Two-way traffic on the road to plasma membrane repair

Abstract

Ca(2+) influx through plasma membrane wounds triggers a rapid-repair response that is essential for cell survival. Earlier studies showed that repair requires the exocytosis of intracellular vesicles. Exocytosis was thought to promote resealing by 'patching' the plasma membrane lesion or by facilitating bilayer restoration through reduction in membrane tension. However, cells also rapidly repair lesions created by pore-forming proteins, a form of injury that cannot be resealed solely by exocytosis. Recent studies indicate that, in cells injured by pores or mechanical abrasions, exocytosis is followed by lesion removal through endocytosis. Describing the relationship between wound-induced exocytosis and endocytosis has implications for the understanding of muscular degenerative diseases that are associated with defects in plasma membrane repair.

Figure 1. Rapid endocytosis is triggered by SLO in the presence of Ca2+

HeLa cells expressing YFP-GPI on the plasma membrane were imaged in a spinning disk confocal microscope in Ca2+-containing medium. (A) Images acquired in the absence of SLO. (B) SLO (100 μg/ml) was locally added with a micropipette 89 s after initiating the imaging. Each image corresponds to 8 optical section confocal stacks acquired at 1 frame /1.4 s. The arrows point to individual endosomes that were detected inside the cells shortly after SLO addition.

Figure 2. Endocytic tracers reveal that the large intracellular vesicles associated with injury sites correspond to rapidly formed endosomes

(A-B) Endothelial cells were injured by passing through a 30-gauge needle in the presence of horse radish peroxidase (HRP), fixed and prepared for transmission EM observation. An accumulation of HRP-positive vesicles was observed at sites near a plasma membrane disruption (arrowheads). Reproduced with permission from Ref. . (C) Transmission EM of NRK cells fixed 4 min after scraping in the presence of Ca²⁺. (D) Transmission EM of NRK cells fixed 4 min after exposure to 200 ng/ml SLO in the presence of Ca²⁺ and the endocytic tracer BSA-gold. The arrows point to large endosomes containing BSA-gold. Bars = 5 μm. (E) Close-up of a BSA-gold containing endosome in a NRK cell wounded by scraping (a procedure that generated numerous plasma membrane lesions, due to disruption of focal contacts). (F) Close-up of a BSA-gold containing endosome in a NRK cell wounded by SLO. Arrows point to individual gold particles. Bars = 200 nm. Reproduced with permission from Ref. .

Figure 3. Model for the rapid membrane traffic events triggered by Ca2+ influx in injured cells

1. Transmembrane pores formed by toxin monomers or mechanical abrasions (microinjection, scratching, laser wounding) wound the plasma membrane. 2. Ca2+ influx triggers lysosomal exocytosis, mediated by v-SNARES and the Ca2+ sensor synaptotagmin VII on lysosomes and t-SNARES on the plasma membrane. Exocytosis results in the release of lysosomal contents extracellularly. 3. Exocytosis is rapidly followed by the formation of large endosomes, which can internalize the lesions and promote plasma membrane repair. Questions that remain to be answered at each of these steps are shown in the right panels.