Dynamics of phagocytosis mediated by phosphatidylserine.

Phagocytosis triggered by the phospholipid phosphatidylserine (PS) is key for the removal of apoptotic cells in development, tissue homeostasis and infection. Modulation of PS-mediated phagocytosis is an attractive target for therapeutic intervention in the context of atherosclerosis, neurodegenerative disease, and cancer. Whereas the mechanisms of target recognition, lipid and protein signalling, and cytoskeletal remodelling in opsonin-driven modes of phagocytosis are increasingly well understood, PS-mediated phagocytosis has remained more elusive. This is partially due to the involvement of a multitude of receptors with at least some redundancy in functioning, which complicates dissecting their contributions and results in complex downstream signalling networks. This review focusses on the receptors involved in PS-recognition, the signalling cascades that connect receptors to cytoskeletal remodelling required for phagocytosis, and recent progress in our understanding of how phagocytic cup formation is coordinated during PS-mediated phagocytosis.

Nanomechanics of Extracellular Vesicles Reveals Vesiculation Pathways.

Extracellular vesicles (EVs) are emerging as important mediators of cell-cell communication as well as potential disease biomarkers and drug delivery vehicles. However, the mechanical properties of these vesicles are largely unknown, and processes leading to microvesicle-shedding from the plasma membrane are not well understood. Here an in depth atomic force microscopy force spectroscopy study of the mechanical properties of natural EVs is presented. It is found that several natural vesicles of different origin have a different composition of lipids and proteins, but similar mechanical properties. However, vesicles generated by red blood cells (RBC) at different temperatures/incubation times are different mechanically. Quantifying the lipid content of EVs reveals that their stiffness decreases with the increase in their protein/lipid ratio. Further, by maintaining RBC at "extreme" nonphysiological conditions, the cells are pushed to utilize different vesicle generation pathways. It is found that RBCs can generate protein-rich soft vesicles, possibly driven by protein aggregation, and low membrane-protein content stiff vesicles, likely driven by cytoskeleton-induced buckling. Since similar cortical cytoskeleton to that of the RBC exists on the membranes of most mammalian cells, our findings help advancing the understanding of the fundamental process of vesicle generation.

The role of the cytoskeleton in sensing changes in gravity by nonspecialized cells.

A large body of evidence indicates that single cells in vitro respond to changes in gravity, and that this response might play an important role for physiological changes at the organism level during spaceflight. Gravity can lead to changes in cell proliferation, differentiation, signaling, and gene expression. At first glance, gravitational forces seem too small to affect bodies with the size of a cell. Thus, the initial response to gravity is both puzzling and important for understanding physiological changes in space. This also offers a unique environment to study the mechanical response of cells. In the past 2 decades, important steps have been made in the field of mechanobiology, and we use these advances to reevaluate the response of single cells to changes in gravity. Recent studies have focused on the cytoskeleton as initial gravity sensor. Thus, we review the observed changes in the cytoskeleton in a microgravity environment, both during spaceflight and in ground-based simulation techniques. We also evaluate to what degree the current experimental evidence supports the cytoskeleton as primary gravity sensor. Finally, we consider how the cytoskeleton itself could be affected by changed gravity. To make the next step toward understanding the response of cells to altered gravity, the challenge will be to track changes quantitatively and on short timescales.

Tim4 enables large peritoneal macrophages to cross-present tumor antigens at early stages of tumorigenesis.

Receptors controlling the cross-presentation of tumor antigens by macrophage subsets in cancer tissues are poorly explored. Here, we show that TIM4+ large peritoneal macrophages efficiently capture and cross-present tumor-associated antigens at early stages of peritoneal infiltration by ovarian cancer cells. The phosphatidylserine (PS) receptor TIM4 promotes maximal uptake of dead cells or PS-coated artificial targets and triggers inflammatory and metabolic gene programs in combination with cytoskeletal remodeling and upregulation of transcriptional signatures related to antigen processing. At the cellular level, TIM4-mediated engulfment induces nucleation of F-actin around nascent phagosomes, delaying the recruitment of vacuolar ATPase, acidification, and cargo degradation. In vivo, TIM4 deletion blunts induction of early anti-tumoral effector CD8 T cells and accelerates the progression of ovarian tumors. We conclude that TIM4-mediated uptake drives the formation of specialized phagosomes that prolong the integrity of ingested antigens and facilitate cross-presentation, contributing to immune surveillance of the peritoneum.

