β2 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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-β aggregates.

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.

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.

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.

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.

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.

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.

Alba shapes the archaeal genome using a delicate balance of bridging and stiffening the DNA.

Architectural proteins have an important role in shaping the genome and act as global regulators of gene expression. How these proteins jointly modulate genome plasticity is largely unknown. In archaea, one of the most abundant proteins, Alba, is considered to have a key role in organizing the genome.

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.