The transition between soluble intrinsically disordered tau protein and aggregated tau in neurofibrillary tangles in Alzheimer's disease is unknown. Here, we propose that soluble tau species can undergo liquid–liquid phase separation (LLPS) under cellular conditions and that phase‐separated tau droplets can serve as an intermediate toward tau aggregate formation. We demonstrate that phosphorylated or mutant aggregation prone recombinant tau undergoes LLPS, as does high molecular weight soluble phospho‐tau isolated from human Alzheimer brain. Droplet‐like tau can also be observed in neurons and other cells. We found that tau droplets become gel‐like in minutes, and over days start to spontaneously form thioflavin‐S‐positive tau aggregates that are competent of seeding cellular tau aggregation. Since analogous LLPS observations have been made for FUS, hnRNPA1, and TDP43, which aggregate in the context of amyotrophic lateral sclerosis, we suggest that LLPS represents a biophysical process with a role in multiple different neurodegenerative diseases.

Understanding the factors that direct tissue organization during development is one of the most fundamental goals in developmental biology. Various hypotheses explain cell sorting and tissue organization on the basis of the adhesive and mechanical properties of the constituent cells. However, validating these hypotheses has been difficult due to the lack of appropriate tools to measure these parameters. Here we use atomic force microscopy (AFM) to quantify the adhesive and mechanical properties of individual ectoderm, mesoderm and endoderm progenitor cells from gastrulating zebrafish embryos. Combining these data with tissue self-assembly in vitro and the sorting behaviour of progenitors in vivo, we have shown that differential actomyosin-dependent cell-cortex tension, regulated by Nodal/TGFbeta-signalling (transforming growth factor beta), constitutes a key factor that directs progenitor-cell sorting. These results demonstrate a previously unrecognized role for Nodal-controlled cell-cortex tension in germ-layer organization during gastrulation.

Atomic force microscopy and single-molecule force spectroscopy were combined to image and manipulate purple membrane patches from Halobacterium salinarum. Individual bacteriorhodopsin molecules were first localized and then extracted from the membrane; the remaining vacancies were imaged again. Anchoring forces between 100 and 200 piconewtons for the different helices were found. Upon extraction, the helices were found to unfold. The force spectra revealed the individuality of the unfolding pathways. Helices G and F as well as helices E and D always unfolded pairwise, whereas helices B and C occasionally unfolded one after the other. Experiments with cleaved loops revealed the origin of the individuality: stabilization of helix B by neighboring helices.

During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round. Mitotic cell rounding is thought to facilitate organization within the mitotic cell and be necessary for the geometric requirements of division. However, the forces that drive this shape change remain poorly understood in the presence of external impediments, such as a tissue environment. Here we use cantilevers to track cell rounding force and volume. We show that cells have an outward rounding force, which increases as cells enter mitosis. We find that this mitotic rounding force depends both on the actomyosin cytoskeleton and the cells' ability to regulate osmolarity. The rounding force itself is generated by an osmotic pressure. However, the actomyosin cortex is required to maintain this rounding force against external impediments. Instantaneous disruption of the actomyosin cortex leads to volume increase, and stimulation of actomyosin contraction leads to volume decrease. These results show that in cells, osmotic pressure is balanced by inwardly directed actomyosin cortex contraction. Thus, by locally modulating actomyosin-cortex-dependent surface tension and globally regulating osmotic pressure, cells can control their volume, shape and mechanical properties.

Pyroptosis is a lytic type of cell death that is initiated by inflammatory caspases. These caspases are activated within multi‐protein inflammasome complexes that assemble in response to pathogens and endogenous danger signals. Pyroptotic cell death has been proposed to proceed via the formation of a plasma membrane pore, but the underlying molecular mechanism has remained unclear. Recently, gasdermin D (GSDMD), a member of the ill‐characterized gasdermin protein family, was identified as a caspase substrate and an essential mediator of pyroptosis. GSDMD is thus a candidate for pyroptotic pore formation. Here, we characterize GSDMD function in live cells and in vitro. We show that the N‐terminal fragment of caspase‐1‐cleaved GSDMD rapidly targets the membrane fraction of macrophages and that it induces the formation of a plasma membrane pore. In vitro, the N‐terminal fragment of caspase‐1‐cleaved recombinant GSDMD tightly binds liposomes and forms large permeability pores. Visualization of liposome‐inserted GSDMD at nanometer resolution by cryo‐electron and atomic force microscopy shows circular pores with variable ring diameters around 20 nm. Overall, these data demonstrate that GSDMD is the direct and final executor of pyroptotic cell death.