ß2 integrins impose a mechanical checkpoint on macrophage phagocytosis.

Phagocytosis is an intensely physical process that depends on the mechanical properties of both the phagocytic cell and its chosen target. Here, we employed differentially deformable hydrogel microparticles to examine the role of cargo rigidity in the regulation of phagocytosis by macrophages. Whereas stiff cargos elicited canonical phagocytic cup formation and rapid engulfment, soft cargos induced an architecturally distinct response, characterized by filamentous actin protrusions at the center of the contact site, slower cup advancement, and frequent phagocytic stalling. Using phosphoproteomics, we identified ß2 integrins as critical mediators of this mechanically regulated phagocytic switch. Macrophages lacking ß2 integrins or their downstream effectors, Talin1 and Vinculin, exhibited specific defects in phagocytic cup architecture and selective suppression of stiff cargo uptake. We conclude that integrin signaling serves as a mechanical checkpoint during phagocytosis to pair cargo rigidity to the appropriate mode of engulfment.

N2FXm, a method for joint nuclear and cytoplasmic volume measurements, unravels the osmo-mechanical regulation of nuclear volume in mammalian cells.

In eukaryotes, cytoplasmic and nuclear volumes are tightly regulated to ensure proper cell homeostasis. However, current methods to measure cytoplasmic and nuclear volumes, including confocal 3D reconstruction, have limitations, such as relying on two-dimensional projections or poor vertical resolution. Here, to overcome these limitations, we describe a method, N2FXm, to jointly measure cytoplasmic and nuclear volumes in single cultured adhering human cells, in real time, and across cell cycles. We find that this method accurately provides joint size over dynamic measurements and at different time resolutions. Moreover, by combining several experimental perturbations and analyzing a mathematical model including osmotic effects and tension, we show that N2FXm can give relevant insights on how mechanical forces exerted by the cytoskeleton on the nuclear envelope can affect the growth of nucleus volume by biasing nuclear import. Our method, by allowing for accurate joint nuclear and cytoplasmic volume dynamic measurements at different time resolutions, highlights the non-constancy of the nucleus/cytoplasm ratio along the cell cycle.

Single-cell topographical profiling of the immune synapse reveals a biomechanical signature of cytotoxicity.

Immune cells have intensely physical lifestyles characterized by structural plasticity and force exertion. To investigate whether specific immune functions require stereotyped mechanical outputs, we used super-resolution traction force microscopy to compare the immune synapses formed by cytotoxic T cells with contacts formed by other T cell subsets and by macrophages. T cell synapses were globally compressive, which was fundamentally different from the pulling and pinching associated with macrophage phagocytosis. Spectral decomposition of force exertion patterns from each cell type linked cytotoxicity to compressive strength, local protrusiveness, and the induction of complex, asymmetric topography. These features were validated as cytotoxic drivers by genetic disruption of cytoskeletal regulators, live imaging of synaptic secretion, and in silico analysis of interfacial distortion. Synapse architecture and force exertion were sensitive to target stiffness and size, suggesting that the mechanical potentiation of killing is biophysically adaptive. We conclude that cellular cytotoxicity and, by implication, other effector responses are supported by specialized patterns of efferent force.

Topographical analysis of immune cell interactions reveals a biomechanical signature for immune cytolysis.

Immune cells live intensely physical lifestyles characterized by structural plasticity, mechanosensitivity, and force exertion. Whether specific immune functions require stereotyped patterns of mechanical output, however, is largely unknown. To address this question, we used super-resolution traction force microscopy to compare cytotoxic T cell immune synapses with contacts formed by other T cell subsets and macrophages. T cell synapses were globally and locally protrusive, which was fundamentally different from the coupled pinching and pulling of macrophage phagocytosis. By spectrally decomposing the force exertion patterns of each cell type, we associated cytotoxicity with compressive strength, local protrusiveness, and the induction of complex, asymmetric interfacial topographies. These features were further validated as cytotoxic drivers by genetic disruption of cytoskeletal regulators, direct imaging of synaptic secretory events, and in silico analysis of interfacial distortion. We conclude that T cell-mediated killing and, by implication, other effector responses are supported by specialized patterns of efferent force.

