrane area asymmetry controls the shape of cellular organelles.

Membrane organelles often have complicated shapes and differ in their volume, surface area and membrane curvature. The ratio between the surface area of the cytosolic and luminal leaflets (trans-membrane area asymmetry (TAA)) determines the membrane curvature within different sites of the organelle. Thus, the shape of the organelle could be critically dependent on TAA. Here, using mathematical modeling and stereological measurements of TAA during fast transformation of organelle shapes, we present evidence that suggests that when organelle volume and surface area are constant, TAA can regulate transformation of the shape of the Golgi apparatus, endosomal multivesicular bodies, and microvilli of brush borders of kidney epithelial cells. Extraction of membrane curvature by small spheres, such as COPI-dependent vesicles within the Golgi (extraction of positive curvature), or by intraluminal vesicles within endosomes (extraction of negative curvature) controls the shape of these organelles. For instance, Golgi tubulation is critically dependent on the fusion of COPI vesicles with Golgi cisternae, and vice versa, for the extraction of membrane curvature into 50-60 nm vesicles, to induce transformation of Golgi tubules into cisternae. Also, formation of intraluminal ultra-small vesicles after fusion of endosomes allows equilibration of their TAA, volume and surface area. Finally, when microvilli of the brush border are broken into vesicles and microvilli fragments, TAA of these membranes remains the same as TAA of the microvilli. Thus, TAA has a significant role in transformation of organelle shape when other factors remain constant.

Locally induced shockwaves for selective perforation of cargo loaded lipid vesicles with temporal and spatial control.

Controlled poration of lipid membranes is crucial for numerous biomimetic applications such as targeted drug delivery. Although several chemical and physical mechanisms have been proposed for the poration of synthetic membranes, achieving good temporal and spatial control remains a challenge. In this study, we introduce a novel method for membrane poration that utilizes the mechanical shockwave generated by the photo-acoustic effect, which occurs when an optically opaque microparticle is illuminated by a near-infrared laser of optical tweezers. We show that the shockwave effectively porates membranes of giant unilamellar vesicles in close proximity to the microparticle without damaging nearby cells, which is a desirable outcome for potential targeted drug delivery. The poration effect is nonspecific and operates on both liquid and gel phase membranes. Since the photo-acoustic effect can be triggered by standard optical tweezers, this method holds broad applicability in various experimental settings within the field of soft matter research.

Real-Time Assessment of the Size Changes of Individual Sub-Visible Protein Particles under Buffer Variations: A Microfluidic Study.

Protein particles in biological drugs can significantly impact drug efficacy and carry the risk of adverse effects. Despite advancements, the understanding and control of particle formation in biopharmaceutical manufacturing remain incomplete. Therefore, further investigation into protein particles is warranted, especially considering that novel formats of biological drugs may be more susceptible to aggregation and particle formation than conventional monoclonal antibodies. In this study, we introduce a microfluidic approach for the real-time analysis of individual sub-visible protein particles during buffer exchange. We find that the modulation of intermolecular forces, achieved by changing the buffer pH or urea concentration, leads to the reversible swelling and shrinkage of particles by up to 50%, which is a consequence of altered intermolecular distances. Additionally, we identify a discrepancy in the biophysical behavior of protein particles compared to monomeric protein. This finding highlights the limited predictive power of commonly applied biophysical characterization methods for particle formation in early formulation development. Moreover, the observed particle swelling may be associated with manufacturing deviations, such as filter clogging. These results highlight the importance of studying individual particles to gain a comprehensive insight into particle behavior and the impact of formulation variations in the biopharmaceutical industry.

Low-cost programable stroboscopic illumination with sub-microsecond pulses for high-throughput microfluidic applications.

To visualize fast-moving objects in microfluidic applications, the image acquisition time must be on the order of a microsecond or less. Commercial imaging systems capable of such short exposure times may be too expensive for many research laboratories. We have therefore developed a low-cost stroboscopic illumination for transmitted-light microscopy based on a high-power LED that can be coupled to a standard industrial camera and provides exposure times on the order of 500 ns. The system is designed to be easily mounted on a standard condenser of an inverted microscope. The illumination is controlled by a fast Arduino-compatible Teensy® 4.0 development board, and the illumination parameters can be set from a PC via a graphical user interface written in Python. The system has been successfully used for high-throughput cell phenotyping using deformability cytometry on a Nikon TE2000 microscope, as well as for droplet microfluidic on an old Olympus inverted microscope.

