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.

Phagocytic clearance of apoptotic, necrotic, necroptotic and pyroptotic cells.

Although millions of cells in the human body will undergo programmed cell death each day, dying cells are rarely detected under homeostatic settings in vivo. The swift removal of dying cells is due to the rapid recruitment of phagocytes to the site of cell death which then recognise and engulf the dying cell. Apoptotic cell clearance - the engulfment of apoptotic cells by phagocytes - is a well-defined process governed by a series of molecular factors including 'find-me', 'eat-me', 'don't eat-me' and 'good-bye' signals. However, in recent years with the rapid expansion of the cell death field, the removal of other necrotic-like cell types has drawn much attention. Depending on the type of death, dying cells employ different mechanisms to facilitate engulfment and elicit varying functional impacts on the phagocyte, from wound healing responses to inflammatory cytokine secretion. Nevertheless, despite the mechanism of death, the clearance of dying cells is a fundamental process required to prevent the uncontrolled release of pro-inflammatory mediators and inflammatory disease. This mini-review summarises the current understandings of: (i) apoptotic, necrotic, necroptotic and pyroptotic cell clearance; (ii) the functional consequences of dying cell engulfment and; (iii) the outstanding questions in the field.

Apoptotic Bodies: Mechanism of Formation, Isolation and Functional Relevance.

In the final stages of apoptosis, apoptotic cells can generate a variety of membrane-bound vesicles known as apoptotic extracellular vesicles (ApoEVs). Apoptotic bodies (ApoBDs), a major subset of ApoEVs, are formed through a process termed apoptotic cell disassembly characterised by a series of tightly regulated morphological steps including plasma membrane blebbing, apoptotic membrane protrusion formation and fragmentation into ApoBDs. To better characterise the properties of ApoBDs and elucidate their function, a number of methods including differential centrifugation, filtration and fluorescence-activated cell sorting were developed to isolate ApoBDs. Furthermore, it has become increasingly clear that ApoBD formation can contribute to various biological processes such as apoptotic cell clearance and intercellular communication. Together, recent literature demonstrates that apoptotic cell disassembly and thus, ApoBD formation, is an important process downstream of apoptotic cell death. In this chapter, we discuss the current understandings of the molecular mechanisms involved in regulating apoptotic cell disassembly, techniques for ApoBD isolation, and the functional roles of ApoBDs in physiological and pathological settings.

Monocyte apoptotic bodies are vehicles for influenza A virus propagation.

The disassembly of apoptotic cells into small membrane-bound vesicles termed apoptotic bodies (ApoBDs) is a hallmark of apoptosis; however, the functional significance of this process is not well defined. We recently discovered a new membrane protrusion (termed beaded apoptopodia) generated by apoptotic monocytes which fragments to release an abundance of ApoBDs. To investigate the function of apoptotic monocyte disassembly, we used influenza A virus (IAV) infection as a proof-of-concept model, as IAV commonly infects monocytes in physiological settings. We show that ApoBDs generated from IAV-infected monocytes contained IAV mRNA, protein and virions and consequently, could facilitate viral propagation in vitro and in vivo, and induce a robust antiviral immune response. We also identified an antipsychotic, Haloperidol, as an unexpected inhibitor of monocyte cell disassembly which could impair ApoBD-mediated viral propagation under in vitro conditions. Together, this study reveals a previously unrecognised function of apoptotic monocyte disassembly in the pathogenesis of IAV infections.

ROCK1 but not LIMK1 or PAK2 is a key regulator of apoptotic membrane blebbing and cell disassembly.