F-actin organization and target constriction during primary macrophage phagocytosis is balanced by competing activity of myosin-I and myosin-II.

Phagocytosis requires rapid remodeling of the actin cytoskeleton for extension of membrane protrusions and force generation to ultimately drive the engulfment of targets. The detailed mechanisms of phagocytosis have almost exclusively been studied in immortalized cell lines. Here, we make use of high-resolution imaging and novel biophysical approaches to determine the structural and mechanical features of phagocytosis by primary bone marrow-derived macrophages. We find that the signature behavior of these primary cells is distinct from macrophage-like cell lines; specifically, it is gentle, with only weak target constriction and modest polarization of the F-actin distribution inside the phagocytic cup. We show that long-tailed myosins 1e/f are critical for this organization. Deficiency of myo1e/f causes dramatic shifts in F-actin localization, reducing F-actin at the phagocytic cup base and enhancing F-actin-mediated constriction at the cup rim. Surprisingly, these changes can be almost fully reverted upon inhibition of another myosin motor protein, myosin-II. Hence, we show that the biomechanics and large-scale organization of phagocytic cups is tightly regulated through competing contributions from myosin-Ie/f and myosin-II.

Phagocytic 'teeth' and myosin-II 'jaw' power target constriction during phagocytosis.

Phagocytosis requires rapid actin reorganization and spatially controlled force generation to ingest targets ranging from pathogens to apoptotic cells. How actomyosin activity directs membrane extensions to engulf such diverse targets remains unclear. Here, we combine lattice light-sheet microscopy (LLSM) with microparticle traction force microscopy (MP-TFM) to quantify actin dynamics and subcellular forces during macrophage phagocytosis. We show that spatially localized forces leading to target constriction are prominent during phagocytosis of antibody-opsonized targets. This constriction is largely driven by Arp2/3-mediated assembly of discrete actin protrusions containing myosin 1e and 1f ('teeth') that appear to be interconnected in a ring-like organization. Contractile myosin-II activity contributes to late-stage phagocytic force generation and progression, supporting a specific role in phagocytic cup closure. Observations of partial target eating attempts and sudden target release via a popping mechanism suggest that constriction may be critical for resolving complex in vivo target encounters. Overall, our findings present a phagocytic cup shaping mechanism that is distinct from cytoskeletal remodeling in 2D cell motility and may contribute to mechanosensing and phagocytic plasticity.

A mechanical perspective on phagocytic cup formation.

Phagocytosis is a widespread and evolutionarily conserved process with diverse biological functions, ranging from engulfment of invading microbes during infection to clearance of apoptotic debris in tissue homeostasis. Along with differences in biochemical composition, phagocytic targets greatly differ in physical attributes, such as size, shape, and rigidity, which are now recognized as important regulators of this process. Force exertion at the cell-target interface and cellular mechanical changes during phagocytosis are emerging as crucial factors underlying sensing of such target properties. With technological developments, mechanical aspects of phagocytosis are increasingly accessible experimentally, revealing remarkable organizational complexity of force exertion. An increasingly high-resolution picture is emerging of how target physical cues and cellular mechanical properties jointly govern important steps throughout phagocytic engulfment.

Mechanical Characterization of Liposomes and Extracellular Vesicles, a Protocol.

Both natural as well as artificial vesicles are of tremendous interest in biology and nanomedicine. Small vesicles (<200 nm) perform essential functions in cell biology and artificial vesicles (liposomes) are used as drug delivery vehicles. Atomic Force Microscopy (AFM) is a powerful technique to study the structural properties of these vesicles. AFM is a well-established technique for imaging at nanometer resolution and for mechanical measurements under physiological conditions. Here, we describe the procedure of AFM imaging and force spectroscopy on small vesicles. We discuss how to image vesicles with minimal structural disturbance, and how to analyze the data for accurate size and shape measurements. In addition, we describe the procedure for performing nanoindentations on vesicles and the subsequent data analysis including mechanical models used for data interpretation.

