The spatial resolution limit of phagocytosis.

Antibody-opsonized bacteria interact with Fc receptors in macrophages and trigger signaling cascades, which induce phagocytosis. The signaling pathways ultimately lead to actin polymerization that induces the protrusion of the membrane around the bacterium until it is completely engulfed. Although many proteins involved in the phagocytic cup formation have already been identified, it is still unclear how far the initial stimulus created by the bacterium-cell contact propagates in the cell. We hypothesize that this spreading distance is closely related to the spatial resolution limit of phagocytosis, the smallest distance in which two stimuli can be differentiated. Here, we probe this resolution limit by using holographic optical tweezers to attach pairs of immunoglobulin G-coated polystyrene microparticles (as models for opsonized bacteria) to murine macrophages in distances ranging from zero to several micrometers. By using 2-µm-sized particles, we found that the particles can be internalized jointly into one phagosome if they are attached to the cell very close together, but that they are taken up separately if they are attached far from each other. To explain this, we developed a model of the signaling process, which predicts the probabilities for separate uptake for different particle sizes and distances using cellular parameters including the average receptor distance. We tested the model by measuring the separate uptake probabilities for particles with a diameter of 1 to 3 µm and for cells with reduced numbers of Fcγ receptors and found very good agreement. Our model shows that the phagocytic uptake behavior can be explained by assuming an effective phagocytic signaling range of about 500 nm. Interestingly, this value corresponds to the lower size limit of phagocytosis. Our work provides quantitative access to spatial parameters of cellular signaling during phagocytosis and thereby contributes to a more quantitative understanding of cellular information processing.

Repulsive Interactions of Eco-corona-Covered Microplastic Particles Quantitatively Follow Modeling of Polymer Brushes.

The environmental fate and toxicity of microplastic particles are dominated by their surface properties. In the environment, an adsorbed layer of biomolecules and natural organic matter forms the so-called eco-corona. A quantitative description of how this eco-corona changes the particles' colloidal interactions is still missing. Here, we demonstrate with colloidal probe-atomic force microscopy that eco-corona formation on microplastic particles introduces a compressible film on the surface, which changes the mechanical behavior. We measure single particle-particle interactions and find a pronounced increase of long-range repulsive interactions upon eco-corona formation. These force-separation characteristics follow the Alexander-de Gennes (AdG) polymer brush model under certain conditions. We further compare the obtained fitting parameters to known systems like polyelectrolyte multilayers and propose these as model systems for the eco-corona. Our results show that concepts of fundamental polymer physics, like the AdG model, also help in understanding more complex systems like biomolecules adsorbed to surfaces, i.e., the eco-corona.

Using blinking optical tweezers to study cell rheology during initial cell-particle contact.

Phagocytosis is an important part of innate immunity and describes the engulfment of bacteria and other extracellular objects on the micrometer scale. The protrusion of the cell membrane around the bacteria during this process is driven by a reorganization of the actin cortex. The process has been studied on the molecular level to great extent during the past decades. However, a deep, fundamental understanding of the mechanics of the process is still lacking, in particular because of a lack of techniques that give access to binding dynamics below the optical resolution limit and cellular viscoelasticity at the same time. In this work, we propose a technique to characterize the mechanical properties of cells in a highly localized manner and apply it to investigate the early stages of phagocytosis. The technique can simultaneously resolve the contact region between a cell and an external object (in our application, a phagocytic target) even below the optical resolution limit. We used immunoglobulin-G-coated microparticles with a size of 2 µm as a model system and attached the particles to the macrophages with holographic optical tweezers. By switching the trap on and off, we were able to measure the rheological properties of the cells in a time-resolved manner during the first few minutes after attachment. The measured viscoelastic cellular response is consistent with power law rheology. The contact radius between particle and cell increased on a timescale of ∼30 s and converged after a few minutes. Although the binding dynamics are not affected by cytochalasin D, we observed an increase of the cellular compliance and a significant fluidization of the cortex after addition of cytochalasin D treatment. Furthermore, we report upper boundaries for the length- and timescale, at which cortical actin has been hypothesized to depolymerize during early phagocytosis.

Measuring Stepwise Binding of Thermally Fluctuating Particles to Cell Membranes without Fluorescence.

