Critical role of lipid membranes in polarization and migration of cells: a biophysical view.

Cell migration plays vital roles in many biologically relevant processes such as tissue morphogenesis and cancer metastasis, and it has fascinated biophysicists over the past several decades. However, despite an increasing number of studies highlighting the orchestration of proteins involved in different signaling pathways, the functional roles of lipid membranes have been essentially overlooked. Lipid membranes are generally considered to be a functionless two-dimensional matrix of proteins, although many proteins regulating cell migration gain functions only after they are recruited to the membrane surface and self-organize their functional domains. In this review, we summarize how the logistical recruitment and release of proteins to and from lipid membranes coordinates complex spatiotemporal molecular processes. As predicted from the classical framework of the Smoluchowski equation of diffusion, lipid/protein membranes serve as a 2D reaction hub that contributes to the effective and robust regulation of polarization and migration of cells involving several competing pathways.

Viscoelasticity of single cells-from subcellular to cellular level.

This review deals with insights into complex cellular structures and processes obtained by measuring viscoelastic impedances of the cell envelope and the cytoplasm by colloidal bead microrheometry. I first introduce a mechanical cell model that allows us to understand their unique ability of mechanical self-stabilization by actin microtubule crosstalk. In the second part, I show how cell movements can be driven by pulsatile or propagating solitary actin gelatin waves (SAGW) that are generated on nascent adhesion domains by logistically controlled membrane recruitment of functional proteins by electrostatic-hydrophobic forces. The global polarization of cell migration is guided by actin-microtubule crosstalk that is mediated by the Ca++ and strain-sensitive supramolecular scaffolding protein IQGAP. In the third part, I introduce the traction force microscopy as a tool to measure the forces between somatic cells and the tissue ´Here I show, how absolute values of viscoelastic impedances of the composite cell envelope can be obtained by deformation field mapping techniques. In the fourth part, it is shown how the dynamic mechanical properties of the active viscoplastic cytoplasmic space can be evaluated using colloidal beads as phantom endosomes. Separate measurements of velocity distributions of directed and random motions of phantom endosomes, yield local values of transport forces, viscosities and life times of directed motion along microtubules. The last part deals with biomimetic experiments allowing us to quantitatively evaluate the mechanical properties of passive and active actin networks on the basis of the percolation theory of gelation.

Probing the Interface Structure of Adhering Cells by Contrast Variation Neutron Reflectometry.

Cellular adhesion is a central element in tissue mechanics, biological cell-cell signaling, and cell motility. In this context, the cell-substrate distance has been investigated in the past by studying natural cells and biomimetic cell models adhering on solid substrates. The amount of water in the membrane substrate gap, however, is difficult to determine. Here, we present a neutron reflectivity (NR) structural study of confluent epithelial cell monolayers on silicon substrates. In order to ensure valid in vitro conditions, we developed a cell culture sample chamber allowing us to grow and cultivate cells under proper cell culture conditions while performing in vitro neutron reflectivity measurements. The cell chamber also enabled perfusion with cell medium and hence allowed for contrast variation in situ by sterile exchange of buffer with different H2O-to-D2O ratio. Contrast variation reduces the ambiguity of data modeling for determining the thickness and degree of hydration of the interfacial cleft between the adherent cells and the substrate. Our data suggest a three-layer interfacial organization. The first layer bound to the silicon surface interface is in agreement with a very dense protein film with a thickness of 9 ± 2 nm, followed by a highly hydrated 24 ± 4 nm thick layer, and a several tens of nanometers thick layer attributed to the composite membrane. Hence, the results provide clear evidence of a highly hydrated intermediate region between the composite cell membrane and the substrate, reminiscent of the basal lamina.

How actin/myosin crosstalks guide the adhesion, locomotion and polarization of cells.