Microparticle traction force microscopy reveals subcellular force exertion patterns in immune cell-target interactions.

Force exertion is an integral part of cellular behavior. Traction force microscopy (TFM) has been instrumental for studying such forces, providing spatial force measurements at subcellular resolution. However, the applications of classical TFM are restricted by the typical planar geometry. Here, we develop a particle-based force sensing strategy for studying cellular interactions. We establish a straightforward batch approach for synthesizing uniform, deformable and tuneable hydrogel particles, which can also be easily derivatized. The 3D shape of such particles can be resolved with superresolution (<50 nm) accuracy using conventional confocal microscopy. We introduce a reference-free computational method allowing inference of traction forces with high sensitivity directly from the particle shape. We illustrate the potential of this approach by revealing subcellular force patterns throughout phagocytic engulfment and force dynamics in the cytotoxic T-cell immunological synapse. This strategy can readily be adapted for studying cellular forces in a wide range of applications.

Multilamellar nanovesicles show distinct mechanical properties depending on their degree of lamellarity.

Small multilamellar vesicles may have benefits over unilamellar vesicles for drug delivery, such as an increased volume for hydrophobic drugs. In addition, their altered mechanical properties might be beneficial for cellular uptake. Here, we show how atomic force microscopy (AFM) can be used to detect and characterize multilamellar vesicles. We quantify the size of each break event occurring during AFM nanoindentations, which shows good agreement with the thickness of supported lipid bilayers. Analyzing the size and number of these events for individual vesicles allows us to distinguish between vesicles consisting of 1 up to 5 bilayers. We validate these results by comparison with correlative cryo-electron microscopy (cryo-EM) data at the vesicle population level. Finally, we quantify the vesicle geometry and mechanical properties, and show that with additional bilayers adherent vesicles are more spherical and stiffer. Surprisingly, at ∼20% stiffening for each additional bilayer, the vesicle stiffness scales only weakly with lamellarity. Our results show the potential of AFM for studying liposomal nanoparticles and suggest that small multilamellar vesicles may have beneficial mechanical properties for cellular uptake.

The fluid membrane determines mechanics of erythrocyte extracellular vesicles and is softened in hereditary spherocytosis.

Extracellular vesicles (EVs) are widely studied regarding their role in cell-to-cell communication and disease, as well as for applications as biomarkers or drug delivery vehicles. EVs contain membrane and intraluminal proteins, affecting their structure and thereby likely their functioning. Here, we use atomic force microscopy for mechanical characterization of erythrocyte, or red blood cell (RBC), EVs from healthy individuals and from patients with hereditary spherocytosis (HS) due to ankyrin deficiency. While these EVs are packed with proteins, their response to indentation resembles that of fluid liposomes lacking proteins. The bending modulus of RBC EVs of healthy donors is ~15 kbT, similar to the RBC membrane. Surprisingly, whereas RBCs become more rigid in HS, patient EVs have a significantly (~40%) lower bending modulus than donor EVs. These results shed light on the mechanism and effects of EV budding and might explain the reported increase in vesiculation of RBCs in HS patients.

Identification of phagocytosis regulators using magnetic genome-wide CRISPR screens.

Phagocytosis is required for a broad range of physiological functions, from pathogen defense to tissue homeostasis, but the mechanisms required for phagocytosis of diverse substrates remain incompletely understood. Here, we developed a rapid magnet-based phenotypic screening strategy, and performed eight genome-wide CRISPR screens in human cells to identify genes regulating phagocytosis of distinct substrates. After validating select hits in focused miniscreens, orthogonal assays and primary human macrophages, we show that (1) the previously uncharacterized gene NHLRC2 is a central player in phagocytosis, regulating RhoA-Rac1 signaling cascades that control actin polymerization and filopodia formation, (2) very-long-chain fatty acids are essential for efficient phagocytosis of certain substrates and (3) the previously uncharacterized Alzheimer's disease-associated gene TM2D3 can preferentially influence uptake of amyloid-ß aggregates. These findings illuminate new regulators and core principles of phagocytosis, and more generally establish an efficient method for unbiased identification of cellular uptake mechanisms across diverse physiological and pathological contexts.