Thermal motions enable a particle to probe the optimal interaction state when binding to a cell membrane. However, especially on the scale of microseconds and nanometers, position and orientation fluctuations are difficult to observe with common measurement technologies. Here, we show that it is possible to detect single binding events of immunoglobulin-G-coated polystyrene beads, which are held in an optical trap near the cell membrane of a macrophage. Changes in the spatial and temporal thermal fluctuations of the particle were measured interferometrically, and no fluorophore labeling was required. We demonstrate both by Brownian dynamic simulations and by experiments that sequential stepwise increases in the force constant of the bond between a bead and a cell of typically 20 pN/µm are clearly detectable. In addition, this technique provides estimates about binding rates and diffusion constants of membrane receptors. The simple approach of thermal noise tracking points out new strategies in understanding interactions between cells and particles, which are relevant for a large variety of processes, including phagocytosis, drug delivery, and the effects of small microplastics and particulates on cells.

Wrapping of Microparticles by Floppy Lipid Vesicles.

Lipid membranes, the barrier defining living cells and many of their subcompartments, bind to a wide variety of nano- and micrometer sized objects. In the presence of strong adhesive forces, membranes can strongly deform and wrap the particles, an essential step in crossing the membrane for a variety of healthy and disease-related processes. A large body of theoretical and numerical work has focused on identifying the physical properties that underly wrapping. Using a model system of micron-sized colloidal particles and giant unilamellar lipid vesicles with tunable adhesive forces, we measure a wrapping phase diagram and make quantitative comparisons to theoretical models. Our data are consistent with a model of membrane-particle interactions accounting for the adhesive energy per unit area, membrane bending rigidity, particle size, and vesicle radius.

Integrin-induced PIP5K1C kinase polarization regulates neutrophil polarization, directionality, and in vivo infiltration.

Neutrophils are important in innate immunity and acute inflammatory responses. However, the regulation of their recruitment to sites of inflammation has not been well characterized. Here, we investigated the kinase PIP5K1C and showed that PIP5K1C deficiency impaired neutrophil recruitment because of an adhesion defect. PIP5K1C regulated the adhesion through facilitating RhoA GTPase and integrin activation by chemoattractants. Integrins could induce polarization of an isoform of PIP5K1C, PIP5K1C-90, in neutrophils through intracellular vesicle transport independently of exogenous chemoattractant. PIP5K1C-90 polarization was required for polarized RhoA activation at uropods and provided an initial directional cue for neutrophil polarization on the endothelium. Importantly, the polarization was also required for circumventing the inhibition of lamellipodium formation by RhoA so that neutrophils could form leading edges required for transendothelial migration. Because integrins are not known to regulate neutrophil polarization, our study revealed a previously underappreciated role of integrin signaling in neutrophil regulation.

Vancomycin Reduces Cell Wall Stiffness and Slows Swim Speed of the Lyme Disease Bacterium.

Borrelia burgdorferi, the spirochete that causes Lyme disease, is a tick-transmitted pathogen that requires motility to invade and colonize mammalian and tick hosts. These bacteria use a unique undulating flat-wave shape to penetrate and propel themselves through host tissues. Previous mathematical modeling has suggested that the morphology and motility of these spirochetes depends crucially on the flagellar/cell wall stiffness ratio. Here, we test this prediction using the antibiotic vancomycin to weaken the cell wall. We found that low to moderate doses of vancomycin (≤2.0 µg/mL for 24 h) produced small alterations in cell shape and that as the dose was increased, cell speed decreased. Vancomycin concentrations >1.0 µg/mL also inhibited cell growth and led to bleb formation on a fraction of the cells. To quantitatively assess how vancomycin affects cell stiffness, we used optical traps to bend unflagellated mutants of B. burgdorferi. We found that in the presence of vancomycin, cell wall stiffness gradually decreased over time, with a 40% reduction in the bending stiffness after 36 h. Under the same conditions, the swimming speed of wild-type B. burgdorferi slowed by ∼15%, with only marginal changes to cell morphology. Interestingly, our biophysical model for the swimming dynamics of B. burgdorferi suggested that cell speed should increase with decreasing cell stiffness. We show that this discrepancy can be resolved if the periplasmic volume decreases as the cell wall becomes softer. These results provide a testable hypothesis for how alterations of cell wall stiffness affect periplasmic volume regulation. Furthermore, since motility is crucial to the virulence of B. burgdorferi, the results suggest that sublethal doses of antibiotics could negatively impact spirochete survival by impeding their swim speed, thereby enabling their capture and elimination by phagocytes.

Simultaneous measurement of the Young's modulus and the Poisson ratio of thin elastic layers.