Cell-tissue-tissue interaction is determined by specific short range forces between cell adhesion molecules (CAMs) and ligands of the tissue, long range repulsion forces mediated by cell surface grafted macromolecules and adhesion-induced elastic stresses in the cell envelope. This interplay of forces triggers the rapid random clustering of tightly coupled linkers. By coupling of actin gel patches to the intracellular domains of the CAMs, these clusters can grow in a secondary process resulting in the formation of functional adhesion microdomains (ADs). The ADs can act as biochemical steering centers by recruiting and activating functional proteins, such as GTPases and associated regulating proteins, through electrostatic-hydrophobic forces with cationic lipid domains that act as attractive centers. First, I summarize physical concepts of cell adhesion revealed by studies of biomimetic systems. Then I describe the role of the adhesion domains as biochemical signaling platforms and force transmission centers promoting cellular protrusions, in terms of a shell string model of cells. Protrusion forces are generated by actin gelation triggered by molecular machines (focal adhesion kinase (FAK), Src-kinases and associated adaptors) which assemble around newly formed integrin clusters. They recruit and activate the GTPases Rac-1 and actin gelation promoters to charged membrane domains via electrostatic-hydrophobic forces. The cell front is pushed forward in a cyclic and stepwise manner and the step-width is determined by the dynamics antagonistic interplay between Rac-1 and RhoA. The global cell polarization in the direction of motion is mediated by the actin-microtubule (MT) crosstalk at adhesion domains. Supramolecular actin-MT assemblies at the front help to promote actin polymerization. At the rear they regulate the dismantling of the ADs through the Ca(++)-mediated activation of the protease calpain and trigger their disruption by RhoA mediated contraction via stress fibers. This article is part of a Special Issue entitled: Mechanobiology.

Association rates of membrane-coupled cell adhesion molecules.

Thus far, understanding how the confined cellular environment affects the lifetime of bonds, as well as the extraction of complexation rates, has been a major challenge in studies of cell adhesion. Based on a theoretical description of the growth curves of adhesion domains, we present a new (to our knowledge) method to measure the association rate k(on) of ligand-receptor pairs incorporated into lipid membranes. As a proof of principle, we apply this method to several systems. We find that the k(on) for the interaction of biotin with neutravidin is larger than that for integrin binding to RGD or sialyl Lewis(x) to E-selectin. Furthermore, we find k(on) to be enhanced by membrane fluctuations that increase the probability for encounters between the binders. The opposite effect on k(on) could be attributed to the presence of repulsive polymers that mimic the glycocalyx, which points to two potential mechanisms for controlling the speed of protein complexation during the cell recognition process.

Endoplasmatic reticulum shaping by generic mechanisms and protein-induced spontaneous curvature.

The endoplasmatic reticulum (ER) comprises flattened vesicles (cisternae) with worm holes dubbed with ribosomes coexisting with a network of interconnected tubes which can extend to the cell periphery or even penetrate nerve axons. The coexisting topologies enclose a continuous luminal space. The complex ER topology is specifically controlled by a group of ER-shaping proteins often called reticulons (discovered by the group of Tom Rapoport). They include atlastin, reticulon, REEP and the MT severing protein spastin. A generic ER shape controlling factor is the necessity to maximize the area-to-volume ratio of ER membranes in the highly crowded cytoplasmic space. I present a model of the ER-shaping function of the reticulons based on the Helfrich bending elasticity concept of soft shell shape changes. Common structural motifs of the reticulons are hydrophobic sequences forming wedge shaped hairpins which penetrate the lipid bilayer of the cell membranes. The wedge-like hydrophobic anchors can both induce the high curvature of the tubular ER fraction and ensure the preferred distribution of the reticulons along the tubules. Tubular junctions may be stabilized by the reticulons forming two forceps twisted by 90°. The ER extensions to the cell periphery and the axons are mediated by coupling of the tubes to the microtubules which is mediated by REEP and spastin. At the end I present a model of the tension driven homotype fusion of ER-membranes by atlastin, based on analogies to the SNARE-complexin-SNARE driven heterotype fusion process.

Physics of cell adhesion: some lessons from cell-mimetic systems.