Competition between Bending and Internal Pressure Governs the Mechanics of Fluid Nanovesicles.

Nanovesicles (∼100 nm) are ubiquitous in cell biology and an important vector for drug delivery. Mechanical properties of vesicles are known to influence cellular uptake, but the mechanism by which deformation dynamics affect internalization is poorly understood. This is partly due to the fact that experimental studies of the mechanics of such vesicles remain challenging, particularly at the nanometer scale where appropriate theoretical models have also been lacking. Here, we probe the mechanical properties of nanoscale liposomes using atomic force microscopy (AFM) indentation. The mechanical response of the nanovesicles shows initial linear behavior and subsequent flattening corresponding to inward tether formation. We derive a quantitative model, including the competing effects of internal pressure and membrane bending, that corresponds well to these experimental observations. Our results are consistent with a bending modulus of the lipid bilayer of ∼14kbT. Surprisingly, we find that vesicle stiffness is pressure dominated for adherent vesicles under physiological conditions. Our experimental method and quantitative theory represents a robust approach to study the mechanics of nanoscale vesicles, which are abundant in biology, as well as being of interest for the rational design of liposomal vectors for drug delivery.

Versatile Quadruple-Trap Optical Tweezers for Dual DNA Experiments.

Optical manipulation techniques provide researchers the powerful ability to directly move, probe and interrogate molecular complexes. Quadruple optical trapping is an emerging method for optical manipulation and force spectroscopy that has found its primary use in studying dual DNA interactions, but is certainly not limited to DNA investigations. The key benefit of quadruple optical trapping is that two molecular strands can be manipulated independently and simultaneously. The molecular geometries of the strands can thus be controlled and their interactions can be quantified by force measurements. Accurate control of molecular geometry is of critical importance for the analysis of, for example, protein-mediated DNA-bridging, which plays an important role in DNA compaction. Here, we describe the design of a dedicated and robust quadruple optical trapping-instrument. This instrument can be switched straightforwardly to a high-resolution dual trap and it is integrated with microfluidics and single-molecule fluorescence microscopy, making it a highly versatile tool for correlative single-molecule analysis of a wide range of biomolecular systems.

Controlled tip wear on high roughness surfaces yields gradual broadening and rounding of cantilever tips.

Tip size in atomic force microscopy (AFM) has a major impact on the resolution of images and on the results of nanoindentation experiments. Tip wear is therefore a key limitation in the application of AFM. Here we show, however, how wear can be turned into an advantage as it allows for directed tip shaping. We studied tip wear on high roughness polycrystalline titanium and diamond surfaces and show that tip wear on these surfaces leads to an increased tip size with a rounded shape of the apex. Next, we fitted single peaks from AFM images in order to track the changes in tip radius over time. This method is in excellent agreement with the conventional blind tip reconstruction method with the additional advantage that we could use it to demonstrate that the increase in tip size is gradual. Moreover, with our approach we can shape and control the tip size, while retaining identical chemical and cantilever properties. This significantly expands the reproducibility of AFM force spectroscopy data and is therefore expected to find a wide applicability.

Generic indicators for loss of resilience before a tipping point leading to population collapse.

Theory predicts that the approach of catastrophic thresholds in natural systems (e.g., ecosystems, the climate) may result in an increasingly slow recovery from small perturbations, a phenomenon called critical slowing down. We used replicate laboratory populations of the budding yeast Saccharomyces cerevisiae for direct observation of critical slowing down before population collapse. We mapped the bifurcation diagram experimentally and found that the populations became more vulnerable to disturbance closer to the tipping point. Fluctuations of population density increased in size and duration near the tipping point, in agreement with the theory. Our results suggest that indicators of critical slowing down can provide advance warning of catastrophic thresholds and loss of resilience in a variety of dynamical systems.