The small molecule raptinal can simultaneously induce apoptosis and inhibit PANX1 activity.

Discovery of new small molecules that can activate distinct programmed cell death pathway is of significant interest as a research tool and for the development of novel therapeutics for pathological conditions such as cancer and infectious diseases. The small molecule raptinal was discovered as a pro-apoptotic compound that can rapidly trigger apoptosis by promoting the release of cytochrome c from the mitochondria and subsequently activating the intrinsic apoptotic pathway. As raptinal is very effective at inducing apoptosis in a variety of different cell types in vitro and in vivo, it has been used in many studies investigating cell death as well as the clearance of dying cells. While examining raptinal as an apoptosis inducer, we unexpectedly identified that in addition to its pro-apoptotic activities, raptinal can also inhibit the activity of caspase-activated Pannexin 1 (PANX1), a ubiquitously expressed transmembrane channel that regulates many cell death-associated processes. By implementing numerous biochemical, cell biological and electrophysiological approaches, we discovered that raptinal can simultaneously induce apoptosis and inhibit PANX1 activity. Surprisingly, raptinal was found to inhibit cleavage-activated PANX1 via a mechanism distinct to other well-described PANX1 inhibitors such as carbenoxolone and trovafloxacin. Furthermore, raptinal also interfered with PANX1-regulated apoptotic processes including the release of the 'find-me' signal ATP, the formation of apoptotic cell-derived extracellular vesicles, as well as NLRP3 inflammasome activation. Taken together, these data identify raptinal as the first compound that can simultaneously induce apoptosis and inhibit PANX1 channels. This has broad implications for the use of raptinal in cell death studies as well as in the development new PANX1 inhibitors.

A common human MLKL polymorphism confers resistance to negative regulation by phosphorylation.

Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. Here we show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKLS132P in biological membranes and MLKLS132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-ß induced death in non-hematopoietic cells. In vivo, MlklS131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease.

Defining the role of cytoskeletal components in the formation of apoptopodia and apoptotic bodies during apoptosis.

During apoptosis, dying cells undergo dynamic morphological changes that ultimately lead to their disassembly into fragments called apoptotic bodies (ApoBDs). Reorganisation of the cytoskeletal structures is key in driving various apoptotic morphologies, including the loss of cell adhesion and membrane bleb formation. However, whether cytoskeletal components are also involved in morphological changes that occur later during apoptosis, such as the recently described generation of thin apoptotic membrane protrusions called apoptopodia and subsequent ApoBD formation, is not well defined. Through monitoring the progression of apoptosis by confocal microscopy, specifically focusing on the apoptopodia formation step, we characterised the presence of F-actin and microtubules in a subset of apoptopodia generated by T cells and monocytes. Interestingly, targeting actin polymerisation and microtubule assembly pharmacologically had no major effect on apoptopodia formation. These data demonstrate apoptopodia as a novel type of membrane protrusion that could be formed in the absence of actin polymerisation and microtubule assembly.

The induction and consequences of Influenza A virus-induced cell death.

Infection with Influenza A virus (IAV) causes significant cell death within the upper and lower respiratory tract and lung parenchyma. In severe infections, high levels of cell death can exacerbate inflammation and comprise the integrity of the epithelial cell barrier leading to respiratory failure. IAV infection of airway and alveolar epithelial cells promotes immune cell infiltration into the lung and therefore, immune cell types such as macrophages, monocytes and neutrophils are readily exposed to IAV and infection-induced death. Although the induction of cell death through apoptosis and necrosis following IAV infection is a well-known phenomenon, the molecular determinants responsible for inducing cell death is not fully understood. Here, we review the current understanding of IAV-induced cell death and critically evaluate the consequences of cell death in aiding either the restoration of lung homoeostasis or the progression of IAV-induced lung pathologies.

A novel mechanism of generating extracellular vesicles during apoptosis via a beads-on-a-string membrane structure.

Disassembly of apoptotic cells into smaller fragments (a form of extracellular vesicle called apoptotic bodies) can facilitate removal of apoptotic debris and intercellular communication. However, the mechanism underpinning this process is unclear. While observing monocytes undergoing apoptosis by time-lapse microscopy, we discovered a new type of membrane protrusion that resembles a 'beads-on-a-string' structure. Strikingly, the 'beads' are frequently sheared off the 'string' to form apoptotic bodies. Generation of apoptotic bodies via this mechanism can facilitate a sorting process and results in the exclusion of nuclear contents from apoptotic bodies. Mechanistically, generation of 'beads-on-a-string' protrusion is controlled by the level of actomyosin contraction and apoptopodia formation. Furthermore, in an unbiased drug screen, we identified the ability of sertraline (an antidepressant) to block the formation of 'beads-on-a-string' protrusions and apoptotic bodies. These data uncover a new mechanism of apoptotic body formation in monocytes and also compounds that can modulate this process.