Cell adhesion is a paradigm of the ubiquitous interplay of cell signalling, modulation of material properties and biological functions of cells. It is controlled by competition of short range attractive forces, medium range repellant forces and the elastic stresses associated with local and global deformation of the composite cell envelopes. We review the basic physical rules governing the physics of cell adhesion learned by studying cell-mimetic systems and demonstrate the importance of these rules in the context of cellular systems. We review how adhesion induced micro-domains couple to the intracellular actin and microtubule networks allowing cells to generate strong forces with a minimum of attractive cell adhesion molecules (CAMs) and to manipulate other cells through filopodia over micrometer distances. The adhesion strength can be adapted to external force fluctuations within seconds by varying the density of attractive and repellant CAMs through exocytosis and endocytosis or protease-mediated dismantling of the CAM-cytoskeleton link. Adhesion domains form local end global biochemical reaction centres enabling the control of enzymes. Actin-microtubule crosstalk at adhesion foci facilitates the mechanical stabilization of polarized cell shapes. Axon growth in tissue is guided by attractive and repulsive clues controlled by antagonistic signalling pathways.

On the mechanical stabilization of filopodia.

We studied force-induced elongation of filopodia by coupling magnetic tweezers to the tip through the bacterial coat protein invasin, which couples the force generator to the actin bundles (through myosin X), thus impeding the growth of the actin plus end. Single force pulses (15-30 s) with amplitudes between 20 and 600 pN and staircase-like force scenarios (amplitudes, ∼50 pN; step widths, 30 s) were applied. In both cases, the responses consist of a fast viscoelastic deflection followed by a linear flow regime. The deflections are reversible after switching off the forces, suggesting a mechanical memory. The elongation velocity exhibits an exponential distribution (half-width , ∼0.02 µm s(-1)) and did not increase systematically with the force amplitudes. We estimate the bending modulus (0.4 × 10(-23) J m) and the number of actin filaments (∼10) by analyzing filopodium bending fluctuations. Sequestering of intracellular Ca(2+) by BAPTA caused a strong reduction in the amplitude of elongation, whereas latrunculin A resulted in loss of the elastic response. We attribute the force-independent velocity to the elongation of actin bundles enabled by the force-induced actin membrane uncoupling and the reversibility by the treadmilling mechanism and an elastic response.

Progress in mimetic studies of cell adhesion and the mechanosensing.

Vesicle-substrate adhesion has been studied for over two decades with the motivation to understand and mimic cell adhesion. In recent years, with progress in theoretical modelling, the development of experimental techniques, and improved data-analysis procedures, considerable advances have been made in the understanding of the adhesion process. It is this progress which constitutes the focus of this review.

Dynamics of specific vesicle-substrate adhesion: from local events to global dynamics.

We present a synergistic combination of simulations and experimental data on the dynamics of membrane adhesion. We show that a change in either the density or the strength of the bonds results in very different dynamics. Such behavior is explained by introducing an effective binding affinity that emerges as a result of the competition between the strength of the chemical bonds and the environment defined by the fluctuating membrane.

Temporal analysis of active and passive transport in living cells.

The cellular cytoskeleton is a fascinating active network, in which Brownian motion is intercepted by distinct phases of active transport. We present a time-resolved statistical analysis dissecting phases of directed motion out of otherwise diffusive motion of tracer particles in living cells. The distribution of active lifetimes is found to decay exponentially with a characteristic time tauA = 0.65 s. The velocity distribution of active events exhibits several peaks, in agreement with a discrete number of motor proteins acting collectively.

Force-induced growth of adhesion domains is controlled by receptor mobility.

In living cells, adhesion structures have the astonishing ability to grow and strengthen under force. Despite the rising evidence of the importance of this phenomenon, little is known about the underlying mechanism. Here, we show that force-induced adhesion-strengthening can occur purely because of the thermodynamic response to the elastic deformation of the membrane, even in the absence of the actively regulated cytoskeleton of the cell, which was hitherto deemed necessary. We impose pN-forces on two fluid membranes, locally pre-adhered by RGD-integrin binding. One of the binding partners is always mobile whereas the mobility of the other can be switched on or off. Immediate passive strengthening of adhesion structures occurs in both cases. When both binding partners are mobile, strengthening is aided by lateral movement of intact bonds as a transient response to force-induced membrane-deformation. By extending our microinterferometric technique to the suboptical regime, we show that the adhesion, as well as the resistance to force-induced de-adhesion, is greatly enhanced when both, rather than only one, of the binding partners are mobile. We formulate a theory that explains our observations by linking the macroscopic shape deformation with the microscopic formation of bonds, which further elucidates the importance of receptor mobility. We propose this fast passive response to be the first-recognition that triggers signaling events leading to mechanosensing in living cells.