Molecular, morphological and functional properties of tunnelling nanotubes between normal and cancer urothelial cells: New insights from the in vitro model mimicking the situation after surgical removal of the urothelial tumor.

Tunnelling nanotubes (TNTs) are membranous connections that represent a unique type of intercellular communication in different cell types. They are associated with cell physiology and cancer pathology. The possible existence of tunnelling nanotubes communication between urothelial cancer and normal cells has not yet been elucidated. Therefore, we analyzed TNTs formed by T24 cells (human invasive cancer urothelial cells) and normal porcine urothelial (NPU) cells, which serve as surrogate models for healthy human urothelial cells. Monocultures and cocultures of NPU and T24 cells were established and analyzed using live-cell imaging, optical tweezers, fluorescence microscopy, and scanning electron microscopy. TNTs of NPU cells differed significantly from tunnelling nanotubes of T24 cells in number, length, diameter, lipid composition, and elastic properties. Membrane domains enriched in cholesterol/sphingomyelin were present in tunnelling nanotubes of T24 cells but not in NPU cells. The tunnelling nanotubes in T24 cells were also easier to bend than the tunnelling nanotubes in NPU cells. The tunnelling nanotubes of both cell types were predominantly tricytoskeletal, and contained actin filaments, intermediate filaments, and microtubules, as well as the motor proteins myosin Va, dynein, and kinesin 5B. Mitochondria were transported within tunnelling nanotubes in living cells, and were colocalized with microtubules and the microtubule-associated protein dynamin 2. In cocultures, heterocellular tunnelling nanotubes were formed between NPU cells and T24 cells and vice versa. The presence of connexin 43 at the end of urothelial tunnelling nanotubes suggests a junctional connection and the involvement of tunnelling nanotube in signal transduction. In this study, we established a novel urothelial cancer-normal coculture model and showed cells in the minority tend to form tunnelling nanotubes with cells in the majority. The condition with cancer cells in the minority is an attractive model to mimic the situation after surgical resection with remaining cancer cells and may help to understand cancer progression and recurrence. Our results shed light on the biological activity of tunnelling nanotubes and have the potential to advance the search for anticancer drugs that target tunnelling nanotubes.

An oomycete NLP cytolysin forms transient small pores in lipid membranes.

Microbial plant pathogens secrete a range of effector proteins that damage host plants and consequently constrain global food production. Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) are produced by numerous phytopathogenic microbes that cause important crop diseases. Many NLPs are cytolytic, causing cell death and tissue necrosis by disrupting the plant plasma membrane. Here, we reveal the unique molecular mechanism underlying the membrane damage induced by the cytotoxic model NLP. This membrane disruption is a multistep process that includes electrostatic-driven, plant-specific lipid recognition, shallow membrane binding, protein aggregation, and transient pore formation. The NLP-induced damage is not caused by membrane reorganization or large-scale defects but by small membrane ruptures. This distinct mechanism of lipid membrane disruption is highly adapted to effectively damage plant cells.

Adhesion and Stiffness of Detached Breast Cancer Cells In Vitro: Co-Treatment with Metformin and 2-Deoxy-d-glucose Induces Changes Related to Increased Metastatic Potential.

Metastatic cancer cells can overcome detachment-induced cell death and can proliferate in anchorage-independent conditions. A recent study revealed that a co-treatment with two drugs that interfere with cell metabolism, metformin and 2-deoxy-D-glucose, promotes detachment of viable MDA-MB-231 breast cancer cells. In the present study, we analyzed if these detached viable MDA-MB-231 cells also exhibit other features related to cancer metastatic potential, i.e., if they are softer and more prone to adhere to epithelial cells. The cell mechanics of attached cells and floating cells were analyzed by optical tweezers and cell deformability cytometry, respectively. The adhesion was assessed on a confluent monolayer of HUVEC cells, with MDA-MB-231 cells either in static conditions or in a microfluidic flow. Additionally, to test if adhesion was affected by the state of the epithelial glycocalyx, HUVEC cells were treated with neuraminidase and tunicamycin. It was found that the treated MDA-MB-231 cells were more prone to adhere to HUVEC cells and that they were softer than the control, both in the floating state and after re-seeding to a substrate. The changes in the HUVEC glycocalyx, however, did not increase the adhesion potential of MDA-MB-231.

Exceptional stability of a perilipin on lipid droplets depends on its polar residues, suggesting multimeric assembly.

