Live Imaging Assay for Assessing the Roles of Ca&lt;sup&gt;2+&lt;/sup&gt; and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Tam, Christina; Flannery, Andrew R.; Andrews, Norma W.

doi:10.3791/50531

Cited by 10 publications

(9 citation statements)

References 26 publications

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“…To test the hypothesis that extracellular proteolysis may facilitate the access of ASM to the cell surface after exocytosis from lysosomes, we took advantage of the earlier observation that exogenously added purified B . cereus sphingomyelinase (SM) can promote PM repair in the absence of extracellular Ca 2+ , a condition not permissive for lysosomal exocytosis [ 39 ]. As expected, NRK cells permeabilized with SLO in Ca 2+ -free medium showed a small increase in the number of PI-negative cells after treatment with SM ( Fig 2B , upper panel).…”

Section: Resultsmentioning

confidence: 99%

Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

et al. 2016

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Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca2+-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.

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Section: Resultsmentioning

confidence: 99%

Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

et al. 2016

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“…Remarkably, exposure to purified ASM or bacterial sphingomyelinase was sufficient to restore membrane integrity in ASM-depleted cells 9 . Exogenous administration of sphingomyelinase promoted plasma membrane repair even in the absence of Ca 2+ , demonstrating that sphingomyelinase activity can bypass the need for Ca 2+ -triggered lysosomal exocytosis 47 . These findings suggest that the essential contribution of lysosomes to plasma membrane repair is to release ASM, which remodels the cell surface thereby promoting lesion removal.…”

Section: Sphingomyelinase Emerges As a Major Regulator Of Lesion Remomentioning

confidence: 98%

Damage control: cellular mechanisms of plasma membrane repair

Andrews

Almeida

Corrotte

2014

Trends in Cell Biology

267

262

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Summary When wounded, eukaryotic cells reseal in a few seconds. Ca2+ 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 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, which are distinct from the “patch” model.

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“…When there is a breach in the membrane, FM1-43 rapidly flows into cells and intercalates into intracellular membranes. Thus, microscopic live imaging of FM1-43 influx coupled to image analysis allows a precise determination of the kinetics of cell injury and resealing in real-time (Tam, Flannery, & Andrews, 2013), complementing end-point assays based on PI detection by flow cytometry. Live imaging of membrane repair is therefore an important complementary assay to the study of membrane repair that can help identify differences between essential actors and regulators of membrane repair.…”

Section: Procedures For Measuring the Extent Of Plasma Membrane Rmentioning

confidence: 99%

Approaches for plasma membrane wounding and assessment of lysosome-mediated repair responses

Corrotte¹,

Castro-Gomes²,

Koushik³

et al. 2015

Methods in Cell Biology

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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.

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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

Cited by 10 publications

References 26 publications

Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

Damage control: cellular mechanisms of plasma membrane repair

Approaches for plasma membrane wounding and assessment of lysosome-mediated repair responses

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