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.
Synopsis
Inflammatory caspases induce a lytic type of cell death known as pyroptosis by cleaving the protein gasdermin D. New findings show that cleaved gasdermin D targets the plasma membrane, where it forms a large permeability pore.
Pyroptosis induction involves the cleavage of gasdermin D by inflammatory caspases.
The N‐terminal fragment of gasdermin D targets cellular membranes and leads to plasma membrane permeabilization.
Gasdermin D targets and permeabilizes liposome membranes after in vitro cleavage by caspase‐1.
A caspase‐1‐processed fragment of gasdermin D inserts into plasma membranes to form large permeable pores, demonstrating the direct role of gasdermin D in pyroptotic cell death.
This article discusses the role of lifestyle in physical material accounting and introduces a new method for simultaneously determining national or regional resource demand and waste generation ...through estimations of the population and its lifestyle, which is manifested in the stocks of service providing goods, their composition and lifetimes. Improving our comprehension of the stocks in use is essential for environmental policy making because (1) they are becoming the most important resource providers, (2) they are important drivers for resource and energy consumption as well as waste and emission generation, and (3) their magnitudes and dynamics are the parts of the material cycles that is usually least understood.
A generic dynamic material flow analysis model is presented and applied for the diffusion of concrete in the Dutch dwelling stock for the period of 1900–2100. Simulation results are illustrated for a standard scenario and a parameter variation. The results show that (1) construction and demolition flows follow a cyclical behaviour, (2) the cycles of construction and demolition flows are phase displaced in the first half of the 21st century, with decreasing construction and increasing demolition, and (3) growth of the dwelling stock is becoming increasingly more material intensive as a growing amount of material is used for replacements.
The presented stock dynamics approach can principally be applied for any anthropogenic material stock; however, it is most useful for the examination of metabolic consequences of diffusion processes of durable and fixed capital stocks.
Cellular membranes are vital for life. They confine cells and cytosolic compartments and are involved in virtually every cellular process. Cellular membranes form cellular contacts and focal ...adhesions, anchor the cytoskeleton, generate energy gradients, transform energy, transduce signals, move cells, and actively form compartments to assemble different membrane proteins into functional entities. But how do cellular membranes perform these tasks? What do the machineries of cellular membranes look like, and how are they controlled and guided? Atomic force microscopy (AFM) allows the observation of biological surfaces in their native environment at a signal-to-noise ratio superior to that of any optical microscopic technique. With a spatial resolution approaching approximately 1 nm, AFM can identify the supramolecular assemblies, characteristic structure, and functional conformation of native membrane proteins. In recent years, AFM has evolved from imaging applications to a multifunctional "laboratory on a tip" that allows observation and manipulation of the machineries of cellular membranes. In the force spectroscopy mode, AFM detects interactions between two single cells at molecular resolution. Force spectroscopy can also be used to probe the local elasticity, chemical groups, and receptor sites of live cells. Other applications locate molecular interactions driving membrane protein folding, assembly, and their switching between functional states. It is also possible to examine the energy landscape of biomolecular reactions, as well as reaction pathways, associated lifetimes, and free energy. In this review, we provide a flavor of the fascinating opportunities offered by the use of AFM as a nanobiotechnological tool in modern membrane biology.
Cell shape and motility are primarily controlled by cellular mechanics. The attachment of the plasma membrane to the underlying actomyosin cortex has been proposed to be important for cellular ...processes involving membrane deformation. However, little is known about the actual function of membrane-to-cortex attachment (MCA) in cell protrusion formation and migration, in particular in the context of the developing embryo. Here, we use a multidisciplinary approach to study MCA in zebrafish mesoderm and endoderm (mesendoderm) germ layer progenitor cells, which migrate using a combination of different protrusion types, namely, lamellipodia, filopodia, and blebs, during zebrafish gastrulation. By interfering with the activity of molecules linking the cortex to the membrane and measuring resulting changes in MCA by atomic force microscopy, we show that reducing MCA in mesendoderm progenitors increases the proportion of cellular blebs and reduces the directionality of cell migration. We propose that MCA is a key parameter controlling the relative proportions of different cell protrusion types in mesendoderm progenitors, and thus is key in controlling directed migration during gastrulation.
