Exocytosis of acid sphingomyelinase by wounded cells promotes endocytosis and plasma membrane repair

Tam, Christina; Idone, Vincent; Devlin, Cecilia M.; Fernandes, Maria Cecília; Flannery, Andrew R.; He, Xingxing; Schuchman, Edward H.; Tabas, Ira; Andrews, Norma W.

doi:10.1083/jcb.201003053

Cited by 318 publications

(436 citation statements)

References 46 publications

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“…These results indicate that sphingomyelinase functions downstream of the Ca 2+ -dependent step of plasma membrane repair. This finding is fully consistent with our previously proposed model, which postulates that Ca 2+ flowing through transmembrane pores triggers exocytosis of lysosomes and ASM release, which cleaves sphingomyelin on the outer leaflet of the plasma membrane, generating ceramide and promoting pore removal by endocytosis 22,19 . The fact that addition of purified SM bypasses the need for Ca 2+ strongly reinforces the view that under physiological conditions Ca 2+ -regulated exocytosis of lysosomes is the source of the sphingomyelinase activity required to promote endocytosis.…”

Section: Representative Resultssupporting

confidence: 81%

“…Using the FM1-43 imaging assay, we previously showed that cells deficient for the lysosomal enzyme ASM and exposed to SLO wounding in the presence of Ca 2+ have a plasma membrane defect is rescued by extracellular addition of the recombinant human enzyme 19 . Since the human lysosomal ASM has a low pH optimum, we investigated whether resealing could also be restored adding SM purified from Bacillus cereus (Sigma), which is fully active at neutral pH.…”

Section: Representative Resultsmentioning

confidence: 99%

“…This assay relies on properties of the lipophilic dye FM1-43, which stably intercalates into the outer leaflet of lipid bilayers increasing in fluorescence intensity. When the plasma membrane bilayer is disrupted extracellular dye gains access to intracellular membranes, providing a sensitive assay to detect plasma membrane injury and repair 18,16,19,20 . To adapt this assay for the assessment of cell resealing after permeabilization with pore-forming proteins, we pre-incubated cells at 4 °C with the bacterial toxin streptolysin O (SLO), which binds to membrane cholesterol 21 .…”

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confidence: 99%

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Live Imaging Assay for Assessing the Roles of Ca<sup>2+</sup> and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Tam

Flannery

Andrews

2013

JoVE

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Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca 2+ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca 2+-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca 2+ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca 2+

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Section: Representative Resultssupporting

confidence: 81%

Section: Representative Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Live Imaging Assay for Assessing the Roles of Ca<sup>2+</sup> and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Tam

Flannery

Andrews

2013

JoVE

Self Cite

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“…Recent studies have revealed that exocytosis of acid sphingomyelinase by wounded cells promotes endocytosis and plasma membrane repair (39). Thus, the local elevation of Cer on the plasma membrane is important for endocytosis.…”

Section: Discussionmentioning

confidence: 99%

Dynamic Modification of Sphingomyelin in Lipid Microdomains Controls Development of Obesity, Fatty Liver, and Type 2 Diabetes

Mitsutake

Zama

Yokota

et al. 2011

Journal of Biological Chemistry

169

170

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Lipid microdomains or caveolae, small invaginations of plasma membrane, have emerged as important elements for lipid uptake and glucose homeostasis. Sphingomyelin (SM) is one of the major phospholipids of the lipid microdomains. In this study, we investigated the physiological function of sphingomyelin synthase 2 (SMS2) using SMS2 knock-out mice, and we found that SMS2 deficiency prevents high fat diet-induced obesity and insulin resistance. Interestingly, in the liver of SMS2 knock-out mice, large and mature lipid droplets were scarcely observed. Treatment with siRNA for SMS2 also decreased the large lipid droplets in HepG2 cells. Additionally, the siRNA of SMS2 decreased the accumulation of triglyceride in liver of leptin-deficient (ob/ob) mice, strongly suggesting that SMS2 is involved in lipid droplet formation. Furthermore, we found that SMS2 exists in lipid microdomains and partially associates with the fatty acid transporter CD36/FAT and with caveolin 1, a scaffolding protein of caveolae. Because CD36/FAT and caveolin 1 exist in lipid microdomains and are coordinately involved in lipid droplet formation, SMS2 is implicated in the modulation of the SM in lipid microdomains, resulting in the regulation of CD36/FAT and caveolae. Here, we established new cell lines, in which we can completely distinguish SMS2 activity from SMS1 activity, and we demonstrated that SMS2 could convert ceramide produced in the outer leaflet of the plasma membrane into SM. Our findings demonstrate the novel and dynamic regulation of lipid microdomains via conformational changes in lipids on the plasma membrane by SMS2, which is responsible for obesity and type 2 diabetes.

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“…Cell surface targeting of P2X4Rs by lysosomes has recently been shown to involve the chemokine CC2 [68]. It is well established that injury to a cell induces lysosome exoctyosis [35,38,57,66]; thus, it is conceivable that cell damage resulting from injury to a nerve might mobilize P2X4Rs from lysosomes to the microglial cell surface.…”

Section: Introductionmentioning

confidence: 99%

P2X4 purinoceptor signaling in chronic pain

Trang

Salter

2012

Purinergic Signalling

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ATP, acting via P2 purinergic receptors, is a known mediator of inflammatory and neuropathic pain. There is increasing evidence that the ATP-gated P2X4 receptor (P2X4R) subtype is a locus through which activity of spinal microglia and peripheral macrophages instigate pain hypersensitivity caused by inflammation or by injury to a peripheral nerve. The present article highlights the recent advances in our understanding of microglia-neuron interactions in neuropathic pain by focusing on the signaling and regulation of the P2X4R. We will also develop a framework for understanding converging lines of evidence for involvement of P2X4Rs expressed on macrophages in peripheral inflammatory pain.

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Exocytosis of acid sphingomyelinase by wounded cells promotes endocytosis and plasma membrane repair

Abstract: Lysosomal enzyme acid sphingomyelinase is released extracellularly when cells are wounded, converting sphingomyelin to ceramide and inducing endosome formation to internalize membrane lesions.

Cited by 318 publications

References 46 publications

Live Imaging Assay for Assessing the Roles of Ca<sup>2+</sup> and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Live Imaging Assay for Assessing the Roles of Ca<sup>2+</sup> and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Dynamic Modification of Sphingomyelin in Lipid Microdomains Controls Development of Obesity, Fatty Liver, and Type 2 Diabetes

P2X4 purinoceptor signaling in chronic pain

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