ESCRT-III Acts Downstream of MLKL to Regulate Necroptotic Cell Death and Its Consequences.

The activation of mixed lineage kinase-like (MLKL) by receptor-interacting protein kinase-3 (RIPK3) results in plasma membrane (PM) disruption and a form of regulated necrosis, called necroptosis. Here, we show that, during necroptosis, MLKL-dependent calcium (Ca2+) influx and phosphatidylserine (PS) exposure on the outer leaflet of the plasma membrane preceded loss of PM integrity. Activation of MLKL results in the generation of broken, PM "bubbles" with exposed PS that are released from the surface of the otherwise intact cell. The ESCRT-III machinery is required for formation of these bubbles and acts to sustain survival of the cell when MLKL activation is limited or reversed. Under conditions of necroptotic cell death, ESCRT-III controls the duration of plasma membrane integrity. As a consequence of the action of ESCRT-III, cells undergoing necroptosis can express chemokines and other regulatory molecules and promote antigenic cross-priming of CD8+ T cells.

The septin cytoskeleton is required for plasma membrane repair.

Plasma membrane repair is a fundamental homeostatic process of eukaryotic cells. Here, we report a new function for the conserved cytoskeletal proteins known as septins in the repair of cells perforated by pore-forming toxins or mechanical disruption. Using a silencing RNA screen, we identified known repair factors (e.g. annexin A2, ANXA2) and novel factors such as septin 7 (SEPT7) that is essential for septin assembly. Upon plasma membrane injury, the septin cytoskeleton is extensively redistributed to form submembranous domains arranged as knob and loop structures containing F-actin, myosin IIA, S100A11, and ANXA2. Formation of these domains is Ca2+-dependent and correlates with plasma membrane repair efficiency. Super-resolution microscopy revealed that septins and F-actin form intertwined filaments associated with ANXA2. Depletion of SEPT7 prevented ANXA2 recruitment and formation of submembranous actomyosin domains. However, ANXA2 depletion had no effect on domain formation. Collectively, our data support a novel septin-based mechanism for resealing damaged cells, in which the septin cytoskeleton plays a key structural role in remodeling the plasma membrane by promoting the formation of SEPT/F-actin/myosin IIA/ANXA2/S100A11 repair domains.

ß2-adrenoceptor agonist formoterol attenuates NLRP3 inflammasome activation and GSDMD-mediated pyroptosis in microglia through enhancing IκBα/NF-κB inhibition, SQSTM1/p62-dependent selective autophagy and ESCRT-III-mediated plasma membrane repair.

Microglia are immune cells that play important roles in the formation of the innate immune response within the central nervous system (CNS). The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is a multiple protein complex that is crucial for innate immunity, and excessive activation of the inflammasome for various reasons contributes to the pathogenesis of neurodegenerative diseases (NDs). ß2-adrenoceptor agonists have become the focus of attention in studies on NDs due to the high synthesis of ß2-adrenoceptors in the central nervous system (CNS). Promising results have been obtained from these studies targeting anti-inflammatory and neuroprotective effects. Formoterol is an effective, safe for long-term use, and FDA-approved ß2-adrenoceptor agonist with demonstrated anti-inflammatory features in the CNS. In this study, we researched the effects of formoterol on LPS/ATP-stimulated NLRP3 inflammasome activation, pyroptosis, NF-κB, autophagy, and ESCRT-III-mediated plasma membrane repair pathways in the N9 microglia cells. The results showed that formoterol, through the IκBα/NF-κB axis, significantly inhibited NLRP3 inflammasome activation, reduced the level of active caspase-1, secretion of IL-1ß and IL-18 proinflammatory cytokine levels, and the levels of pyroptosis. Additionally, we showed that formoterol activates autophagy, autophagosome formation, and ESCRT-III-mediated plasma membrane repair, which are significant pathways in the inhibition of NLRP3 inflammasome activation and pyroptosis. Our study suggests that formoterol efficaciously prevents the NLRP3 inflammasome activation and pyroptosis in microglial cells regulation through IκBα/NF-κB, autophagy, autophagosome formation, and ESCRT-III-mediated plasma membrane repair.

Cellular and molecular mechanisms underlying plasma membrane functionality and integrity.

