Inflammatory caspases (caspases 1, 4, 5 and 11) are activated in response to microbial infection and danger signals. When activated, they cleave mouse and human gasdermin D (GSDMD) after Asp276 and Asp275, respectively, to generate an N-terminal cleavage product (GSDMD-NT) that triggers inflammatory death (pyroptosis) and release of inflammatory cytokines such as interleukin-1β. Cleavage removes the C-terminal fragment (GSDMD-CT), which is thought to fold back on GSDMD-NT to inhibit its activation. However, how GSDMD-NT causes cell death is unknown. Here we show that GSDMD-NT oligomerizes in membranes to form pores that are visible by electron microscopy. GSDMD-NT binds to phosphatidylinositol phosphates and phosphatidylserine (restricted to the cell membrane inner leaflet) and cardiolipin (present in the inner and outer leaflets of bacterial membranes). Mutation of four evolutionarily conserved basic residues blocks GSDMD-NT oligomerization, membrane binding, pore formation and pyroptosis. Because of its lipid-binding preferences, GSDMD-NT kills from within the cell, but does not harm neighbouring mammalian cells when it is released during pyroptosis. GSDMD-NT also kills cell-free bacteria in vitro and may have a direct bactericidal effect within the cytosol of host cells, but the importance of direct bacterial killing in controlling in vivo infection remains to be determined.

/pdf/inflammasome-activated-gasdermin-d-causes-pyroptosis-by-38h3azswtu.pdf

Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores

Recent studies suggest both normal and cancerous cells secrete vesicles into the extracellular space. These extracellular vesicles (EVs) contain materials that mirror the genetic and proteomic content of the secreting cell. The identification of cancer-specific material in EVs isolated from the biofluids (e.g., serum, cerebrospinal fluid, urine) of cancer patients suggests EVs as an attractive platform for biomarker development. It is important to recognize that the EVs derived from clinical samples are likely highly heterogeneous in make-up and arose from diverse sets of biologic processes. This article aims to review the biologic processes that give rise to various types of EVs, including exosomes, microvesicles, retrovirus like particles, and apoptotic bodies. Clinical pertinence of these EVs to neuro-oncology will also be discussed.

/pdf/biogenesis-of-extracellular-vesicles-ev-exosomes-56u837qalm.pdf

Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies.

In all animal cells, phospholipids are asymmetrically distributed between the outer and inner leaflets of the plasma membrane. This asymmetrical phospholipid distribution is disrupted in various biological systems. For example, when blood platelets are activated, they expose phosphatidylserine (PtdSer) to trigger the clotting system. The PtdSer exposure is believed to be mediated by Ca(2+)-dependent phospholipid scramblases that transport phospholipids bidirectionally, but its molecular mechanism is still unknown. Here we show that TMEM16F (transmembrane protein 16F) is an essential component for the Ca(2+)-dependent exposure of PtdSer on the cell surface. When a mouse B-cell line, Ba/F3, was treated with a Ca(2+) ionophore under low-Ca(2+) conditions, it reversibly exposed PtdSer. Using this property, we established a Ba/F3 subline that strongly exposed PtdSer by repetitive fluorescence-activated cell sorting. A complementary DNA library was constructed from the subline, and a cDNA that caused Ba/F3 to expose PtdSer spontaneously was identified by expression cloning. The cDNA encoded a constitutively active mutant of TMEM16F, a protein with eight transmembrane segments. Wild-type TMEM16F was localized on the plasma membrane and conferred Ca(2+)-dependent scrambling of phospholipids. A patient with Scott syndrome, which results from a defect in phospholipid scrambling activity, was found to carry a mutation at a splice-acceptor site of the gene encoding TMEM16F, causing the premature termination of the protein.

/pdf/calcium-dependent-phospholipid-scrambling-by-tmem16f-4b8wxeevrp.pdf

Calcium-dependent phospholipid scrambling by TMEM16F

Mechanotransduction has an important role in physiology. Biological processes including sensing touch and sound waves require as-yet-unidentified cation channels that detect pressure. Mouse Piezo1 (MmPiezo1) and MmPiezo2 (also called Fam38a and Fam38b, respectively) induce mechanically activated cationic currents in cells; however, it is unknown whether Piezo proteins are pore-forming ion channels or modulate ion channels. Here we show that Drosophila melanogaster Piezo (DmPiezo, also called CG8486) also induces mechanically activated currents in cells, but through channels with remarkably distinct pore properties including sensitivity to the pore blocker ruthenium red and single channel conductances. MmPiezo1 assembles as a ∼1.2-million-dalton homo-oligomer, with no evidence of other proteins in this complex. Purified MmPiezo1 reconstituted into asymmetric lipid bilayers and liposomes forms ruthenium-red-sensitive ion channels. These data demonstrate that Piezo proteins are an evolutionarily conserved ion channel family involved in mechanotransduction. Large transmembrane proteins of the Piezo family assemble as tetramers to form a new class of ion channel that can be activated by mechanical force. Many tissues are able to detect and respond to mechanical forces, and this mechanical sensitivity has been implicated in many biological processes and diseases, including touch, pain, deafness and hypertension. The conversion of mechanical force into biological signals, or 'mechanotransduction', is thought to involve specialized cation channels. In a pair of papers, Ardem Patapoutian and colleagues establish that the large transmembrane proteins of the 'Piezo' family — conserved from animals to plants and protozoa — are among the long-sought-after mechanically activated ion channels. Coste et al. show that the Drosophila melanogaster Piezo protein induces mechanically activated cationic currents in human embryonic kidney cells, establishing functional conservation. Comparison of the mechanically activated currents induced by mouse and fly Piezos reveals ion-channel activities with unique pore properties, suggesting that Piezos are bona fide ion channels. Kim et al. show that D. melanogaster Piezo is essential for sensing mechanical pain in fruitflies, giving the first demonstration that Piezos are physiologically relevant mechanosensors in vivo.

/pdf/piezo-proteins-are-pore-forming-subunits-of-mechanically-549ou07mxb.pdf

Piezo proteins are pore-forming subunits of mechanically activated channels

Technological developments, especially in mass spectrometry and bioinformatics, have revealed that living cells contain thousands rather than dozens of different lipids [for classification and nomenclature, see Fahy et al. ([Fahy et al., 2009][1])]. Now, the resulting questions are what is the

Lipid Map of the Mammalian Cell

Phosphatidylserine (PS) is the most abundant negatively charged phospholipid in eukaryotic membranes. PS directs the binding of proteins that bear C2 or gamma-carboxyglutamic domains and contributes to the electrostatic association of polycationic ligands with cellular membranes. Rather than being evenly distributed, PS is found preferentially in the inner leaflet of the plasma membrane and in endocytic membranes. The loss of PS asymmetry is an early indicator of apoptosis and serves as a signal to initiate blood clotting. This review discusses the determinants and functional implications of the subcellular distribution and membrane topology of PS.

The distribution and function of phosphatidylserine in cellular membranes.

Phosphatidylserine (PS) is the most abundant negatively charged phos- pholipid in eukaryotic membranes. PS directs the binding of proteins that bear C2 or gamma-carboxyglutamic domains and contributes to the electrostatic association of polycationic ligands with cellular mem- branes. Rather than being evenly distributed, PS is found preferentially in the inner leaflet of the plasma membrane and in endocytic mem- branes. The loss of PS asymmetry is an early indicator of apoptosis and serves as a signal to initiate blood clotting. This review discusses the determinants and functional implications of the subcellular distribution and membrane topology of PS.

Peter A. Leventis

Papers

The distribution and function of phosphatidylserine in cellular membranes.

in Cellular Membranes