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.
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.
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.
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.
Tau pathology is known to spread in a hierarchical pattern in Alzheimer's disease (AD) brain during disease progression, likely by trans-synaptic tau transfer between neurons. However, the tau ...species involved in inter-neuron propagation remains unclear. To identify tau species responsible for propagation, we examined uptake and propagation properties of different tau species derived from postmortem cortical extracts and brain interstitial fluid of tau-transgenic mice, as well as human AD cortices. Here we show that PBS-soluble phosphorylated high-molecular-weight (HMW) tau, though very low in abundance, is taken up, axonally transported, and passed on to synaptically connected neurons. Our findings suggest that a rare species of soluble phosphorylated HMW tau is the endogenous form of tau involved in propagation and could be a target for therapeutic intervention and biomarker development.
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.
Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we ...show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes.
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.
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.
Polymyxins are last-resort antibiotics with potent activity against multi-drug resistant pathogens. They interact with lipopolysaccharide (LPS) in bacterial membranes, but mechanistic details at the ...molecular level remain unclear. Here, we characterize the interaction of polymyxins with native, LPS-containing outer membrane patches of Escherichia coli by high-resolution atomic force microscopy imaging, along with structural and biochemical assays. We find that polymyxins arrange LPS into hexagonal assemblies to form crystalline structures. Formation of the crystalline structures is correlated with the antibiotic activity, and absent in polymyxin-resistant strains. Crystal lattice parameters alter with variations of the LPS and polymyxin molecules. Quantitative measurements show that the crystalline structures decrease membrane thickness and increase membrane area as well as stiffness. Together, these findings suggest the formation of rigid LPS-polymyxin crystals and subsequent membrane disruption as the mechanism of polymyxin action and provide a benchmark for optimization and de novo design of LPS-targeting antimicrobials.
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