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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.

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.

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.

Plasma membrane repair (PMR) is an important process for cell homeostasis, especially for cells under constant physical stress. Repair involves a sequence of Ca2+-dependent events, including lysosomal exocytosis and subsequent compensatory endocytosis. Cholesterol sequestration from plasma membrane causes actin cytoskeleton reorganization and polymerization, increasing cell stiffness, which leads to exocytosis and reduction of a peripheral pool of lysosomes involved in PMR. These changes in mechanical properties are similar to those observed in cells exposed to oxidized Low Density Lipoprotein (oxLDL), a key molecule during atherosclerosis development. Using a human umbilical vein endothelial cell line (EAhY926) we evaluated the influence of mechanical modulation induced by oxLDL in PMR and its effect in endothelial fragility. Similar to MβCD (a drug capable of sequestering cholesterol) treatment, oxLDL exposure led to actin reorganization and de novo polymerization, as well as an increase in cell rigidity and lysosomal exocytosis. Additionally, for both MβCD and oxLDL treated cells, there was an initial increase in endocytic events, likely triggered by the peak of exocytosis induced by both treatments. However, no further endocytic events were observed, suggesting that constitutive endocytosis is blocked upon treatment and that the reorganized cytoskeleton function as a mechanical barrier to membrane traffic. Finally, the increase in cell rigidity renders cells more prone to mechanical injury. Together, these data show that mechanical modulation induced by oxLDL exposure not only alters membrane traffic in cells, but also makes them more susceptible to mechanical injury, which may likely contribute to the initial steps of atherosclerosis development.

Intracellular parasites of the genus Leishmania are the causative agents of human leishmaniasis, a widespread emergent tropical disease. The parasite is transmitted by the bite of a hematophagous sandfly vector that inoculates motile flagellated promastigote forms into the dermis of the mammalian host. After inoculation, parasites are ultimately captured by macrophages and multiply as round-shaped amastigote forms. Macrophages seem not to be the first infected cells since parasites were observed invading neutrophils first whose leishmania-containing apoptotic bodies were latter captured by macrophages, thereby becoming infected. The fact that Leishmania spp are able to live and replicate inside immune phagocytic cells and that macrophages are the main cell type found infected in chronicity created the perception that Leishmania spp are passive players waiting to be captured by phagocytes. However, several groups have described the infection of non-phagocytic cells in vivo and in vitro. The objective of this work was to study the cellular mechanisms involved in the invasion of non-professional phagocytes by Leishmania. We show that promastigotes of L.amazonensis actively induces invasion in fibroblasts without cytoskeleton activity, thus by a mechanism that is distinct from phagocytosis. Inside fibroblasts parasites transformed in amastigotes, remained viable for at least two weeks and re-transformed in promastigotes when returned to insect vector conditions. Similarly to what was observed for T. cruzi, infection involves calcium signaling, recruitment and exocytosis of lysosomes involved in plasma membrane repair and lysosome-triggered endocytosis. Conditions that alter lysosomal function such as cytochalasin-D and brefeldin-A treatment or the knockout of host cell lysosomal proteins LAMP-1 and 2 dramatically affected invasion. Likewise, triggering of lysosomal exocytosis and lysosome-dependent plasma membrane repair by low doses of streptolysin-O dramatically increased parasite entry. Together our results show that L.amazonensis promastigotes are able to take advantage of calcium-dependent lysosomal exocytosis and lysosome-induced endocytosis to invade and persist in non-phagocytic cells.AUTHOR SUMMARYIntracellular 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 on Leishmania infection is that non-phagocytic cells have also been found infected by amastigotes. Nevertheless, the mechanisms by which Leishmania invades non-phagocytic cells were not studied to date. Here we show that L. amazonensis can actively induce their own entry into fibroblasts independently of actin cytoskeleton activity, thus by a mechanism that is distinct from phagocytosis. Invasion involves subversion of host cell functions such as calcium signaling and recruitment and exocytosis of host cell lysosomes involved in plasma membrane repair and whose positioning and content interfere in invasion. Parasites were able to replicate and remained viable in fibroblasts, suggesting that cell invasion trough the mechanism demonstrated here could serve as a parasite hideout and reservoir, facilitating infection amplification and persistence.

Gaucher disease (GD) is caused by bi-allelic mutations in GBA1, the gene that encodes acid β-glucocerebrosidase (GCase). Individuals affected by GD have hematologic, visceral and bone abnormalities, and in severe cases there is also neurodegeneration. To shed light on the mechanisms by which mutant GBA1 causes bone disease, we examined the ability of human induced pluripotent stem cells (iPSC) derived from patients with Types 1, 2 and 3 GD, to differentiate to osteoblasts and carry out bone deposition. Differentiation of GD iPSC to osteoblasts revealed that these cells had developmental defects and lysosomal abnormalities that interfered with bone matrix deposition. Compared with controls, GD iPSC-derived osteoblasts exhibited reduced expression of osteoblast differentiation markers, and bone matrix protein and mineral deposition were defective. Concomitantly, canonical Wnt/β catenin signaling in the mutant osteoblasts was downregulated, whereas pharmacological Wnt activation with the GSK3β inhibitor CHIR99021 rescued GD osteoblast differentiation and bone matrix deposition. Importantly, incubation with recombinant GCase (rGCase) rescued the differentiation and bone-forming ability of GD osteoblasts, demonstrating that the abnormal GD phenotype was caused by GCase deficiency. GD osteoblasts were also defective in their ability to carry out Ca(2+)-dependent exocytosis, a lysosomal function that is necessary for bone matrix deposition. We conclude that normal GCase enzymatic activity is required for the differentiation and bone-forming activity of osteoblasts. Furthermore, the rescue of bone matrix deposition by pharmacological activation of Wnt/β catenin in GD osteoblasts uncovers a new therapeutic target for the treatment of bone abnormalities in GD.