Numerous proteins target lipid droplets (LDs) through amphipathic helices (AHs). It is generally assumed that AHs insert bulky hydrophobic residues in packing defects at the LD surface. However, this model does not explain the targeting of perilipins, the most abundant and specific amphipathic proteins of LDs, which are weakly hydrophobic. A striking example is Plin4, whose gigantic and repetitive AH lacks bulky hydrophobic residues. Using a range of complementary approaches, we show that Plin4 forms a remarkably immobile and stable protein layer at the surface of cellular or in vitro generated oil droplets, and decreases LD size. Plin4 AH stability on LDs is exquisitely sensitive to the nature and distribution of its polar residues. These results suggest that Plin4 forms stable arrangements of adjacent AHs via polar/electrostatic interactions, reminiscent of the organization of apolipoproteins in lipoprotein particles, thus pointing to a general mechanism of AH stabilization via lateral interactions.

Cortical stiffness of keratinocytes measured by lateral indentation with optical tweezers.

Keratin intermediate filaments are the principal structural element of epithelial cells. Their importance in providing bulk cellular stiffness is well recognized, but their role in the mechanics of cell cortex is less understood. In this study, we therefore compared the cortical stiffness of three keratinocyte lines: primary wild type cells (NHEK2), immortalized wild type cells (NEB1) and immortalized mutant cells (KEB7). The cortical stiffness was measured by lateral indentation of cells with AOD-steered optical tweezers without employing any moving mechanical elements. The method was validated on fixed cells and Cytochalasin-D treated cells to ensure that the observed variations in stiffness within a single cell line were not a consequence of low measurement precision. The measurements of the cortical stiffness showed that primary wild type cells were significantly stiffer than immortalized wild type cells, which was also detected in previous studies of bulk elasticity. In addition, a small difference between the mutant and the wild type cells was detected, showing that mutation of keratin impacts also the cell cortex. Thus, our results indicate that the role of keratins in cortical stiffness is not negligible and call for further investigation of the mechanical interactions between keratins and elements of the cell cortex.

Equilibrium mechanics of monolayered epithelium.

In order to fully understand the epithelial mechanics it is essential to integrate different levels of epithelial organization. In this work, we propose a theoretical approach for connecting the macroscopic mechanical properties of a monolayered epithelium to the mechanical properties at the cellular level. The analysis is based on the established mechanical models-at the macroscopic scale the epithelium is described within the mechanics of thin layers, while the cellular level is modeled in terms of the cellular surface (cortical) tension and the intercellular adhesion. The macroscopic elastic energy of the epithelium is linked to the energy of an average epithelial cell. The epithelial equilibrium state is determined by energy minimization and the macroscopic elastic moduli are calculated from deformations around the equilibrium. The results indicate that the epithelial equilibrium state is defined by the ratio between the adhesion strength and the cellular surface tension. The lower and the upper bounds for this ratio are estimated. If the ratio is small, the epithelium is cuboidal, if it is large, the epithelium becomes columnar. Importantly, it is found that the cellular cortical tension and the intercellular adhesion alone cannot produce the flattened squamous epithelium. Any difference in the surface tension between the apical and basal cellular sides bends the epithelium towards the side with the larger surface tension. Interestingly, the analysis shows that the epithelial area expansivity modulus and the shear modulus depend only on the cellular surface tension and not on the intercellular adhesion. The results are presented in a general analytical form, and are thus applicable to a variety of monolayered epithelia, without relying on the specifics of numerical finite-element methods. In addition, by using the standard theoretical tools for multi-laminar systems, the results can be applied to epithelia consisting of layers with different mechanical properties.

Migration of blood cells and phospholipid vesicles induced by concentration gradients in microcavities.

Microcavities provide a well-controlled flow-free microenvironment and play an important role in many microfluidic systems, for example as cell-culturing microchambers. Here we show that transient concentration gradients that emerge during diffusive exchange of solutes in microcavities induce passive migration (diffusiophoresis) of blood cells and synthetic phospholipid vesicles. The passive migration is observed in various concentration gradients comprising non-electrolytes and electrolytes, i.e., glucose, sucrose, sodium chloride, potassium chloride, potassium benzoate, and potassium sulfate. The results add to prior reports, where gradients of non-electrolytes and monovalent salts, produced by micropipette injection, did not induce a noticeable migration of vesicles. The migration distances measured depended on the solution and the cell or vesicle type, and were in the range of several tens of micrometers. The results show that diffusiophoresis of cells and vesicles is a notable phenomenon in a flow-free environment and has to be taken into account when an accurate spatiotemporal control of cells or vesicles in microcavities is required.