The behavior of cells and tissue is greatly influenced by the mechanical properties of their environment. For studies on the interactions between cells and soft matrices, especially those applying traction force microscopy the characterization of the mechanical properties of thin substrate layers is essential. Various techniques to measure the elastic modulus are available. Methods to accurately measure the Poisson ratio of such substrates are rare and often imply either a combination of multiple techniques or additional equipment which is not needed for the actual biological studies. Here we describe a novel technique to measure both parameters, the Youngs's modulus and the Poisson ratio in a single experiment. The technique requires only a standard inverted epifluorescence microscope. As a model system, we chose cross-linked polyacrylamide and poly-N-isopropylacrylamide hydrogels which are known to obey Hooke's law. We place millimeter-sized steel spheres on the substrates which indent the surface. The data are evaluated using a previously published model which takes finite thickness effects of the substrate layer into account. We demonstrate experimentally for the first time that the application of the model allows the simultaneous determination of both the Young's modulus and the Poisson ratio. Since the method is easy to adapt and comes without the need of special equipment, we envision the technique to become a standard tool for the characterization of substrates for a wide range of investigations of cell and tissue behavior in various mechanical environments as well as other samples, including biological materials.

Optical Pushing: A Tool for Parallelized Biomolecule Manipulation.

The ability to measure and manipulate single molecules has greatly advanced the field of biophysics. Yet, the addition of more single-molecule tools that enable one to measure in a parallel fashion is important to diversify the questions that can be addressed. Here we present optical pushing (OP), a single-molecule technique that is used to exert forces on many individual biomolecules tethered to microspheres using a single collimated laser beam. Forces ranging from a few femtoNewtons to several picoNewtons can be applied with a submillisecond response time. To determine forces exerted on the tethered particles by the laser, we analyzed their measured Brownian motion using, to our knowledge, a newly derived analytical model and numerical simulations. In the model, Brownian rotation of the microspheres is taken into account, which proved to be a critical component to correctly determine the applied forces. We used our OP technique to map the energy landscape of the protein-induced looping dynamics of DNA. OP can be used to apply loading rates in the range of 10(-4)-10(6) pN/s to many molecules at the same time, which makes it a tool suitable for dynamic force spectroscopy.

Nominally identical microplastic models differ greatly in their particle-cell interactions.

Due to the abundance of microplastics in the environment, research about its possible adverse effects is increasing exponentially. Most studies investigating the effect of microplastics on cells still rely on commercially available polystyrene microspheres. However, the choice of these model microplastic particles can affect the outcome of the studies, as even nominally identical model microplastics may interact differently with cells due to different surface properties such as the surface charge. Here, we show that nominally identical polystyrene microspheres from eight different manufacturers significantly differ in their ζ-potential, which is the electrical potential of a particle in a medium at its slipping plane. The ζ-potential of the polystyrene particles is additionally altered after environmental exposure. We developed a microfluidic microscopy platform to demonstrate that the ζ-potential determines particle-cell adhesion strength. Furthermore, we find that due to this effect, the ζ-potential also strongly determines the internalization of the microplastic particles into cells. Therefore, the ζ-potential can act as a proxy of microplastic-cell interactions and may govern adverse effects reported in various organisms exposed to microplastics.

Nano- and microplastics: a comprehensive review on their exposure routes, translocation, and fate in humans.

Contamination of the environment with nano-and microplastic particles (NMPs) and its putative adverse effects on organisms, ecosystems, and human health is gaining increasing scientific and public attention. Various studies show that NMPs occur abundantly within the environment, leading to a high likelihood of human exposure to NMPs. Here, different exposure scenarios can occur. The most notable exposure routes of NMPs into the human body are via the airways and gastrointestinal tract (GIT) through inhalation or ingestion, but also via the skin due to the use of personal care products (PCPs) containing NMPs. Once NMPs have entered the human body, it is possible that they are translocated from the exposed organ to other body compartments. In our review article, we combine the current knowledge on the (1) exposure routes of NMPs to humans with the basic understanding of the potential (2) translocation mechanisms into human tissues and, consequently, their (3) fate within the human body. Regarding the (1) exposure routes, we reviewed the current knowledge on the occurrence of NMPs in food, beverages, personal care products and the air (focusing on indoors and workplaces) and found that the studies suggest an abundant presence of MPs within the exposure scenarios. The overall abundance of MPs in exposure matrices relevant to humans highlights the importance of understanding whether NMPs have the potential for tissue translocation. Therefore, we describe the current knowledge on the potential (2) translocation pathways of NMPs from the skin, GIT and respiratory systems to other body compartments. Here, particular attention was paid to how likely NMPs can translocate from the primary exposed organs to secondary organs due to naturally occurring defence mechanisms against tissue translocation. Based on the current understanding, we conclude that a dermal translocation of NMPs is rather unlikely. In contrast, small MPs and NPs can generally translocate from the GIT and respiratory system to other tissues. Thus, we reviewed the existing literature on the (3) fate of NMPs within the human body. Based on the current knowledge of the contamination of human exposure routes and the potential translocation mechanisms, we critically discuss the size of the detected particles reported in the fate studies. In some cases, the particles detected in human tissue samples exceed the size of a particle to overcome biological barriers allowing particle translocation into tissues. Therefore, we emphasize the importance of critically reading and discussing the presented results of NMP in human tissue samples.