Repair of plasma membrane wounds in B lymphocytes that lack caveolin requires lysosome exocytosis and lipid raft–mediated endocytosis and inhibits activation of the B cell receptor by sequestering lipid rafts., Cells rapidly repair plasma membrane (PM) damage by a process requiring Ca2+-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 Ca2+-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.

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.

The study of plasma membrane repair is coming of age. Mirroring human adolescence, the field shows at the same time signs of maturity and significant uncertainty, confusion and skepticism. Here we discuss concepts that emerged from experimental data over the years, some of which are solidly established while others are still subject to different interpretations. The firmly established concepts include the critical requirement for Ca(2+) in wound repair, and the role of rapid exocytosis of intracellular vesicles. Lysosomes are being increasingly recognized as the major vesicles involved in injury-induced exocytosis in many cell types, as a growing number of laboratories detect markers for these organelles on the cell surface and lysosomal hydrolases in the supernatant of wounded cells. The more recent observation of massive endocytosis following Ca(2+)-triggered exocytosis initially came as a surprise, but this finding is also being increasingly reported by different groups, shifting the discussion to the mechanisms by which endocytosis promotes repair, and whether it operates or not in parallel with the shedding of membrane blebs. We discuss how the abundant intracellular vesicles that undergo homotypic fusion close to wound sites, previously interpreted as exocytic membrane patches, actually acquire extracellular tracers demonstrating their endocytic origin. We also suggest that an initial, temporary patch that prevents cytosol loss until the bilayer is restored might result not from vesicular fusion, but from rapid Ca(2+)-dependent crosslinking and aggregation of cytosolic proteins. Finally, we propose that cell surface remodeling, orchestrated by the extracellular release of lysosomal hydrolases and perhaps also cytosolic molecules, may represent a key aspect of the plasma membrane repair mechanism that has received little attention so far.

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.

Rapid plasma membrane repair is essential to restore cellular homeostasis and improve cell survival after injury. Several mechanisms for plasma membrane repair have been proposed, including formation of an intracellular vesicle patch, reduction of plasma membrane tension, lesion removal by endocytosis, and/or shedding of the wounded membrane. Under all conditions studied to date, plasma membrane repair is strictly dependent on the entry of calcium into cells, from the extracellular medium. Calcium-dependent exocytosis of lysosomes is an important early step in the plasma membrane repair process, and defects in plasma membrane repair have been observed in cells carrying mutations responsible for serious lysosomal diseases, such as Chediak–Higashi (Huynh, Roth, Ward, Kaplan, & Andrews, 2004) and Niemann–Pick Disease type A (Tam et al., 2010). A functional role for release of the lysosomal enzyme acid sphingomyelinase, which generates ceramide on the cell surface and triggers endocytosis, has been described (Corrotte et al., 2013; Tam et al., 2010). Therefore, procedures for measuring the extent of lysosomal fusion with the plasma membrane of wounded cells are important indicators of the cellular repair response. The importance of carefully selecting the methodology for experimental plasma membrane injury, in order not to adversely impact the membrane repair machinery, is becoming increasingly apparent. Here, we describe physiologically relevant methods to induce different types of cellular wounds, and sensitive assays to measure the ability of cells to secrete lysosomes and reseal their plasma membrane.

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.

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 Ca2+-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, eLife digest Cells must be able to rapidly repair damage to their outer membranes. This is particularly important in the case of muscle cells, which are vulnerable to damage, and the failure of these cells to repair their outer membranes leads to the muscle wastage seen in muscular dystrophy. Researchers do not fully understand how cells repair membrane, but one popular theory is that they use the membranes of specialized vesicles to ‘patch’ areas that have been damaged. A group of proteins called caveolins have also been implicated in membrane repair but, again, the details have not been worked out. These proteins are best known for their role in the formation of caveolae — small pouches formed by invaginated sections of the plasma membrane. Now, Corrotte et al. have obtained evidence that membrane repair relies not on patching, but on endocytosis (the process by which substances are taken into the cell in small vesicles that ‘pinch’ from the plasma membrane) of these caveolae pouches. Corrotte et al. treated cells with streptolysin O, a toxin that forms pores in the membrane that cannot be repaired using patches, and found that this led to the formation of small membrane-derived vesicles that looked just like caveolae. Further tests confirmed that these vesicles contained caveolar proteins, and that they removed the toxin from the plasma membrane by endocytosis. Similar effects were seen in response to mechanical damage caused by tiny glass beads. Moreover, blocking the expression of caveolar genes prevented cells from repairing membrane damage. Based on their findings, Corrotte et al. propose an alternative model for the repair process; namely that cellular damage triggers an influx of calcium ions, which causes vesicles called lysosomes to release chemicals that promote the formation of caveolae. These then remove the damaged area through endocytosis, restoring the integrity of the membrane. The results offer new insights into why mutations in caveolar proteins are associated with muscle disorders, including muscular dystrophy and cardiac dysfunction. DOI: http://dx.doi.org/10.7554/eLife.00926.002