Adhesion of giant vesicles mediated by weak binding of sialyl-LewisX to E-selectin in the presence of repelling poly(ethylene glycol) molecules.

Prior to establishing tight contact with the endothelium, cells such as leukocytes or cancer cells use the recognition between sialyl-LewisX ligands and E-selectin receptors to establish weak, reversible adhesion and to roll along the vessel wall. We study the physical aspects of this process by constructing a mimetic system that consists of a giant fluid vesicle with incorporated lipid-anchored sialyl-LewisX molecules that bind to E-selectin that is immobilized on the flat substrate. The vesicles also carry a certain fraction of repelling PEG2000 molecules. We analyze the equilibrium state of adhesion in detail by means of reflection interference contrast microscopy and find that the adhesion process relies purely on the formation of one or more adhesion domains within the vesicle-substrate contact zone. We find that the content of ligands in the vesicle must be above 5 mol % to establish specific contacts. All concentrations of sialyl-LewisX above 8 mol % provide a very similar final state of adhesion. However, the size and shape of the adhesion domains strongly depend on both the concentrations of E-selectin (0-3500 molecules/microm2) and PEG2000 (0-5 mol %). At 3500 E-selectin molecules/microm2 and small concentrations of PEG2000, the vesicle-substrate contact is maximized and fully occupied by a single adhesion domain. At concentrations of 5 mol %, PEG2000 completely impedes the specific binding to any substrate. Lastly, an increase in the adhesion strength is observed in systems with identical compositions if the reduced volume of the vesicles is larger.

Biomimetic models of the actin cytoskeleton.

The cytoskeleton is a complex polymer network that plays an essential role in the functionality of eukaryotic cells. It endows cells with mechanical stability, adaptability, and motility. To identify and understand the mechanisms underlying this large variety of capabilities and to possibly transfer them to engineered networks makes it necessary to have in vitro and in silico model systems of the cytoskeleton. These models must be realistic representatives of the cellular network and at the same time be controllable and reproducible. Here, an approach to design complementary experimental and numerical model systems of the actin cytoskeleton is presented and some of their properties discussed.

Control of frictional coupling of transmembrane cell receptors in model cell membranes with linear polymer spacers.

The planar plasma membrane model with linear polymer spacers with defined lengths enables the control of the frictional coupling between incorporated transmembrane proteins (human platelet integrin) and the solid substrate. This mimics the viscous environment provided by the extracellular matrix of cells. The friction coefficient can be calculated quantitatively from the diffusion coefficient of integrin, measured by fluorescence recovery after photobleaching. The obtained results demonstrate a clear influence of the length and lateral density of polymer chains on the mobility of transmembrane proteins.

Structural characterization of an elevated lipid bilayer obtained by stepwise functionalization of a self-assembled alkenyl silane film.

This work reports a novel tethered lipid membrane supported on silicon oxide providing an improved model cell membrane. There is an increasing need for robust solid supported fluid model membranes that can be easily deposited on soft cushions. In such architecture the space between the membrane and the substrate should be tunable in the nanometer range. For this purpose a SiO(2) surface was functionalized with poly(ethylene glycol) (PEG)-lipid tethers and further modified with poly(ethylene glycol) making a biologically passivated substrate available for lipid bilayer deposition. First, a short chain self-assembled alkenyl silane film was oxidized to yield terminal COOH groups and then functionalized with amino-terminated PEG-lipids via N-hydroxysuccinimide chemistry. The functionalized silane film was then additionally passivated by functionalization of unreacted COOH groups with amino-terminated PEG of variable chain length. X-ray photoelectron spectroscopy (XPS) analysis of dry films, carried out near the C 1s ionization edge to characterize chemical groups formed in the near-surface region, confirmed binding of PEG-lipid tethers to the silane film. XPS further indicated that backfilling with PEG caused the lipid tails to stick up above the PEG layer which was confirmed by the x-ray reflectivity measurements. Lipid vesicle fusion on these surfaces in the presence of excess water resulted in the formation of supported membranes characterized by very high homogeneity and long range mobility, as confirmed by fluorescence bleaching experiments. Even after repeated drying-hydrating cycles, these robust surfaces provided good templates for high fluidity elevated membranes. X-ray reflectivity measurements of the tethered membranes, with a resolution of 0.6 nm in water, showed that these fluid membranes are elevated up to 8 nm above the silicon oxide surface.