The plasma membrane not only protects the cell from the extracellular environment, acting as a selective barrier, but also regulates cellular events that originate at the cell surface, playing a key role in various biological processes that are essential for the preservation of cell homeostasis. Therefore, elucidation of the mechanisms involved in the maintenance of plasma membrane integrity and functionality is of utmost importance. Cells have developed mechanisms to ensure the quality of proteins that inhabit the cell surface, as well as strategies to cope with injuries inflicted to the plasma membrane. Defects in these mechanisms can lead to the development or onset of several diseases. Despite the importance of these processes, a comprehensive and holistic perspective of plasma membrane quality control is still lacking. To tackle this gap, in this Review, we provide a thorough overview of the mechanisms underlying the identification and targeting of membrane proteins that are to be removed from the cell surface, as well as the membrane repair mechanisms triggered in both physiological and pathological conditions. A better understanding of the mechanisms underlying protein quality control at the plasma membrane can reveal promising and unanticipated targets for the development of innovative therapeutic approaches.

Phenothiazines alter plasma membrane properties and sensitize cancer cells to injury by inhibiting annexin-mediated repair.

Repair of damaged plasma membrane in eukaryotic cells is largely dependent on the binding of annexin repair proteins to phospholipids. Changing the biophysical properties of the plasma membrane may provide means to compromise annexin-mediated repair and sensitize cells to injury. Since, cancer cells experience heightened membrane stress and are more dependent on efficient plasma membrane repair, inhibiting repair may provide approaches to sensitize cancer cells to plasma membrane damage and cell death. Here, we show that derivatives of phenothiazines, which have widespread use in the fields of psychiatry and allergy treatment, strongly sensitize cancer cells to mechanical-, chemical-, and heat-induced injury by inhibiting annexin-mediated plasma membrane repair. Using a combination of cell biology, biophysics, and computer simulations, we show that trifluoperazine acts by thinning the membrane bilayer, making it more fragile and prone to ruptures. Secondly, it decreases annexin binding by compromising the lateral diffusion of phosphatidylserine, inhibiting the ability of annexins to curve and shape membranes, which is essential for their function in plasma membrane repair. Our results reveal a novel avenue to target cancer cells by compromising plasma membrane repair in combination with noninvasive approaches that induce membrane injuries.

Evidence of an Annexin A4 mediated plasma membrane repair response to biomechanical strain associated with glaucoma pathogenesis.

Glaucoma is a common neurodegenerative blinding disease that is closely associated with chronic biomechanical strain at the optic nerve head (ONH). Yet, the cellular injury and mechanosensing mechanisms underlying the resulting damage have remained critically unclear. We previously identified Annexin A4 (ANXA4) from a proteomic analyses of human ONH astrocytes undergoing pathological biomechanical strain that mimics glaucomatous conditions. Annexins are a family of calcium-dependent phospholipid binding proteins with key functions in plasma membrane repair (PMR); an active mechanism to limit and mend cellular injury that involves membrane and cytoskeletal reorganizations. However, a role for direct membrane damage and PMR has not been well studied in the context of biomechanical strain, such as that associated with glaucoma. Here we report that this moderate strain surprisingly damages cell membranes to increase permeability in a calcium-dependent manner, and induces rapid aggregation of ANXA4 at injury sites. ANXA4 loss-of-function increases permeability, while exogenous ANXA4 reduces it. Furthermore, ANXA4 aggregation is associated with F-actin dynamics in vitro, and remarkably this interaction and aggregation signature is also observed in the glaucomatous ONH in patient samples. Together these studies link moderate biomechanical strain with direct membrane damage and actin dynamics, and identify an active PMR role for ANXA4 in new model of cell injury associated with glaucoma pathogenesis.

Endophilin-A2-dependent tubular endocytosis promotes plasma membrane repair and parasite invasion.

Endocytosis of caveolae has previously been implicated in the repair of plasma membrane wounds. Here, we show that caveolin-1-deficient fibroblasts lacking caveolae upregulate a tubular endocytic pathway and have a reduced capacity to reseal after permeabilization with pore-forming toxins compared with wild-type cells. Silencing endophilin-A2 expression inhibited fission of endocytic tubules and further reduced plasma membrane repair in cells lacking caveolin-1, supporting a role for tubular endocytosis as an alternative pathway for the removal of membrane lesions. Endophilin-A2 was visualized in association with cholera toxin B-containing endosomes and was recruited to recently formed intracellular vacuoles containing Trypanosoma cruzi, a parasite that utilizes the plasma membrane wounding repair pathway to invade host cells. Endophilin-A2 deficiency inhibited T. cruzi invasion, and fibroblasts deficient in both caveolin-1 and endophilin-A2 did not survive prolonged exposure to the parasites. These findings reveal a novel crosstalk between caveolin-1 and endophilin-A2 in the regulation of clathrin-independent endocytosis and plasma membrane repair, a process that is subverted by T. cruzi parasites for cell invasion.

