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.

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.

Tissue wound repair has been studied extensively. It involves the coordinated activation of several intracellular and intercellular pathways, as well as remodeling from the sequential recruitment of different cell types to the wound site. There is, however, an equally important process that happens at the single cell level, when the integrity of the plasma membrane is compromised. Individual eukaryotic cells can rapidly repair their plasma membrane after injury, through a process that restores internal homeostasis and prevents cell death. Despite its importance, investigations of this fascinating mechanism have been limited. Only recently have we begun to understand that plasma-membrane repair resembles tissue healing, in the sense that it also involves sequential, highly localized remodeling steps that ultimately eliminate all traces of the injury.

Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes.

Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca(2+)-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D.

Lipid raft-dependent plasma membrane repair interferes with the activation of B lymphocytes.

Cells rapidly repair plasma membrane (PM) damage by a process requiring Ca(2+)-dependent lysosome exocytosis. Acid sphingomyelinase (ASM) released from lysosomes induces endocytosis of injured membrane through caveolae, membrane invaginations from lipid rafts. How B lymphocytes, lacking any known form of caveolin, repair membrane injury is unknown. Here we show that B lymphocytes repair PM wounds in a Ca(2+)-dependent manner. Wounding induces lysosome exocytosis and endocytosis of dextran and the raft-binding cholera toxin subunit B (CTB). Resealing is reduced by ASM inhibitors and ASM deficiency and enhanced or restored by extracellular exposure to sphingomyelinase. B cell activation via B cell receptors (BCRs), a process requiring lipid rafts, interferes with PM repair. Conversely, wounding inhibits BCR signaling and internalization by disrupting BCR-lipid raft coclustering and by inducing the endocytosis of raft-bound CTB separately from BCR into tubular invaginations. Thus, PM repair and B cell activation interfere with one another because of competition for lipid rafts, revealing how frequent membrane injury and repair can impair B lymphocyte-mediated immune responses.

The exocyst is required for trypanosome invasion and the repair of mechanical plasma membrane wounds.

The process of host cell invasion by Trypanosoma cruzi shares mechanistic elements with plasma membrane injury and repair. Both processes require Ca(2+)-triggered exocytosis of lysosomes, exocytosis of acid sphingomyelinase and formation of ceramide-enriched endocytic compartments. T. cruzi invades at peripheral sites, suggesting a need for spatial regulation of membrane traffic. Here, we show that Exo70 and Sec8 (also known as EXOC7 and EXOC4, respectively), components of the exocyst complex, accumulate in nascent T. cruzi vacuoles and at sites of mechanical wounding. Exo70 or Sec8 depletion inhibits T. cruzi invasion and Ca(2+)-dependent resealing of mechanical wounds, but does not affect the repair of smaller lesions caused by pore-forming toxins. Thus, T. cruzi invasion and mechanical lesion repair share a unique requirement for the exocyst, consistent with a dependence on targeted membrane delivery.

Damage control: cellular mechanisms of plasma membrane repair.

When wounded, eukaryotic cells reseal in a few seconds. Ca(2+) influx induces exocytosis of lysosomes, a process previously thought to promote repair by 'patching' wounds. New evidence suggests that resealing involves direct wound removal. Exocytosis of lysosomal acid sphingomyelinase (ASM) triggers endocytosis of lesions followed by intracellular degradation. Characterization of injury-induced endosomes revealed a role for caveolae, sphingolipid-enriched plasma membrane invaginations that internalize toxin pores and are abundant in mechanically stressed cells. These findings provide a novel mechanistic explanation for the muscle pathology associated with mutations in caveolar proteins. Membrane remodeling by the ESCRT complex was also recently shown to participate in small-wound repair, emphasizing that cell resealing involves previously unrecognized mechanisms for lesion removal that are distinct from the patch model.

Caveolae internalization repairs wounded cells and muscle fibers.

Rapid repair of plasma membrane wounds is critical for cellular survival. Muscle fibers are particularly susceptible to injury, and defective sarcolemma resealing causes muscular dystrophy. Caveolae accumulate in dystrophic muscle fibers and caveolin and cavin mutations cause muscle pathology, but the underlying mechanism is unknown. Here we show that muscle fibers and other cell types repair membrane wounds by a mechanism involving Ca(2+)-triggered exocytosis of lysosomes, release of acid sphingomyelinase, and rapid lesion removal by caveolar endocytosis. Wounding or exposure to sphingomyelinase triggered endocytosis and intracellular accumulation of caveolar vesicles, which gradually merged into larger compartments. The pore-forming toxin SLO was directly visualized entering cells within caveolar vesicles, and depletion of caveolin inhibited plasma membrane resealing. Our findings directly link lesion removal by caveolar endocytosis to the maintenance of plasma membrane and muscle fiber integrity, providing a mechanistic explanation for the muscle pathology associated with mutations in caveolae proteins. DOI:http://dx.doi.org/10.7554/eLife.00926.001.

Genetically encoded probes for phosphatidic acid.

In addition to forming bilayers to separate cellular compartments, lipids participate in vesicular trafficking and signal transduction. Among others, phosphatidic acid (PA) is emerging as an important signaling molecule. The spatiotemporal distribution of cellular PA appears to be tightly regulated by localized synthesis and a rapid metabolism. Although PA has been long proposed as a pleiotropic bioactive lipid, when and where PA is produced in the living cells have only recently been explored using biosensors that specifically bind to PA. The probes that we have generated are composed of the PA-binding domains of either Spo20p or Raf1 directly fused to GFP. In this chapter, we will describe the expression and purification of GST-fusion proteins of these probes, and the use of phospholipid strips to validate the specificity of their interaction with PA. We will then illustrate the use of GFP-tagged probes to visualize the synthesis of PA in the neurosecretory PC12 cells and RAW 267.4 macrophages. Interestingly, the two probes show a differential distribution in these cell types, indicating that they may have different affinities for PA or recognize different pools of PA. In conclusion, the development of a broader choice of probes may be required to adequately follow the complex dynamics of PA in different cell types, in order to determine the cellular distribution of PA and its role in various cellular processes.

