ESCRT Machinery Is Required
for Plasma Membrane Repair
Ana Joaquina Jimenez,1,2 Paolo Maiuri,1,2 Julie Lafaurie-Janvore,1,2 Séverine Divoux,1,2
Matthieu Piel,1,2 Franck Perez1,2†
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.
Endosomal sorting complex required for transport (ESCRT) has been implicated in multivesicular body (MVB) biogenesis,
viral budding, cytokinesis, and spontaneous bud-
ding of the plasma membrane (1). They are in-
volved in local membrane deformation and scission.
The topology of the bent membrane is very char-
acteristic, with its concave side facing the cy-
toplasm. ESCRT subunits are classified in five
complexes: ESCRT-0, ESCRT-I, ESCRT-II,
ESCRT-III, and ESCRT disassembly subcomplex,
which are recruited in a serial fashion during MVB
biogenesis (2–4). ESCRT-III and ESCRT disas-
sembly proteins appear to be involved in every
ESCRT-associated function. ESCRT-0 and ESCRT-II
Extracellular budding events have been de-
scribed during cellular response to plasma mem-
brane wounding by pore-forming toxins (15, 16).
Because the topology of these budded membranes
was reminiscent of ESCRT-dependent membrane
deformation, we decided to explore the role of
ESCRTs in plasma membrane repair.
We first analyzed the behavior of the ESCRT-III
complex upon localized plasma membrane damage. We followed the dynamics of CHMP4B, an
ESCRT-III protein necessary for all known ESCRT
functions in mammals. The plasma membrane of
HeLa cells was wounded using three different
means. In the first assay, we incubated HeLa cells
expressing CHMP4B-EGFP (enhanced green fluorescent protein) at endogenous levels (17), with
two pore-forming molecules (digitonin at 250 mM
or saponin at 0.05%) added to the cells for a short
time. In both cases, we observed a redistribution
of cytosolic CHMP4B-EGFP to localized spots
at the cell membrane less than 3 min after the addition of the pore-forming molecules (Fig. 1A).
Similar results were obtained with the pore-forming
toxins streptolysin O and listeriolysin O (Fig. 1B).
Second, we mechanically damaged the plasma
membrane using a micropipette in a standard
1Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05, France.
2CNRS UMR 144, 26 rue d’Ulm, 75248 Paris Cedex 05, France.
*These authors contributed equally to this work.
†Corresponding author. E-mail: firstname.lastname@example.org
Fig. 1. CHMP4B is recruited to the wounded
plasma membrane. (A to C) HeLa cells stably
expressing CHMP4B-EGFP at endogenous levels (
referred below as HeLa CHMP4B-EGFP) were used.
Cells were treated with digitonin or saponin for
1 min and fixed for 3 min after the beginning of
the treatment (A) or were treated with streptolysin
O or listeriolysin O for 2.5 min before fixation (B).
Cells were fixed and immunolabeled with antibodies
to GFP (for better visualization of CHMP4B-EGFP
recruitment) (A to C). Plasma membrane wounding
with a glass needle (highlighted with dashed lines)
was followed by differential interference contrast
(DIC) microscopy imaging, and CHMP4B-EGFP recruitment was observed by confocal microscopy in
6 of 20 cells analyzed (C). (D) HeLa cells cotransfected
with CHMP4B-mCherry and MyrPalm-EGFP constructs
were wounded at the plasma membrane with a
UV laser/spinning disc microscope system. Scale
bars, 10 mm.