Probing the cell membrane by magnetic particle actuation and Euler angle tracking.

The mechanical properties of the cell membrane and the subjacent actin cortex are determinants of a variety of processes in immunity and cell division. The lipid bilayer itself and its connection to the actin cortex are anisotropic. An accurate description of the mechanical structure of the cell membrane and the involved dynamics therefore necessitates a measurement technique that can capture the inherent anisotropy of the system. Here, we combine magnetic particle actuation with rotational and translational particle tracking to simultaneously measure the mechanical stiffness of monocytic cells in three rotational and two translational directions. When using particles that bind via integrins to the cell membrane and the subjacent cortex, we measured an isotropic stiffness and a characteristic power-law dependence of the shear modulus on the applied frequency. When using particles functionalized with immunoglobulin G, we measured an anisotropic stiffness with a 10-fold-reduced value in one dimension. We suggest that the observed reduced stiffness in the plane of the cell membrane is caused by a local detachment of the lipid bilayer from the subjacent cytoskeletal cortex. We expect that our technique will enable new insights into the mechanical properties of the cell membrane that will help us to better understand membrane processes such as phagocytosis and blebbing.

Cell stimulation with optically manipulated microsources.

Molecular gradients are important for various biological processes including the polarization of tissues and cells during embryogenesis and chemotaxis. Investigations of these phenomena require control over the chemical microenvironment of cells. We present a technique to set up molecular concentration patterns that are chemically, spatially and temporally flexible. Our strategy uses optically manipulated microsources, which steadily release molecules. Our technique enables the control of molecular concentrations over length scales down to about 1 microm and timescales from fractions of a second to an hour. We demonstrate this technique by manipulating the motility of single human neutrophils. We induced directed cell polarization and migration with microsources loaded with the chemoattractant formyl-methionine-leucine-phenylalanine. Furthermore, we triggered highly localized retraction of lamellipodia and redirection of polarization and migration with microsources releasing cytochalasin D, an inhibitor of actin polymerization.

In-depth characterization revealed polymer type and chemical content specific effects of microplastic on Dreissena bugensis.

In aquatic ecosystems, filter feeders like mussels are particularly vulnerable to microplastics (MP). However, little is known about how the polymer type and the associated properties (like additives or remaining monomers) of MP impact organisms, as the predominant type of MP used for effect studies on the organismic level are micron grade polystyrene spheres, without considering their chemical composition. Therefore, we exposed the freshwater mussel Dreissena bugensis (D. bugensis) to in-depth characterized fragments in the same concentration and size range (20-120 µm): recycled polyethylene terephthalate from drinking bottles, polyamide, polystyrene, polylactic acid, and mussel shell fragments as natural particle control. Real-time valvometry, used to study behavioral responses via the movement of the mussels' valves, showed that mussels cannot distinguish between natural and MP particles, and therefore do not cease their filtration, as when exposed to dissolved pollutants. This unintentional ingestion led to polymer type-dependent adverse effects (activity of antioxidant enzymes and proteomic alterations), related to chemicals and residual monomers found in MP. Overall, recycled PET elicited the strongest negative effects, likely caused by anthranilamide, anthranilonitrile and butylated hydroxytoluene, contained in the fragments, which are toxic to aquatic organisms. As PET is among the most abundant MP in the environment, sublethal effects may gradually manifest at the population level, leading to irreversible ecosystem changes.

From properties to toxicity: Comparing microplastics to other airborne microparticles.

Microplastic (MP) debris is considered as a potentially hazardous material. It is omnipresent in our environment, and evidence that MP is also abundant in the atmosphere is increasing. Consequently, the inhalation of these particles is a significant exposure route to humans. Concerns about potential effects of airborne MP on human health are rising. However, currently, there are not enough studies on the putative toxicity of airborne MP to adequately assess its impact on human health. Therefore, we examined potential drivers of airborne MP toxicity. Physicochemical properties like size, shape, ζ-potential, adsorbed molecules and pathogens, and the MP's bio-persistence have been proposed as possible drivers of MP toxicity. Since their role in MP toxicity is largely unknown, we reviewed the literature on toxicologically well-studied non-plastic airborne microparticles (asbestos, silica, soot, wood, cotton, hay). We aimed to link the observed health effects and toxicology of these microparticles to the abovementioned properties. By comparing this information with studies on the effects of airborne MP, we analyzed possible mechanisms of airborne MP toxicity. Thus, we provide a basis for a mechanistic understanding of airborne MP toxicity. This may enable the assessment of risks associated with airborne MP pollution, facilitating effective policymaking and product design.