Many cell types are known to undergo a series of morphological changes during the progression of apoptosis, leading to their disassembly into smaller membrane-bound vesicles known as apoptotic bodies (ApoBDs). In particular, the formation of circular bulges called membrane blebs on the surface of apoptotic cells is a key morphological step required for a number of cell types to generate ApoBDs. Although apoptotic membrane blebbing is thought to be regulated by kinases including ROCK1, PAK2 and LIMK1, it is unclear whether these kinases exhibit overlapping roles in the disassembly of apoptotic cells. Utilising both pharmacological and CRISPR/Cas9 gene editing based approaches, we identified ROCK1 but not PAK2 or LIMK1 as a key non-redundant positive regulator of apoptotic membrane blebbing as well as ApoBD formation. Functionally, we have established an experimental system to either inhibit or enhance ApoBD formation and demonstrated the importance of apoptotic cell disassembly in the efficient uptake of apoptotic materials by various phagocytes. Unexpectedly, we also noted that ROCK1 could play a role in regulating the onset of secondary necrosis. Together, these data shed light on both the mechanism and function of cell disassembly during apoptosis.

Plexin B2 Is a Regulator of Monocyte Apoptotic Cell Disassembly.

Billions of cells undergo apoptosis daily and often fragment into small, membrane-bound extracellular vesicles termed apoptotic bodies (ApoBDs). We demonstrate that apoptotic monocytes undergo a highly coordinated disassembly process and form long, beaded protrusions (coined as beaded apoptopodia), which fragment to release ApoBDs. Here, we find that the protein plexin B2 (PlexB2), a transmembrane receptor that regulates axonal guidance in neurons, is enriched in the ApoBDs of THP1 monocytes and is a caspase 3/7 substrate. To determine whether PlexB2 is involved in the disassembly of apoptotic monocytes, we generate PlexB2-deficient THP1 monocytes and demonstrate that lack of PlexB2 impairs the formation of beaded apoptopodia and ApoBDs. Consequently, the loss of PlexB2 in apoptotic THP1 monocytes impairs their uptake by both professional and non-professional phagocytes. Altogether, these data identify PlexB2 as a positive regulator of apoptotic monocyte disassembly and demonstrate the importance of this process in apoptotic cell clearance.

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

The original version of the article unfortunately contained a typo in the fourth author name. The author name was incorrectly listed as Rochelle Tixeria. The correct name should be Rochelle Tixeira. The original article has been corrected.

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.

Moving beyond size and phosphatidylserine exposure: evidence for a diversity of apoptotic cell-derived extracellular vesicles in vitro.

Apoptosis is a form of programmed cell death that occurs throughout life as part of normal development as well as pathologic processes including chronic inflammation and infection. Although the death of a cell is often considered as the only biological outcome of a cell committed to apoptosis, it is becoming increasingly clear that the dying cell can actively communicate with other cells via soluble factors as well as membrane-bound extracellular vesicles (EVs) to regulate processes including cell clearance, immunity and tissue repair. Compared to EVs generated from viable cells such as exosomes and microvesicles, apoptotic cell-derived EVs (ApoEVs) are less well defined and the basic criteria for ApoEV characterization have not been established in the field. In this study, we will examine the current understanding of ApoEVs, in particular, the ApoEV subtype called apoptotic bodies (ApoBDs). We described that a subset of ApoBDs can be larger than 5 µm and smaller than 1 µm based on flow cytometry and live time-lapse microscopy analysis, respectively. We also described that a subset of ApoBDs can expose a relatively low level of phosphatidylserine on its surface based on annexin A5 staining. Furthermore, we characterized the presence of caspase-cleaved proteins (in particular plasma membrane-associated or cytoplasmic proteins) in samples enriched in ApoBDs. Lastly, using a combination of biochemical-, live imaging- and flow cytometry-based approaches, we characterized the progressive lysis of ApoBDs. Taken together, these results extended our understanding of ApoBDs.

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.

Detection and Isolation of Apoptotic Bodies to High Purity.