Polymer-tethered membranes as quantitative models for the study of integrin-mediated cell adhesion.

Here we report a remarkable enhancement in the adhesion strength of transmembrane cell receptors, human platelet integrin, in a new class of supported lipid membranes, which are separated from the solid substrates by linear polymer spacers. The amphiphilic polymer tether consists of linear hydrophilic poly(2-oxazoline) chains of defined length (degree of polymerization n = 104, MW/Mn = 1.30), whose chain termini are functionalized with the tri-functional silane surface coupling group and hydrophobic n-alkyl chains as membrane anchors (lipopolymers). As a model of test cells, giant lipid vesicles were functionalized with synthetic ligand molecules containing the RGD sequence, and the free energy of adhesion Δgad between the integrin-doped tethered membrane and the vesicle was measured using a micro-interferometry technique. It has been demonstrated that the adhesion function of integrin receptors in these polymer-tethered membranes is 30 times stronger than those incorporated into membranes directly deposited onto solid substrates (solid-supported membranes). The obtained results demonstrate that linear lipopolymer spacers provide a fluid and non-denaturing environment for the incorporated cell receptors and allow quantitative modelling of cell adhesion processes.

Active mechanical stabilization of the viscoplastic intracellular space of Dictyostelia cells by microtubule-actin crosstalk.

The micro-viscoelasticity of the intracellular space of Dictyostelium discoideum cells is studied by evaluating the intracellular transport of magnetic force probes and their viscoelastic responses to force pulses of 20-700 pN. The role of the actin cortex, the microtubule (MT) aster and their crosstalk is explored by comparing the behaviour of wild-type cells, myosin II null mutants, latrunculin A and benomyl treated cells. The MT coupled beads perform irregular local and long range directed motions which are characterized by measuring their velocity distributions (P(v)). The correlated motion of the MT and the centrosome are evaluated by microfluorescence of GFP-labelled MTs. P(v) can be represented by log-normal distributions with long tails and it is determined by random sweeping motions (v approximately 0.5 microm/s) of the MTs (caused by tangential forces on the filament ends coupled to the actin cortex) and by intermittent bead transports parallel to the MTs (v(max) approximately 1.5 microm/s). The tails are due to spontaneous filament deflections (with speeds up to 10 microm/s) attributed to pre-stressing of the MT by local cortical tensions, generated by dynactin motors generating plus-end directed forces in the MTs. The viscoelastic responses are strongly non-linear and are mostly directed opposite or perpendicular to the force, showing that the cytoplasm behaves as an active viscoplastic body with time and force dependent drag coefficients. Nano-Newton loads exerted on the soft MT are balanced by traction forces arising at the MT ends coupled to the actin cortex and the centrosome, respectively. The mechanical coupling between the soft microtubules and the viscoelastic actin cortex provides cells with high mechanical stability despite the softness of the cytoplasm.

Coupling artificial actin cortices to biofunctionalized lipid monolayers.

We report the assembly of protein supramolecular structures at an air-water interface and coupling of artificial actin cortices to such structures. The coupling strategies adopted include electrostatic binding of actin to monolayers doped with lipids, exposing positively charged poly(ethylene glycol) headgroups; binding of biotinylated actin to lipids carrying biotin headgroups through avidin; binding of actin to membranes through biotinylated hisactophilin (a cellular actin-membrane coupler) using an avidin-biotin linkage; and coupling of actin to membranes carrying chelating lipids through a 15-nm-diameter protein capsid (bacterial lumazine synthase or LuSy) exhibiting histidine tags (which bind both to actin and to the chelating lipid). The distribution of the proteins in a direction normal to the interface was measured by neutron reflectivity under different conditions of pH and ionic strength. In the case of the first three binding methods, the thickness of the actin film was found to correspond to a single actin filament. Multilayers of actin could be formed only by using the multifunctional LuSy couplers that exhibit 60 hexahistidine tags and can thus act as actin cross-linkers. The LuSy-mediated binding can be reversibly switched by pH variations.