ESCRT Machinery Is Required for Plasma Membrane Repair

Introduction Plasma membrane damage can result from numerous threats, including mechanical stress or biochemical agents such as pore-forming toxins. Different mechanisms for plasma membrane repair have been described in a variety of cellular models, including patching with endomembranes, endocytosis, and extracellular budding. We found that the endosomal sorting complex required for transport (ESCRT), which is implicated in numerous membrane fission events (such as during cytokinesis or for the budding of several viruses) was also required for the rapid closure of small wounds made at the plasma membrane. ESCRT recruitment mediates pinching out of wounded plasma membrane. (A) Cells expressing the ESCRT subunit CHMP4B-EGFP and wounded (arrow) in the presence of propidium iodide (PI) were observed by means of fluorescence imaging. (B) Model for ESCRT-mediated detection and shedding of wounded plasma membrane. Methods We used micropipettes, detergents, pore-forming toxins, and laser wounding to damage the plasma membrane of mammalian cells in tissue culture. Ultraviolet or two-photon lasers were used to induce small, localized wounds, and cell reactions were followed with time-lapse imaging. Propidium iodide (PI) entry in wounded cells was used to allow imaging of the plasma membrane opening and to quantify the rate of closure of single wounds. Mathematical fit of PI entry kinetics was used to estimate the diameter and the rate of closure of individual wounds. Characterization of PI fluorescence and diffusion gave us an estimation of wound sizes. Transfection of small interfering RNA or dominant-negative mutants of ESCRT subunits allowed us to assess their importance during plasma membrane repair. Last, using correlative-scanning electron microscopy we examined the ultrastructure of wounded plasma membranes. Results The various wounding methods used here revealed a systematic recruitment of ESCRTs to the plasma membrane. Wounding with a laser beam showed that ESCRTs—and in particular, ESCRT-III proteins—were specifically recruited to wound sites and were accumulated until wound closure. This recruitment depended on calcium, which is known to be a crucial signaling molecule for wound repair. The depletion of important ESCRT subunits such as CHMP4B, CHMP2A, or Vps4 was deleterious for a subpopulation of cells bearing small wounds (less than 100 nm in diameter). Correlative scanning electron microscopy and time-lapse imaging revealed that wounding was followed by ESCRT-positive membrane budding and shedding. Energy depletion did not prevent—and rather increased—ESCRT accumulation but prevented both membrane shedding and repair. Discussion These results show that ESCRT proteins play an important role in the detection and removal through the extracellular shedding of small wounds present at the plasma membrane. We propose that different mechanisms for membrane repair (patching, budding, or endocytosis) can be used by cells depending on the type and size of the wound. These mechanisms are stimulated by common early signaling events, such as calcium, but downstream events are likely to depend on the physiochemical characteristics of the wounds. ESCRT-positive plasma membrane shedding has been observed in a variety of normal and pathological conditions. It remains unclear whether these phenomena are linked to local plasma membrane damage and whether ESCRT-III proteins are involved in these processes. ESCRT Your Wound Away The ESCRT (endosomal sorting complex required for transport) protein complex plays a role in budding into multivesicular bodies, in cytokinesis, and in HIV budding. Now, Jimenez et al. (p. 10.1126/science.1247136, published online 30 January) propose a role for ESCRT proteins in wound repair at the plasma membrane. In vivo imaging, modeling, and electron microscopy were used to reveal how the ESCRTs participate in a rapid energy-independent, calcium-dependent, membrane-shedding process at the plasma membrane that reseals small wounds caused by toxins or laser treatment. ESCRT proteins repair small wounds in the plasma membrane by shearing off damaged portions. Plasma membrane damage can be triggered by numerous phenomena, and efficient repair is essential for cell survival. Endocytosis, membrane patching, or extracellular budding can be used for plasma membrane repair. We found that endosomal sorting complex required for transport (ESCRT), involved previously in membrane budding and fission, plays a critical role in plasma membrane repair. ESCRT proteins were recruited within seconds to plasma membrane wounds. Quantitative analysis of wound closure kinetics coupled to mathematical modeling suggested that ESCRTs are involved in the repair of small wounds. Real-time imaging and correlative scanning electron microscopy (SEM) identified extracellular buds and shedding at the site of ESCRT recruitment. Thus, the repair of certain wounds is ensured by ESCRT-mediated extracellular shedding of wounded portions.