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. It has been proposed that ApoBDs can aid cell clearance as well as intercellular communication through trafficking biomolecules. Conventional approaches used for the identification and isolation of ApoBDs are often limited by the lack of accurate quantification and low sample purity. Here, we describe a workflow to confirm the induction of apoptosis, validate ApoBD formation, and isolate ApoBDs to high purity. We will also outline and compare fluorescence-activated cell sorting (FACS) and differential centrifugation based approaches to isolate ApoBDs. Furthermore, the purity of isolated ApoBDs will be confirmed using a previously establish flow cytometry-based staining and analytical method. Taken together, using the described approach, THP-1 monocyte apoptosis and apoptotic cell disassembly was induced and validated, and ApoBD generated from THP-1 monocytes were isolated to a purity of 97-99%.

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Determining the contents and cell origins of apoptotic bodies by flow cytometry.

Over 200 billion cells undergo apoptosis every day in the human body in order to maintain tissue homeostasis. Increased apoptosis can also occur under pathological conditions including infection and autoimmune disease. During apoptosis, cells can fragment into subcellular membrane-bound vesicles known as apoptotic bodies (ApoBDs). We recently developed a flow cytometry-based method to accurately differentiate ApoBDs from other particles (e.g. cells and debris). In the present study, we aim to further characterize subsets of ApoBDs based on intracellular contents and cell type-specific surface markers. Utilizing a flow cytometry-based approach, we demonstrated that intracellular contents including nuclear materials and mitochondria are distributed to some, but not all ApoBDs. Interestingly, the mechanism of ApoBD formation could affect the distribution of intracellular contents into ApoBDs. Furthermore, we also showed that ApoBDs share the same surface markers as their cell of origin, which can be used to distinguish cell type-specific ApoBDs from a mixed culture. These studies demonstrate that ApoBDs are not homogeneous and can be divided into specific subclasses based on intracellular contents and cell surface markers. The described flow cytometry-based method to study ApoBDs could be used in future studies to better understand the function of ApoBDs.

Isolation of cell type-specific apoptotic bodies by fluorescence-activated cell sorting.

Apoptotic bodies (ApoBDs) are membrane-bound extracellular vesicles that can mediate intercellular communication in physiological and pathological settings. By combining recently developed analytical strategies with fluorescence-activated cell sorting (FACS), we have developed a method that enables the isolation of ApoBDs from cultured cells to 99% purity. In addition, this approach also enables the identification and isolation of cell type-specific ApoBDs from tissue, bodily fluid and blood-derived samples.

Disassembly of the Dying: Mechanisms and Functions.

The disassembly of an apoptotic cell into subcellular fragments, termed apoptotic bodies (ApoBDs), is a hallmark of apoptosis. Although the generation of ApoBDs is generally understood as being stochastic, it is becoming increasingly clear that ApoBD formation is a highly regulated process involving distinct morphological steps and molecular factors. Functionally, ApoBDs could facilitate the efficient clearance of apoptotic material by surrounding phagocytes as well as mediate the transfer of biomolecules including microRNAs and proteins between cells to aid in intercellular communications. Therefore, the formation of ApoBDs is an important process downstream from apoptotic cell death. We discuss here the mechanisms and functions of apoptotic cell disassembly.

Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry.

The use of annexin A5 (A5) and either propidium iodide or 7-aminoactinomycin D (PI/7-AAD) stains to measure cell death by flow cytometry has been considered the gold standard by most investigators. However, this widely used method often makes the assumption that there are only three types of particles in a sample: viable, apoptotic and necrotic cells. To study the progression of cell death in greater detail, in particular how apoptotic cells undergo fragmentation to generate membrane-bound vesicles known as apoptotic bodies, we established a flow cytometry-based protocol to accurately and rapidly measure the cell death process. This protocol uses a combination of A5 and TO-PRO-3 (a commercially available nucleic acid-binding dye that stains early apoptotic and necrotic cells differentially), and a logical seven-stage analytical approach to distinguish six types of particles in a sample, including apoptotic bodies and cells at three different stages of cell death. The protocol requires 1-5 h for sample preparation (including induction of cell death), 20 min for staining and 5 min for data analysis.

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.