Naturally occurring and tumor-associated variants of RNF167 promote lysosomal exocytosis and plasma membrane resealing.

Lysosomal exocytosis and resealing of damaged plasma membrane are essential for cellular homeostasis and tumor invasion. However, very little is known of the molecular machinery that regulates these physiological processes. Moreover, no mutations in any of the known regulators of lysosomal exocytosis in primary tumors of patients have been characterized. Here we demonstrate that RNF167-a, a lysosomal-associated ubiquitin ligase, negatively regulates lysosomal exocytosis by inducing perinuclear clustering of lysosomes. Importantly, we also characterized a set of novel natural mutations in RNF167-a, which are commonly found in diverse tumor types. We found that RNF167-a-K97N mutant, unlike the wild type, localizes in the cytoplasm and does not promote perinuclear lysosomal clustering. Furthermore, cells expressing RNF167-a-K97N exhibit dispersed lysosomes, increased exocytosis and enhanced plasma membrane repair. Interestingly, these functional features of RNF167-a-K97N were shared with a naturally occurring short version of RNF167 (isoform RNF167-b). In brief, the results presented here reveal a novel role of RNF167-a, as well as its natural variants RNF167-a-K97N and RNF167-b, as an upstream regulator of lysosomal exocytosis and plasma membrane resealing.

Clathrin-mediated trafficking of phospholipid flippases is required for local plasma membrane/cell wall damage repair in budding yeast.

Plasma membrane damage and repair frequently happen in cells. A critical process underlying plasma membrane repair is to redirect repair factors, such as protein kinase C and the exocyst complex, from the polarized site to the damage site. However, the mechanism underlying the repair factor delivery to the damage site remains unknown. Here, we demonstrate that clathrin-mediated trafficking of repair factors is involved in plasma membrane/cell wall repair in budding yeast. Using laser-induced plasma membrane/cell wall damage assay, we identified phospholipid flippases, Lem3-Dnf1/Dnf2 and Cdc50-Drs2, as essential clathrin cargos for plasma membrane/cell wall repair. We found that flippase impairment significantly compromised the recruitment of exocyst Exo70 to the damage site. In contrast, the recruitment of protein kinase C (Pkc1) was only mildly compromised. Taken together, clathrin-mediated trafficking of the phospholipid flippases is critical for the recruitment of exocyst to the damage site. Mechanisms to redirect exocyst via the clathrin and flippase-mediated pathways may be a general feature of effective plasma membrane repair in polarized cells.

Leishmania amazonensis hijacks host cell lysosomes involved in plasma membrane repair to induce invasion in fibroblasts.

Intracellular parasites of the genus Leishmania are the causative agents of leishmaniasis. The disease is transmitted by the bite of a sand fly vector, which inoculates the parasite into the skin of mammalian hosts, including humans. During chronic infection the parasite lives and replicates inside phagocytic cells, notably the macrophages. An interesting, but overlooked finding, is that other cell types and even non-phagocytic cells have been found to be infected by Leishmania spp. Nevertheless, the mechanisms by which Leishmania invades such cells had not been previously studied. Here, we show that L. amazonensis can induce their own entry into fibroblasts independently of actin cytoskeleton activity, and, thus, through a mechanism that is distinct from phagocytosis. Invasion involves subversion of host cell functions, such as Ca2+ signaling and recruitment and exocytosis of host cell lysosomes involved in plasma membrane repair.This article has an associated First Person interview with the first author of the paper.

Bacterial pore-forming toxin pneumolysin: Cell membrane structure and microvesicle shedding capacity determines differential survival of cell types.

Bacterial infectious diseases can lead to death or to serious illnesses. These outcomes are partly the consequence of pore-forming toxins, which are secreted by the pathogenic bacteria (eg, pneumolysin of Streptococcus pneumoniae). Pneumolysin binds to cholesterol within the plasma membrane of host cells and assembles to form trans-membrane pores, which can lead to Ca2+ influx and cell death. Membrane repair mechanisms exist that limit the extent of damage. Immune cells which are essential to fight bacterial infections critically rely on survival mechanisms after detrimental pneumolysin attacks. This study investigated the susceptibility of different immune cell types to pneumolysin. As a model system, we used the lymphoid T-cell line Jurkat, and myeloid cell lines U937 and THP-1. We show that Jurkat T cells are highly susceptible to pneumolysin attack. In contrast, myeloid THP-1 and U937 cells are less susceptible to pneumolysin. In line with these findings, human primary T cells are shown to be more susceptible to pneumolysin attack than monocytes. Differences in susceptibility to pneumolysin are due to (I) preferential binding of pneumolysin to Jurkat T cells and (II) cell type specific plasma membrane repair capacity. Myeloid cell survival is mostly dependent on Ca2+ induced expelling of damaged plasma membrane areas as microvesicles. Thus, in myeloid cells, first-line defense cells in bacterial infections, a potent cellular repair machinery ensures cell survival after pneumolysin attack. In lymphoid cells, which are important at later stages of infections, less efficient repair mechanisms and enhanced toxin binding renders the cells more sensitive to pneumolysin.