Toxin pores endocytosed during plasma membrane repair traffic into the lumen of MVBs for degradation.

Cells permeabilized by the bacterial pore-forming toxin streptolysin O (SLO) reseal their plasma membrane in a Ca(2+) -dependent manner. Resealing involves Ca(2+) -dependent exocytosis of lysosomes, release of acid sphingomyelinase and rapid formation of endosomes that carry the transmembrane pores into the cell. The intracellular fate of the toxin-carrying endocytic vesicles, however, is still unknown. Here, we show that SLO pores removed from the plasma membrane by endocytosis are sorted into the lumen of lysosomes, where they are degraded. SLO-permeabilized cells contain elevated numbers of total endosomes, which increase gradually in size while transitioning from endosomes with flat clathrin coats to large multivesicular bodies (MVBs). Under conditions that allow endocytosis and plasma membrane repair, SLO is rapidly ubiquitinated and gradually degraded, in a process sensitive to inhibitors of lysosomal hydrolysis but not of proteasomes. The endosomes induced by SLO permeabilization become increasingly acidified and promote SLO degradation under normal conditions, but not in cells silenced for expression of Vps24, an ESCRT-III complex component required for the release of intraluminal vesicles into MVBs. Thus, cells dispose of SLO transmembrane pores by ubiquitination/ESCRT-dependent sorting into the lumen of late endosomes/lysosomes.

Ral isoforms are implicated in Fc gamma R-mediated phagocytosis: activation of phospholipase D by RalA.

Phagocytosis is an essential element of the immune response permitting the elimination of pathogens, cellular debris, apoptotic cells, and tumor cells. Recently, both phospholipase D (PLD) isoforms, PLD1 and PLD2, were shown to be necessary for efficient FcgammaR-mediated phagocytosis. In this study, we investigated the role of a potential PLD regulator, the Ral GTPases RalA and RalB, in murine RAW 264.7 macrophages. Both Ral isoforms are expressed in macrophages and are transiently activated following FcgammaR stimulation. When Ral expression levels were varied using Ral mutants or interference RNA, phagocytosis assays revealed that Ral isoforms have antagonistic effects; RalA is a positive modulator, whereas RalB plays a negative role. We then focused on RalA and its possible relationship with PLD. The increase in PLD activity that occurs when phagocytosis is stimulated was inhibited in cells with reduced RalA protein, but it was unaffected by reduced levels of RalB. Furthermore, in macrophages transfected with dsRed-RalA and GFP-PLD1 or GFP-PLD2, RalA colocalized with PLD1 and PLD2 at the phagocytic cup during phagosome formation. Additional results obtained from immunoprecipitation of PLD from macrophages transfected with myc-RalA and hemagglutinin-tagged PLD1 or PLD2 indicated an enhanced interaction of RalA with both PLD isoforms during phagocytic stimulation. The increase in RalA and PLD1 interaction was transient and correlated with the time course of RalA activation. These findings reveal a novel pathway involving RalA and PLD in the regulation of FcgammaR-mediated phagocytosis.

Dynamics and function of phospholipase D and phosphatidic acid during phagocytosis.

Phospholipase D (PLD) produces phosphatidic acid (PA), an established intracellular signalling lipid that has been also implicated in vesicular trafficking, and as such, PLD could play multiple roles during phagocytosis. Using an RNA interference strategy, we show that endogenous PLD1 and PLD2 are necessary for efficient phagocytosis in murine macrophages, in line with results obtained with wild-type constructs and catalytically inactive PLD mutants which, respectively, enhance and inhibit phagocytosis. Furthermore, we found that PA is transiently produced at sites of phagosome formation. Macrophage PLD1 and PLD2 differ in their subcellular distributions. PLD1 is associated with cytoplasmic vesicles, identified as a late endosomal/lysosomal compartment, whereas PLD2 localizes at the plasma membrane. In living cells undergoing phagocytosis, PLD1 vesicles are recruited to nascent and internalized phagosomes, whereas PLD2 is only observed on nascent phagosomes. These results provide evidence that both PLD isoforms are required for phagosome formation, but only PLD1 seems to be implicated in later stages of phagocytosis occurring after phagosomal internalization.

Fibrillar prion peptide (106-126) and scrapie prion protein hamper phagocytosis in microglia.

The inflammatory response in prion diseases is dominated by microglial activation. As macrophages of the central nervous system, the phagocytic capacity of microglia is well recognized, and it is possible that microglia are involved in the removal and processing of amyloid fibrils, thus preventing their harmful effect. We have analyzed the effects of a synthetic peptide of the human prion protein, PrP(106-126), which can form fibrils, and the pathogenic form of prion protein, PrPsc, on phagocytosis in microglia isolated from neonatal rat brain cultures. To some extent, fibrillar PrP(106-126) is internalized and processed. However, both synthetic prion peptide PrP(106-126) in a fibrillar form and pathogenic prion protein PrPsc severely hamper the phagocytic activity as measured by the uptake of beads by microglia. At a concentration that does not induce microglial death, PrP(106-126) reduced the number of beads internalized and altered their cytoplasmic distribution. This effect was not due to decreased binding of beads to the cell surface, nor restricted to specific classes of receptors. Although the PrP(106-126) did not prevent F-actin and Rac1 accumulation at sites of particle engulfment, it appeared to interfere with a later step of the internalization process.