Nanomaterial interactions with biomembranes: Bridging the gap between soft matter models and biological context.

Synthetic polymers, nanoparticles, and carbon-based materials have great potential in applications including drug delivery, gene transfection, in vitro and in vivo imaging, and the alteration of biological function. Nature and humans use different design strategies to create nanomaterials: biological objects have emerged from billions of years of evolution and from adaptation to their environment resulting in high levels of structural complexity; in contrast, synthetic nanomaterials result from minimalistic but controlled design options limited by the authors' current understanding of the biological world. This conceptual mismatch makes it challenging to create synthetic nanomaterials that possess desired functions in biological media. In many biologically relevant applications, nanomaterials must enter the cell interior to perform their functions. An essential transport barrier is the cell-protecting plasma membrane and hence the understanding of its interaction with nanomaterials is a fundamental task in biotechnology. The authors present open questions in the field of nanomaterial interactions with biological membranes, including: how physical mechanisms and molecular forces acting at the nanoscale restrict or inspire design options; which levels of complexity to include next in computational and experimental models to describe how nanomaterials cross barriers via passive or active processes; and how the biological media and protein corona interfere with nanomaterial functionality. In this Perspective, the authors address these questions with the aim of offering guidelines for the development of next-generation nanomaterials that function in biological media.

Control of relative radiation pressure in optical traps: application to phagocytic membrane binding studies.

We show how to control the relative radiation pressure and thereby the stable trap position of an optically trapped bead by variation of the mean incident axial photon momentum. The thermal position fluctuations of a trapped bead are recorded by a three-dimensional back-focal-plane interferometry. The interferometric detection signals are in agreement with predictions based on an extended Mie theory. Depending on the application, the unique and linear range of such a detection system can be optimized by controlling the trap position of the bead. We use this method to investigate in three dimensions the binding of beads to membranes of living cells during phagocytosis. We found that independent of the bead coating (IgG, complement, LPS, avidin) the most frequent initial mechanical response of the cell was a downward pulling of the bead into the cell. The time delay between binding and response was on average 2 s.

A method for time-resolved measurements of the mechanics of phagocytic cups.

The internalization of matter by phagocytosis is of key importance in the defence against bacterial pathogens and in the control of cancerous tumour growth. Despite the fact that phagocytosis is an inherently mechanical process, little is known about the forces and energies that a cell requires for internalization. Here, we use functionalized magnetic particles as phagocytic targets and track their motion while actuating them in an oscillating magnetic field, in order to measure the translational and rotational stiffnesses of the phagocytic cup as a function of time. The measured evolution of stiffness reveals a characteristic pattern with a pronounced peak preceding the finalization of uptake. The measured stiffness values and their time dependence can be interpreted with a model that describes the phagocytic cup as a prestressed membrane connected to an elastically deformable actin cortex. In the context of this model, the stiffness peak is a direct manifestation of a previously described mechanical bottleneck, and a comparison of model and data suggests that the membrane advances around the particle at a speed of about 20 nm s(-1). This approach is a novel way of measuring the progression of emerging phagocytic cups and their mechanical properties in situ and in real time.

Elastic coupling of nascent apCAM adhesions to flowing actin networks.

Adhesions are multi-molecular complexes that transmit forces generated by a cell's acto-myosin networks to external substrates. While the physical properties of some of the individual components of adhesions have been carefully characterized, the mechanics of the coupling between the cytoskeleton and the adhesion site as a whole are just beginning to be revealed. We characterized the mechanics of nascent adhesions mediated by the immunoglobulin-family cell adhesion molecule apCAM, which is known to interact with actin filaments. Using simultaneous visualization of actin flow and quantification of forces transmitted to apCAM-coated beads restrained with an optical trap, we found that adhesions are dynamic structures capable of transmitting a wide range of forces. For forces in the picoNewton scale, the nascent adhesions' mechanical properties are dominated by an elastic structure which can be reversibly deformed by up to 1 µm. Large reversible deformations rule out an interface between substrate and cytoskeleton that is dominated by a number of stiff molecular springs in parallel, and favor a compliant cross-linked network. Such a compliant structure may increase the lifetime of a nascent adhesion, facilitating signaling and reinforcement.