Atomic force microscopy (AFM) is a powerful, multifunctional imaging platform that allows biological samples, from single molecules to living cells, to be visualized and manipulated. Soon after the instrument was invented, it was recognized that in order to maximize the opportunities of AFM imaging in biology, various technological developments would be required to address certain limitations of the method. This has led to the creation of a range of new imaging modes, which continue to push the capabilities of the technique today. Here, we review the basic principles, advantages and limitations of the most common AFM bioimaging modes, including the popular contact and dynamic modes, as well as recently developed modes such as multiparametric, molecular recognition, multifrequency and high-speed imaging. For each of these modes, we discuss recent experiments that highlight their unique capabilities.

The controlled adhesion of cells to each other and to the extracellular matrix is crucial for tissue development and maintenance. Numerous assays have been developed to quantify cell adhesion. Among these, the use of atomic force microscopy (AFM) for single-cell force spectroscopy (SCFS) has recently been established. This assay permits the adhesion of living cells to be studied in near-physiological conditions. This implementation of AFM allows unrivaled spatial and temporal control of cells, as well as highly quantitative force actuation and force measurement that is sufficiently sensitive to characterize the interaction of single molecules. Therefore, not only overall cell adhesion but also the properties of single adhesion-receptor–ligand interactions can be studied. Here we describe current implementations and applications of SCFS, as well as potential pitfalls, and outline how developments will provide insight into the forces, energetics and kinetics of cell-adhesion processes.

Mechanobiology focuses on how physical forces and the mechanical properties of proteins, protein assemblies, cells and tissues contribute to signalling, development, cell division, differentiation and sorting, physiology and disease 1-4. On virtually any scale, ranging from organisms 2,4 to components such as organs 5,6 , tissues 3,7 , cells 8-10 , viruses 11,12 , complex extracellular or intracellular architecture (including vesicles, the extracellular matrix or actin network 13,14) or single proteins 15-17 , biological systems respond to mechanical forces and generate mechanical cues. In mechanobiology, living systems are described by cycles of mechanosensation, mechanotransduction and mechanoresponse 2,18. In addition to its state, the functional response of a living system depends on the nature of the mechanical signal, whether it is applied at the nanometre or micrometre scale, for a short or long time, with low or high magnitude, and on whether it is scalar or vectorial. Nanotechnological and microtechnological approaches have enabled tremendous progress in quantifying the mechanical properties of biological systems. The links between mechanical response, morphology and function, however, are conspicuously ill understood. The most widely used approaches to structurally map the mechanical properties and responses of biological systems, ranging from millimetre to sub-nanometre resolution and from micronewton to piconewton sensitivity, are based on atomic force microscopy (AFM) 19,20. In this Review, we survey the exciting developments in AFM-based approaches towards the morphological mapping of a wide variety of mechanical properties and the characterization of the functional response of biological systems under physiologically relevant conditions. We further discuss key challenges and caveats that have to be taken into account to overcome the limitations of AFM-based approaches to more fully describe the mechanical properties of living systems and highlight how complementary techniques can contribute to directly linking the functional responses of complex biological systems to mechanical cues. Characterizing biosystems by AFM The introduction of AFM in 1986 opened the door to imaging and manipulating matter at the atomic, molecular and cellular scales and was central to the nascent nanotechnological revolution 21,22. Of particular importance for the characterization of biological systems, atomic force microscopes can operate in aqueous environments and at physiological temperatures. In an atomic force microscope, a cantilever that is several micrometres long and has a molecularly sharp probe at the end is used to trace the sample topography, detecting