Repairing plasma membrane damage in regulated necrotic cell death.

The plasma membrane performs a central role in maintaining cellular homeostasis and viability by acting as a semi-permeable barrier separating the cell from its surroundings. Under physiological conditions, it is constantly exposed to different kinds of stress, such as from pore-forming proteins/toxins and mechanical activity, that compromises its integrity resulting in cells developing various ways to cope with these dangers to survive. These plasma membrane repair mechanisms are initiated by the rapid influx of extracellular Ca2+ ions and are thus hinged on the activity of various Ca2+-binding proteins. The cell's response to membrane damage also depends on the nature and extent of the stimuli as well as the cell type, and the mechanisms involved are believed to be not mutually exclusive. In regulated necrotic cell death, specifically necroptosis, pyroptosis, and ferroptosis, plasma membrane damage ultimately causes cell lysis and the release of immunomodulating damage-associated molecular patterns. Here, I will discuss how these three cell death pathways are counterbalanced by the action of ESCRT (Endosomal Sorting Complex Required for Transport)-III-dependent plasma membrane repair mechanism, that eventually affects the profile of released cytokines and cell-to-cell communication. These highlight a crucial role that plasma membrane repair play in regulated necrosis, and its potential as a viable target to modulate the immune responses associated with these pathways in the context of the various human pathologies where these cell death modalities are implicated.

Actin Cytoskeletal Dynamics in Single-Cell Wound Repair.

The plasma membrane protects the eukaryotic cell from its surroundings and is essential for cell viability; thus, it is crucial that membrane disruptions are repaired quickly to prevent immediate dyshomeostasis and cell death. Accordingly, cells have developed efficient repair mechanisms to rapidly reseal ruptures and reestablish membrane integrity. The cortical actin cytoskeleton plays an instrumental role in both plasma membrane resealing and restructuring in response to damage. Actin directly aids membrane repair or indirectly assists auxiliary repair mechanisms. Studies investigating single-cell wound repair have often focused on the recruitment and activation of specialized repair machinery, despite the undeniable need for rapid and dynamic cortical actin modulation; thus, the role of the cortical actin cytoskeleton during wound repair has received limited attention. This review aims to provide a comprehensive overview of membrane repair mechanisms directly or indirectly involving cortical actin cytoskeletal remodeling.

Mechanisms protecting host cells against bacterial pore-forming toxins.

Pore-forming toxins (PFTs) are key virulence determinants produced and secreted by a variety of human bacterial pathogens. They disrupt the plasma membrane (PM) by generating stable protein pores, which allow uncontrolled exchanges between the extracellular and intracellular milieus, dramatically disturbing cellular homeostasis. In recent years, many advances were made regarding the characterization of conserved repair mechanisms that allow eukaryotic cells to recover from mechanical disruption of the PM membrane. However, the specificities of the cell recovery pathways that protect host cells against PFT-induced damage remain remarkably elusive. During bacterial infections, the coordinated action of such cell recovery processes defines the outcome of infected cells and is, thus, critical for our understanding of bacterial pathogenesis. Here, we review the cellular pathways reported to be involved in the response to bacterial PFTs and discuss their impact in single-cell recovery and infection.

Lysosomes and plasma membrane repair.

The ability of repairing damages on the plasma membrane is crucial for cell survival. When damaged, eukaryotic cells are able to recover plasma membrane integrity within a few seconds, thus avoiding cytoplasm leakage and cell death. The process is driven by the influx of extracellular calcium which triggers a multitude of intracellular effects that participate in the process of plasma membrane resealing. One of the landmarks of plasma membrane repair is the triggering of intracellular vesicles recruitment and their exocytosis at damage sites. Since lysosomes are able to respond to calcium influx and that some of the lysosomal enzymes exocytosed after plasma membrane permeabilization are essential to restore cell integrity, these organelles have emerged as essential for the maintenance of plasma membrane integrity. Here we summarize the scientific evidences showing the involvement of lysosomes in plasma membrane repair that allowed researchers to propose a totally different function for this famous organelle.

