ESCRT Machinery Is Required for
Plasma Membrane Repair
Ana Joaquina Jimenez, Paolo Maiuri, Julie Lafaurie-Janvore, Séverine Divoux, Matthieu Piel,
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 endo-membranes, 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.
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.
FIGURES IN THE FULL ARTICLE
Fig. 1. CHMP4B is recruited to wounded
Fig. 2. CHMP4B-recruitment occurs before
Fig. 3. CHMP2, CHMP3, and the ESCRT-associated protein ALIX are recruited to
Fig. 4. ESCRT-III proteins and the ESCRT-associated protein ALIX are recruited
in a calcium-dependent manner.
Fig. 5. Perturbation of wound resealing
after depletion of ESCRT-III and ESCRT
Fig. 6. ESCRT-positive cell surface budding
Fig. 7. Energy-independent ESCRT recruitment
and energy-dependent shedding of wounded
Materials and Methods
Figs. S1 to S17
Full Reference List
Movies S1 to S8
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.
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Cite this article as A. J. Jimenez et al.,
Science 343, 1247136 (2014).
The list of author affiliations is available in the full article online.
*Corresponding author. E-mail: email@example.com
RESEARCH ARTICLE SUMMARY
28 FEBRUARY 2014 VOL 343 SCIENCE www.sciencemag.org 986