GPMVs in variable physiological conditions: could they be used for therapy delivery?

BACKGROUND: Cell based carriers are increasingly recognized as a good system for cargo delivery to cells. One of the reasons is their biocompatibility and low toxicity compared to artificial systems. Giant plasma membrane vesicles (GPMV) derive from the cell plasma membrane. Thus they offer the closest approximation to it, which makes them good candidates for potential drug delivery systems. To evaluate the applicability of GPMVs as carriers, we analyzed their basic biophysical properties to test their robustness in the face of changeable physiological conditions, as well as their ability to translocate across the membrane into cells. RESULTS: GPMVs formed from human umbilical vein endothelial cells (HUVEC) sustain a drastic osmotic challenge (50-500 mOsmoL/kg) unlike giant unilamelar vesicles (GUVs). In hyper-osmotic solutions the average volume decreases and membrane invaginations form, while in the hypo-osmolar buffer the volume of GPMVs increases and these changes were not reversible. The membranes of flaccid GPMVs started to wrinkle unevenly giving rise to buds after exposure to lipopolysaccharide (LPS). The shape changes in GUVs are reversible in contrast to GPMVs after LPS removal. GPMVs exposed to fluorescent LPS exhibited a signal that remained visible in some GPMVs even after LPS removal, which was never the case with GUVs. Calcein penetrated both into GUVs and GPMVs, however after the removal from the bulk solution some of the GPMVs still exhibited very bright signal, while in GUVs only a weak fluorescent signal was detected. We could also see that practically all GPMVs incorporated dextran initially, but after the dextran solution was changed with the initial non-fluorescent solution it remained only in 20% of them. The majority of HUVEC cells displayed a fluorescent signal after the incubation with GPMVs that contained fluorescently labeled dextran. CONCLUSION: Our findings indicate that GPMVs behave quite differently from artificially made giant phospholipid vesicles and the changes induced by the different treatments we subjected them to are not reversible. We also demonstrate that different substances can be both loaded into them and delivered into cells, so GPMVs may be of potential use as cargo/therapy delivery systems.

Curvature-driven lateral segregation of membrane constituents in Golgi cisternae.

Lateral segregation of mobile membrane constituents (e.g. lipids, proteins or membrane domains) into the regions of their preferred curvature relaxes stresses in the membrane. The equilibrium distribution of the constituents in the membrane is thus a balance between the gains in the membrane elastic energy and the segregation-induced loss of entropy. The membrane in the Golgi cisternae is particularly susceptible to the curvature-driven segregation because it possesses two very different curvatures-the highly curved membrane in the cisternal rims and the flat membrane in the cisternal sides. In this work, we calculate the extent of lateral segregation in the Golgi cisternae in the case where the segregation is driven by the Helfrich bending energy. It is assumed that the membrane bending constant and spontaneous curvature depend on the local membrane composition. A simple analytical expression for the extent of the lateral segregation is derived. The results show that the segregation depends on the ratio between the bending constant and the thermal energy, the difference of the preferred curvatures of the constituents and the sizes of the constituents. Applying the model to a typical Golgi cisterna, it was found that entropy can effectively limit the extent of the curvature-driven lateral segregation.

Blastula wall invagination examined on the basis of shape behavior of vesicular objects with laminar envelopes.

A complete description of the blastula shape transformations which occur in the process of gastrulation involves the mechanics of the blastula wall. It is inferred that the mechanical properties of this wall can, to a first approximation, be described in terms of elastic deformational modes which are equivalent to those of simple vesicular systems such as phospholipid vesicles and red blood cells. For membranes composed of an arbitrary number of aligned, laterally unconnected thin layers, these are area expansivity and local and non-local bending. Stable shapes of vesicular objects with laminar envelopes are defined as shapes which correspond to the minimum of the system elastic energy. The shape behavior of these objects is briefly reported. The criterion for the stability of the spherical shape of a vesicular object is derived. In particular we analyze the shape transition from a sphere to an invaginated gastrula. Conditions are demonstrated under which this shape transition can occur through a series of continuous shape transformations. A possible example of such a shape transformation is given. The blastula wall of the sea urchin is modeled as a closed laminar membranous system with three layers, the monocellular epithelial sheet and the extracellular apical lamina and hyaline layers. Experimentally measured elastic constants of some of these layers are shown to be in accord with the restrictions on their values imposed by the requirement for continuous shape transformations.