Opioids are mainly used to treat both acute and chronic pain. Several opioids are metabolized to some extent by CYP2D6 (codeine, tramadol, hydrocodone, oxycodone, and methadone). Polymorphisms in ...CYP2D6 have been studied for an association with the clinical effect and safety of these drugs. Other genes that have been studied for their association with opioid clinical effect or adverse events include OPRM1 (mu receptor) and COMT (catechol‐O‐methyltransferase). This guideline updates and expands the 2014 Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline for CYP2D6 genotype and codeine therapy and includes a summation of the evidence describing the impact of CYP2D6, OPRM1, and COMT on opioid analgesia and adverse events. We provide therapeutic recommendations for the use of CYP2D6 genotype results for prescribing codeine and tramadol and describe the limited and/or weak data for CYP2D6 and hydrocodone, oxycodone, and methadone, and for OPRM1 and COMT for clinical use.
Using pharmacogenetics in guiding drug therapy experiences a steady increase in uptake, although still leads to discussions as to its clinical use. Psychiatry constitutes a field where ...pharmacogenomic testing might help in guiding drug therapy. To address current challenges, this minireview provides an update regarding genotyping (SNP analysis/arrays/NGS), structural variant detection (star-alleles/CNVs/hybrid alleles), genotype-to-phenotype translations, cost-effectiveness, and actionability of results (FDA/CPIC/PharmGKB) regarding clinical importance of pre-emptive pharmacogenomic testing for prescription of antidepressants and antipsychotics.
A current challenge in the life sciences is to understand how biological systems change their structural, biophysical and chemical properties to adjust functionality. Addressing this issue has been ...severely hampered by the lack of methods capable of imaging biosystems at high resolution while simultaneously mapping their multiple properties. Recent developments in force-distance (FD) curve-based atomic force microscopy (AFM) now enable researchers to combine (sub)molecular imaging with quantitative mapping of physical, chemical and biological interactions. Here we discuss the principles and applications of advanced FD-based AFM tools for the quantitative multiparametric characterization of complex cellular and biomolecular systems under physiological conditions.
Atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS) enables the quantitative study of cell adhesion under physiological conditions. SCFS probes adhesive interactions of single ...living cells with substrates such as extracellular matrix (ECM) proteins and other cells. Here we present a protocol to study integrin-mediated adhesion of HeLa cells to collagen type I using SCFS. We describe procedures for (i) functionalization of AFM cantilevers with the lectin concanavalin A and supports with collagen, (ii) cell handling and attachment to the AFM cantilever, (iii) measurement of adhesion forces and (iv) data analysis and interpretation. Although designed to measure HeLa cell adhesion to collagen, the protocol can be modified for other cell lines and ECM proteins. Compared with other SCFS assays (for example, optical tweezer, biomembrane force probe), AFM-based SCFS has a more versatile force detection range, and it can therefore be used to address a broader range of biological questions. The protocol can be completed in 2-3 d.
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.
A current challenge in the life sciences is to understand how the properties of individual molecular machines adjust in order to meet the functional requirements of the cell. Recent developments in ...force-distance (FD) curve-based atomic force microscopy (FD-based AFM) enable researchers to combine sub-nanometer imaging with quantitative mapping of physical, chemical and biological properties. Here we present a protocol to apply FD-based AFM to the multiparametric imaging of native proteins under physiological conditions. We describe procedures for experimental FD-based AFM setup, high-resolution imaging of proteins in the native unperturbed state with simultaneous quantitative mapping of multiple parameters, and data interpretation and analysis. The protocol, which can be completed in 1-3 d, enables researchers to image proteins and protein complexes in the native unperturbed state and to simultaneously map their biophysical and biochemical properties at sub-nanometer resolution.
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