Plasma membrane repair involvement in parasitic and other pathogen infections.

Intracellular pathogens depend on specific mechanisms to be able to gain entry and survive into their host cells. For this, they subvert pathways involved in physiological cellular processes. Here we are going to focus on how two protozoan parasites, Trypanosoma cruzi and Leishmania sp, which may cause severe diseases in humans, use plasma membrane repair (PMR) mechanisms to gain entry in host intracellular environment. T. cruzi is the causative agent of Chagas disease, a disease originally endemic of central and South America, but that has become widespread around the globe. T. cruzi is able to invade any nucleated cell, but muscle cells are usually the main targets during chronic disease. During host cell contact, the parasite interacts with proteins at the host cell surface and may cause damage to their membrane, which has been shown to be responsible for inducing intracellular calcium increase and PMR-related events that culminate with parasite internalization. The same was recently observed for Leishmania sp, when infecting nonprofessional phagocytic cells, such as fibroblasts. Other pathogens, such as viruses or bacteria may also use PMR-related events for invasion and vacuole escape/maturation. In some cases, PMR may also be responsible to modulate pathogen intracellular development. These other PMR roles in pathogen infections will also be briefly discussed.

Actin dynamics and myosin contractility during plasma membrane repair and restoration: Does one ring really heal them all?

In order to survive daily insults, cells have evolved various mechanisms that detect, stabilize and repair damages done to their plasma membrane and cytoskeletal structures. Damage to the PM endangers wounded cells by exposing them to uncontrolled exchanges with the extracellular milieu. The processes and molecular machinery enabling PM repair are therefore at the center of the bulk of the investigations into single-cell repair program. Wounds are repaired by dynamically remodeling the composition and shape of the injured area through exocytosis-mediated release of intracellular membrane components to the wounded area, endocytosis-mediated removal of the injured area, or the shedding of the injury. The wound healing program of Xenopus oocytes and early Drosophila embryos is by contrast, mostly characterized by the rapid formation of a large membrane patch over the wound that eventually fuse with the plasma membrane which restores plasma membrane continuity and lead to the shedding of patch material into the extracellular space. Formation and contraction of actomyosin ring restores normal plasma membrane composition and organizes cytoskeletal repairs. The extend of the contributions of the cytoskeleton to the wound healing program of somatic cells have comparatively received little attention. This review offers a survey of the current knowledge on how actin dynamics, myosin-based contraction and other cytoskeletal structures affects PM and cortical cytoskeleton repair of somatic cells.

ESCRT: nipping the wound in the bud?

Rapid repair of plasma membrane wounds is critical for cellular survival. Exocytic patches, membrane tension reduction and endocytosis were previously proposed to mediate resealing. A recent study implicating the ESCRT complex adds to the growing evidence that repair involves removal of damaged plasma membrane, and not simply patching the wound.

Cell healing: Calcium, repair and regeneration.

Cell repair is attracting increasing attention due to its conservation, its importance to health, and its utility as a model for cell signaling and cell polarization. However, some of the most fundamental questions concerning cell repair have yet to be answered. Here we consider three such questions: (1) How are wound holes stopped? (2) How is cell regeneration achieved after wounding? (3) How is calcium inrush linked to wound stoppage and cell regeneration?

Defying death: Cellular survival strategies following plasmalemmal injury by bacterial toxins.

The perforation of the plasmalemma by pore-forming toxins causes an influx of Ca(2+) and an efflux of cytoplasmic constituents. In order to ensure survival, the cell needs to identify, plug and remove lesions from its membrane. Quarantined by membrane folds and isolated by membrane fusion, the pores are removed from the plasmalemma and expelled into the extracellular space. Outward vesiculation and microparticle shedding seem to be the strategies of choice to eliminate toxin-perforated membrane regions from the plasmalemma of host cells. Depending on the cell type and the nature of injury, the membrane lesion can also be taken up by endocytosis and degraded internally. Host cells make excellent use of an initial, moderate rise in intracellular [Ca(2+)], which triggers containment of the toxin-inflicted damage and resealing of the damaged plasmalemma. Additional Ca(2+)-dependent defensive cellular actions range from the release of effector molecules in order to warn neighbouring cells, to the activation of caspases for the initiation of apoptosis in order to eliminate heavily damaged, dysregulated cells. Injury to the plasmalemma by bacterial toxins can be prevented by the early sequestration of bacterial toxins. Artificial liposomes can act as a decoy system preferentially binding and neutralizing bacterial toxins.